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 fine painting brush (see Table of Materials) to collect about 50 mustard aphids into a 50 mL centrifuge tube from cruciferous crops in the field and bring the sample to the laboratory within 3 h.</li>
		<li>Prepare a temporary maintenance Petri dish with excised cruciferous leaves and water-infiltrated filter paper at the bottom.</li>
		<li>Place the five aphids on the temporary maintenance Petri dish under room temperature (25 &#177; 2 &#176;C) with a relative humidity of 70% and 12:12 h (light:dark) photoperiod for 14 days to confirm that aphid spare natural enemy-free before further rearing. 
		NOTE: To ensure that field-collected aphids are free from natural enemies such as parasitoid wasps or entomopathogenic fungi, it is crucial to confirm the absence of these organisms before proceeding with further rearing.</li>
	</ol>
	</li>
	<li>Maintainance of mustard aphids 
	NOTE: To maintain mustard aphids, use pesticide-free (including biopesticide) cruciferous leaves.
	<ol>
		<li>Provide a stable and sufficient supply of cruciferous leaves (about 25 cm2). 
		NOTE: Obtain the cruciferous leaves either from the market or grow the plants. Before long-term, massive rearing of mustard aphids, several different kinds of crucifer could be tested to find a suitable crucifer. Once the species of the cruciferous leaf is fixed, do not change it. In this study, Komatsuna (Brassica rapa var. perviridis) at 6-7-leaf stage was used (see Table of Materials). Additionally, dispose of the 2-3 oldest leaves.</li>
		<li>Add water before the filter paper at the bottom is completely dry.</li>
		<li>Observe the population density of the mustard aphids daily. The population should grow to 2-3-fold after 7 days.</li>
		<li>When the number of aphids increases, cut the originally excised leaves with mustard aphids into 4-6 smaller pieces and put each small leaf piece with mustard aphids into one Petri dish with fresh excised leaf and water-infiltrated filter paper.</li>
		<li>Place the Petri dish in an incubator at 25 &#176;C with a 12:12 h (light:dark) photoperiod and observe the population density of mustard aphids daily.</li>
		<li>Remove the original excised leaves after most of the aphids migrate to fresh excised leaves before the original leaves rot.</li>
	</ol>
	</li>
</ol>
2. Molecular identification of mustard aphid
NOTE: To confirm the species of field-collected mustard aphid, molecular identification was performed using two molecular markers: the sequence characterized amplified region (SCAR) based A05Le designed by Lu et al.21, and mustard aphid cytochrome oxidase subunit 1 (COI) region of the mustard aphid.
<ol>
	<li>Extraction of mustard aphid genomic DNA
	<ol>
		<li>Collect approximately 50 aphids in a 1.5 mL centrifuge tube. 
		NOTE: Aphids used for molecular identification should be descendants of one single aphid, and various stages could also be pooled in the same tube for DNA extraction.</li>
		<li>Homogenize the aphids with a pellet pestle and extract the DNA using Gene-Spin Genomic DNA Isolation Kit following the manufacturer&#39;s instructions (see Table of Materials).</li>
		<li>Elute the genomic DNA with 50 &#181;L of preheated nuclease-free water.</li>
	</ol>
	</li>
	<li>PCR amplification and DNA sequencing
	<ol>
		<li>Amplify the target DNA sequences from aphid genomic DNA using PCR Master Mix (2x) with primer pairs A05LeF/A05LeR and LeCO1F/LeCO1R (Table 1) with different PCR programs21. 
		NOTE: The PCR reaction with a total volume of 20 &#181;L is composed of 2 &#181;L aphid genomic DNA template, 1 &#181;L forward and 1 &#181;L reversed primers, 10 &#181;L PCR Master Mix (see Table of Materials), and 6 &#181;L ddH2O.</li>
		<li>Perform the PCR in a thermal cycler (see Table of Materials) with the following program: 94 &#176;C for 5 min, 25 cycles of 94 &#176;C for 45 s, 61 &#176;C (for A05LeF/A05LeR) and 58 &#176;C (for LeCO1F/LeCO1R) for 45 s, 72 &#176;C for 1 min, followed by a final extension of 72 &#176;C for 5 min.</li>
		<li>Analyze the PCR product by 1% agarose gel electrophoresis to confirm the identity of the mustard aphid (Figure 2). 
		&#8203;NOTE: If primer pair A05LeF/A05LeR successfully amplifies the correct size of the DNA fragment, it has convincingly identified the field-collected aphid as mustard aphid21. The primer pairs are listed in Table 2.</li>
		<li>Purify the PCR product of mustard aphid COI amplicon using a commercially available gel/PCR kit following the manufacturer&#39;s instructions (see Table of Materials), and sequence the PCR product using a commercial sequencing service.</li>
	</ol>
	</li>
	<li>Sequence analysis 
	NOTE: According to Lu et al.21, the DNA bending of A05Le is a mustard aphid-specific DNA fragment; therefore, the A05Le PCR product does not need to be further sequenced.
	<ol>
		<li>Check the read quality by Chromas software (see Table of Materials) after obtaining the LeCO1 sequence.</li>
		<li>Trim upstream and downstream low-quality reads by opening a fasta (or txt) file and directly removing the low-quality reads.</li>
		<li>Submit the trimmed sequence to NCBI web BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) using default parameters.&#160; 
		&#8203;NOTE: If the amplicon sizes of A05Le and LeCO1 primer sets are correct, theoretically, the sequence blast search against NCBI standard databases must match mustard aphid.</li>
	</ol>
	</li>
</ol>
3. Preparation of Entomopathogenic fungi
NOTE: The EPF used in this study is listed in Table 1.
<ol>
	<li>Recovery of EPF from fungal library
	<ol>
		<li>Thaw the preserved fungi stock (conidia suspended in 30% glycerin) from the -80 &#176;C freezer.</li>
		<li>Transfer a small amount (approximately 10 &#181;L) of the conidia suspension to 6 cm &#188; Sabouraud dextrose agar (SDA) plate (0.75 g Sabouraud dextrose broth with 1.5 g agar per 100 mL ddH2O) and spread it evenly using a cell spreader in a laminar flow hood.</li>
		<li>Seal the Petri dish with paraffin film, and incubate it in darkness at 25 &#176;C for 10-14 days.</li>
		<li>Re-culture the fungal isolates by streaking fungal mass on a &#188; SDA plate and incubating fungi in darkness at 25 &#176;C for 10-14 days before harvesting the conidia22. 
		NOTE: The fungal culture should be used approximately 10 days before inoculating the aphids. EPF culture that is more than 14 days old is not recommended for use in the virulence test.</li>
	</ol>
	</li>
	<li>Preparation of conidia suspension
	<ol>
		<li>Scrape the conidia from the 10-14 days old fungal culture on &#188; SDA plate by inoculating loop after adding 2-3 mL 0.03% Tween 80 (see Table of Materials).</li>
		<li>Collect the fungal suspension into a centrifuge tube.</li>
		<li>Vortex the solution at the highest speed, count the number of conidia using a hemocytometer under a light microscope, and adjust the concentration of the conidia suspension to 108 conidia/mL.</li>
		<li>Transfer the conidia suspension into a UV-sterilized micro-sprayer. 
		&#8203;NOTE: Vortex the conidia suspension again before transferring it, as the conidia are prone to precipitate. The micro-sprayer should be exposed to ultraviolet radiation in a laminar flow hood for 30 min before use.</li>
	</ol>
	</li>
</ol>
4. Virulence screening against mustard aphid
<ol>
	<li>Preparation of newly emerged adults
	<ol>
		<li>Move apterous (without wing) and alate (winged) adults from the rearing Petri dish to freshly excised leaves to reproduce new progenies of the same age. Remove the adults after 24 h to avoid overcrowding and minimize the production of alated progenies23,24. Only apterous adults will be used in further experiments.</li>
		<li>Rear progenies to the adult stage and remove the progenies that do not develop into the adult stage after 48 h (Figure 3) to prevent age variation between aphids for the experiment. Additionally, remove alated adults.</li>
	</ol>
	</li>
	<li>Preparation of inoculation chamber 
	NOTE: It is recommended to prepare 9 cm diameter cruciferous leaves first, so that the leaf disc can be embedded into the water agar before solidification.
	<ol>
		<li>Clean the cruciferous leaves with tap water, remove dirt and residue, and any other arthropod if present.</li>
		<li>Wipe away any excess water with a paper towel and check for any other arthropods on the surface of the cruciferous leaves using a stereomicroscope. Remove any arthropods if present.</li>
		<li>Cut a 9 cm diameter leaf disc either by directly pressing the bottom Petri dish on the leaf as a mold or by using scissors. 
		NOTE: If the leaves are smaller than 9 cm in diameter, combine a couple of excised leaves.</li>
		<li>Prepare 1.5% water agar for each Petri dish by heating and dissolving 450 mg of agar (see Table of Materials) in 30 mL ddH2O using microwave. 
		NOTE: The amount of 1.5% water agar can be adjusted depending on the experiment scale.</li>
		<li>Pour 30 mL of 1.5% water agar in the 9 cm Petri dish before solidification in the laminar flow hood, and then wait for the agar to cool down to ~40 &#176;C until the surface of the agar is semi-solidified. 
		NOTE: Check if the surface is semi-solidified by gently horizontally shaking the Petri dish.</li>
		<li>Place the leaf disc, abaxial surface up, on top of the water agar, embedding the leaf disc in the agar. 
		NOTE: Minimize the exposure of the agar surface.</li>
		<li>After the water agar has solidified completely, close the cover of the Petri dish. 
		NOTE: Open the cover for water vaporization if lots of droplets are condensing on the cover immediately.</li>
		<li>Place 20 apterous adults on the leaf disc. 
		NOTE: It is recommended to operate under a stereomicroscope to prevent damage to their mouthparts.</li>
	</ol>
	</li>
	<li>EPF inoculation and observation
	<ol>
		<li>Open the cover of the inoculation chamber and spray 0.3 mL of conidia suspension (from step 3.2) directly onto the aphids and leaf disc from about 15 cm above the leaf disc. 
		NOTE: Vortex the conidia suspension again before spraying. The spraying areas should be tested before the experiment as they can vary between different sprayers. In this case, 0.3 mL with 15 cm distance is recommended. The spraying area should cover the entire leaf disc, and a thin layer of conidia suspension should cover the leaf disc. If droplets converge into standing water on the leaf, the inoculation volume should be decreased. Clean the table with 70% ethanol before inoculation and between using different isolates.</li>
		<li>Wait for the conidia suspension to dry, and then close the cover to prevent mustard aphids from drowning. 
		NOTE: Aphids seldom roam around during the experiment; however, ensuring that the aphids do not escape is still necessary.</li>
		<li>Seal the inoculation chamber with paraffin film to maintain high relative humidity and place the inoculation chamber in an incubator at 25 &#176;C with 12:12 h (light:dark) photoperiod.</li>
		<li>Open the cover of the inoculation chamber to count mortality under a stereomicroscope every 12 h for 5 days.</li>
		<li>Wipe out the honeydew adhering to the cover with a paper towel and remove the newly emerged aphids with a fine painting brush. Clean the cover with tap water every 24 h. 
		NOTE: The observation period might vary for different species of aphids based on adult longevity.</li>
		<li>Keep the cadaver on the leaf disc for mycosis to confirm successful inoculation.</li>
	</ol>
	</li>
	<li>Confirmation of conidia germination rate 
	NOTE: This step is optional. To confirm the conidia germination rate pairwise when performing the virulence screening to ensure the activity of conidia.
	<ol>
		<li>Drop one droplet of 5 &#181;L conidia suspension on &#188; SDA for three replicates.</li>
		<li>After 18 h, calculate the germination rate with the light microscope. Count at least 100 spores randomly in a single droplet to confirm the fungal germination rate. 
		&#8203;NOTE: Normally, the germination rate of the isolates used in the experiment should be higher than 90%.</li>
	</ol>
	</li>
</ol>
5. Bioassay of selected EPF isolates
NOTE: EPF isolates that showed high virulence, which was selected from step 4, were subjected to a bioassay against mustard aphids using four concentrations of conidia suspensions (ranging from 104 to 107 conidia/mL).
<ol>
	<li>Preparation of conidia suspension
	<ol>
		<li>Repeat step 3.1 to recover the selected EPF isolates.</li>
		<li>Repeat step 3.2 to prepare four concentrations of conidia suspension, including 104, 105,106, and 107 conidia/mL.</li>
	</ol>
	</li>
	<li>Preparation of newly emerged adults
	<ol>
		<li>Repeat step 4.1 to prepare newly emerged adults.</li>
	</ol>
	</li>
	<li>Preparation of inoculation chamber
	<ol>
		<li>Repeat step 4.1. to prepare the inoculation chamber.</li>
	</ol>
	</li>
	<li>EPF inoculation and observation
	<ol>
		<li>Repeat step 4.3. to perform the EPF inoculation and observation with the four concentrations of conidia suspension, respectively.</li>
	</ol>
	</li>
</ol>
6. Statistical analysis
<ol>
	<li>Calculation of corrected mortality
	<ol>
		<li>Calculate corrected mortality using Abbott&#39;s formula25, as shown below: 
		Corrected mortality (%) = [(MT&#8722; MC) &#215; 100%] / (100&#8722; MC) 
		MT represents the mortality of the treatment group, and MC represents the mortality of the control group. 
		&#8203;NOTE: The mortality of the control group should not be higher than 20%.</li>
		<li>Open the SPSS software platform (see Table of Materials), create variables including &#34;treatment&#34; and &#34;cor_mor&#34; (representing corrected mortality), and enter the calculated results for the inoculation of different isolates at the same time point with the same inoculated concentration.</li>
		<li>Select Analyze, Compare Means and Proportions, Independent-Samples T Test in SPSS for independent t-test analysis.</li>
		<li>In SPSS, input treatment into the &#34;Grouping Variable&#34; box, and cor_mor into the &#34;Test Variable(s)&#34;. Define groups by different fungal isolates. Press OK to analyze.</li>
	</ol>
	</li>
	<li>Calculation of median lethal time (LT50) and median lethal concentration (LC50)
	<ol>
		<li>Open the SPSS, create variables &#34;total&#34;, &#34;response&#34; and &#34;duration&#34;/&#34;concentration&#34;, and input the recorded result into the spreadsheet.</li>
		<li>Select Analyze, Regression, Probit in SPSS to calculate LT50 and/or LC50. 
		NOTE: If cumulative mortality does not exceed 50% eventually, LT50 and/or LC50 cannot be estimated.</li>
		<li>Input total into the &#34;Total Observed&#34; box, response into &#34;Response Frequency&#34; box, duration/concentration into &#34;Covariate(s)&#34; box. Press OK to analyze. 
		NOTE: Generally, press Options and set the &#34;Significance level for use of heterogeneity factor&#34; to 0.05.</li>
	</ol>
	</li>
</ol>

Optimized Bioassay for Entomopathogenic Fungi Against Mustard Aphid

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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. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+evaluation+of+temperature+effects+on+the+germination+and+growth+of+entomopathogenic+fungi+and+on+their+pathogenicity+to+two+aphid+species.">Laboratory evaluation of temperature effects on the germination and growth of entomopathogenic fungi and on their pathogenicity to two aphid species.</a> Pest Management Science. 59 (2), 156-165 (2003).</li><li>Erdos, Z., Chandler, D., Bass, C., Raymond, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+insecticide+resistant+clones+of+the+aphid,+Myzus+persicae,+using+the+entomopathogenic+fungus+Akanthomyces+muscarius:+fitness+cost+of+resistance+under+pathogen+challenge.">Controlling insecticide resistant clones of the aphid, Myzus persicae, using the entomopathogenic fungus Akanthomyces muscarius: fitness cost of resistance under pathogen challenge.</a> Pest Management Science. 77 (11), 5286-5293 (2021).</li></ol>

The authors declare there is no conflict of interest involved in this work.

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.

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

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 insects.

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, ...

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1.7K 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 

Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; agar-block). It is well-accepted that soil-block microcosms yield higher decay rates, fewer moisture issues, lower variability among studies, and higher thresholds of preservative toxicity. Soil-block testing is thus the more utilized technique and has been standardized by American Society for Testing and Materials (ASTM) (method D 1413-07). The soil-block design has drawbacks, however, using locally-variable soil sources and in limiting the control of nutrients external (exogenous) to the decaying tissues. These drawbacks have emerged as a problem in applying this method to other, increasingly popular research aims. These modern aims include degrading lignocellulosics for bioenergy research, testing bioremediation of co-metabolized toxics, evaluating oxidative mechanisms, and tracking translocated elements along hyphal networks. Soil-blocks do not lend enough control in these applications. A refined agar-block approach is necessary.
Here, we use the brown rot wood-degrading fungus Serpula lacrymans to degrade wood in agar-block microcosms, using deep Petri dishes with low-calcium agar. We test the role of exogenous gypsum on decay in a time-series, to demonstrate the utility and expected variability. Blocks from a single board rip (longitudinal cut) are conditioned, weighed, autoclaved, and introduced aseptically atop plastic mesh. Fungal inoculations are at each block face, with exogenous gypsum added at interfaces. Harvests are aseptic until the final destructive harvest. These microcosms are designed to avoid block contact with agar or Petri dish walls. Condensation is minimized during plate pours and during incubation. Finally, inoculum/gypsum/wood spacing is minimized but without allowing contact. These less technical aspects of agar-block design are also the most common causes of failure and the key source of variability among studies. Video publication is therefore useful in this case, and we demonstrate low-variability, high-quality results.

This video demonstrates a controlled environment approach to study degradation of lignocellulosic plant tissues by aerobic fungi. The ability to control nutrient sources and moisture is a key advantage of agar-block microcosms, but the approach often yields mixed success. We address critical pitfalls to yield reproducible, low-variability results.

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; ...

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, ...

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of these disease vectors. We have recently identified a bitter sensing neuron in the labellum of female Aedes aegypti that responds to DEET and other repellents, as well as bitter quinine, through direct electrophysiological investigation. These gustatory receptor neuron responses prompted our sequencing of total mRNA from both male and female labella and tarsi samples to elucidate the putative chemoreception genes expressed in these contact chemoreception tissues. Samples of tarsi were divided into pro-, meso- and metathoracic subtypes for both sexes. We then validated our dataset by conducting qRT-PCR on the same tissue samples and used statistical methods to compare results between the two methods. Studies addressing molecular function may now target specific genes to determine those involved in repellent perception by mosquitoes. These receptor pathways may be used to screen novel repellents towards disruption of host-seeking behavior to curb the spread of harmful viruses.

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Using two methods to estimate gene expression in the major gustatory appendages of Aedes aegypti, we have identified the set of genes putatively underlying the neuronal responses to bitter and repulsive compounds, as determined by electrophysiological examination.

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of ...

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It also describes an imaging method for studying the 3D structure of thick biological specimens by scanning transmission electron tomography. The sample preparation protocol is based on conventional methods in which the sample is fixed using chemical agents, treated with a heavy atom salt contrasting agent, dehydrated in a series of ethanol baths, and embedded in resin. The specific imaging conditions for observing thick samples by scanning transmission electron microscopy are then described. Sections of the sample are observed using a through-focus method involving the collection of several images at various focal planes. This enables the recovery of in-focus information at various heights throughout the sample. This particular collection pattern is performed at each tilt angle during tomography data collection. A single image is then generated, merging the in-focus information from all the different focal planes. A classic tilt-series dataset is then generated. The advantage of the method is that the tilt-series alignment and reconstruction can be performed using standard tools. The collection of through-focal images allows the reconstruction of a 3D volume that contains all of the structural details of the sample in focus.

This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to study the functions and evolution of bacterial symbionts in their host. As most symbionts cannot be cultured in vitro, methods to isolate an adequate quantity of bacteria for genome sequencing are very important. In the whitefly Bemisia tabaci, a number of endosymbionts have been identified and are predicted to be of importance in the development and reproduction of the pests through multiple approaches. However, the mechanism underpinning the associations remains largely unknown. The obstacle partially comes from the fact that the endosymbionts in whitefly, mostly restrained in bacteriocytes, are hard to separate from the host cells. Here we report a step-by-step protocol for the identification, extraction and purification of endosymbionts from the whitefly B. tabaci mainly by dissection and filtration. Endosymbiont samples prepared by this method, although still a mixture of different endosymbiont species, are suitable for subsequent genome sequencing and analysis of the possible roles of endosymbionts in B. tabaci. This method may also be used to isolate endosymbionts from other insects.

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Here, we present a protocol to isolate endosymbionts from the whitefly Bemisia tabaci through dissection and filtration. After amplification, the DNA samples are suitable for subsequent sequencing and study of the mutualism between endosymbionts and whitefly.

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...

Application of Membrane and Cell Wall Selective Fluorescent Dyes for Live-Cell Imaging of Filamentous Fungi

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two decades ago and since then continues to contribute greatly to our understanding of the filamentous fungal lifestyle. This paper provides a practical guide for the utilization of the two membrane dyes FM 1-43 and FM 4-64 and the four cell wall stains Calcofluor White M2R, Solophenyl Flavine 7GFE 500, Pontamine Fast Scarlet 48 and Congo Red. The focus is on their low-dose application to ascertain artefact-free staining, their co-imaging properties, and their quantitative evaluation. The presented methods are applicable to all filamentous fungal samples that can be prepared in the described ways. The fundamental staining approaches can serve as starting points for adaptations to species that might require different cultivation conditions. First, biophysical and biochemical properties are reviewed as their understanding is essential for using these dyes as truly vital fluorescent stains. Secondly, step-by-step protocols are presented that detail the preparation of various fungal sample types for fluorescent live-cell imaging. Finally, example experiments illustrate different approaches to: (1) identify defects in the spatio-temporal organization of endocytosis in genetic mutants, (2) comparatively characterize shared and distinct co-localization of GFP-labeled target proteins in the endocytic pathway, (3) identify morphogenetic cell wall defects in a genetic mutant, and (4) monitor cell wall biogenesis in real time.

Vital fluorescent dyes are essential tools for live-cell imaging analyses in modern fungal cell biology. This paper details the application of established and lesser-known fluorescent dyes for tracking plasma membrane dynamics, endo-/exocytosis and cell wall morphogenesis in filamentous fungi.

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two ...

A 3D Printed Pollen Trap for Bumble Bee (Bombus) Hive Entrances

To verify the plant sources from which bumble bees forage for pollen, individuals must be collected to remove their corbicular pollen loads for analysis. This has traditionally been done by netting foragers at nest entrances or on flowers, chilling the bees on ice, and then removing the pollen loads from the corbiculae with forceps or a brush. This method is time and labor intensive, may alter normal foraging behavior, and can result in stinging incidents for the worker performing the task. Pollen traps, such as those used on honey bee hives, collect pollen by dislodging corbicular pollen loads from the legs of workers as they pass through screens at the nest entrance. Traps can remove a large quantity of pollen from returning forager bees with minimal labor, yet to date no such trap is available for use with bumble bee colonies. Workers within a bumble bee colony can vary in size making size selection of entrances difficult to adapt this mechanism to commercially reared bumble bee hives. Using 3D printing design programs, we created a pollen trap that successfully removes the corbicular pollen loads from the legs of returning bumble bee foragers. This method significantly reduces the amount of time required by researchers to collect pollen from bumble bee foragers returning to the colony. We present the design, results of pollen removal efficiency tests, and suggest areas of modifications for investigators to adapt traps to a variety of bumble bee species or nest box designs.

A 3D Printed Pollen Trap for Bumble Bee Bombus Hive Entrances

We present a non-lethal and automated mechanism to collect pollen from bumble bee (Bombus) workers returning to a hive. Instructions for producing, preparing, installing and using the devices are included. By using 3D-printed objects, modification to the design was timely, efficient and allowed for quick turnaround for testing.

To verify the plant sources from which bumble bees forage for pollen, individuals must be collected to remove their corbicular pollen loads for analysis.  ...

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by filarial nematodes such as canine heartworm and elephantasis. Recently, CRISPR/Cas9 genome editing has been used to induce site-directed mutations by injecting a Cas9 protein that has been complexed with a guide RNA (gRNA) into freshly laid embryos of several insect species, including mosquitoes that belong to the genera Anopheles and Aedes. Manipulating and injecting Culex mosquitoes is slightly more difficult as these mosquitoes lay their eggs upright in rafts rather than individually like other species of mosquitoes. Here we describe how to design gRNAs, complex them with Cas9 protein, induce female mosquitoes of Culex pipiens to lay eggs, and how to prepare and inject newly laid embryos for microinjection with Cas9/gRNA. We also describe how to rear and screen injected mosquitoes for the desired mutation. The representative results demonstrate that this technique can be used to induce site-directed mutations in the genome of Culex mosquitoes and, with slight modifications, can be used to generate null-mutants in other mosquito species as well.&#160;

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

CRISPR/Cas9 is increasingly used to characterize gene function in non-model organisms. This protocol describes how to generate knock-out lines of Culex pipiens, from preparing injection mixes, to obtaining and injecting mosquito embryos, as well as how to rear, cross, and screen injected mosquitoes and their progeny for desired mutations.

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by ...

Immunofluorescent Labeling of Plant Virus and Insect Vector Proteins in Hemipteran Guts

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are highly efficient at virus transmission plays a key role in virus epidemics in fields. Studying virus-insect vector interactions can advance our understanding of virus transmission and epidemics with the aim of designing novel strategies to control plant viruses and their vector insects. Immunofluorescence labeling has been widely used to analyze interactions between pathogens and hosts and is used here in the white-backed planthopper (WBPH, Sogatella furcifera), which efficiently transmits the southern rice black streaked dwarf virus (SRBSDV, genus Fijivirus, family Reoviridae), to locate the virions and insect proteins in the midgut epithelial cells. Using laser scanning confocal microscopy, we studied the morphological characteristics of midgut epithelial cells, cellular localization of insect proteins, and the colocalization of virions and an insect protein. This protocol can be used to study virus activities in insects, functions of insect proteins, and interactions between virus and vector insect.

This protocol for immunofluorescent labeling of both plant virus proteins and vector insect proteins in excised insect guts can be used to study interactions among virus and vector insects, insect protein functions and molecular mechanisms underlying virus transmission.

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are ...

Isolation and Selection of Entomopathogenic Fungi from Soil Samples and Evaluation of Fungal Virulence against Insect Pests

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important to isolate and select indigenous EPF. Therefore, the soil bait method combined with the insect bait (mealworm, Tenebrio molitor) system was used in this study with some modifications. The isolated EPF were then subjected to the virulence test against the agricultural pest Spodoptera litura. Furthermore, the potential EPF strains were subjected to morphological and molecular identifications. In addition, the conidia production and thermotolerance assay were performed for the promising EPF strains and compared; these data were further substituted into the formula of effective conidia number (ECN) for laboratory ranking. The soil bait-mealworm system and the ECN formula can be improved by replacing insect species and integrating more stress factors for the evaluation of commercialization and field application. This protocol provides a quick and efficient approach for EPF selection and will improve the research on biological control agents.

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 field.

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important ...