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

NOTE: The whole flowchart is shown in Figure 1.
1. Isolation and selection of potential Entomopathogenic fungi (EPF)
<ol>
	<li>Collect the soil sample
	<ol>
		<li>Remove 1 cm of the surface soil, and then collect the soil within the 5-10 cm depth using a shovel from each sampling site. 
		NOTE:Sampling sites would be a mountain, forest, or sparsely populated areas to avoid the contamination of artificially sprayed EPF strains. Ensure that areas for the soil samples collection are covered with weed on the surface. Dry soil or damp soil is not suitable for this experiment.</li>
		<li>Record the details of each sampling site, including GPS, elevation, type of field, annual temperature, yearly precipitation, collection time (season), soil type, and pH value.</li>
		<li>Collect 100 g of the soil sample into a plastic bag and maintain it at room temperature if subjecting it to the fungal isolation protocol in the laboratory within 3 h. 
		NOTE: If the sample cannot be used within 3 h, store the soil at room temperature in dark conditions. If the experiment is not performed immediately, the soil sample can be stored at 4 &#176;C for 1 week until the start of the protocol18,19.</li>
	</ol>
	</li>
	<li>Bait and isolate the EPF with mealworm (Tenebrio molitor)
	<ol>
		<li>Place 100 g of the soil sample in a plastic cup (cap diameter = 8.5 cm, height = 12.5 cm), and then place 5 mealworms on the surface of the soil at room temperature in the dark for 2 weeks. 
		NOTE: Other types of plastic cup containers can also be used. If soils are too dry (cracked or sandy), spray sterilized ddH2O (about 5-10 mL) on the soils. The body length c.a. 2.5 cm (14th instar) of T. molitor larvae helps in fungal isolate screens20.</li>
		<li>Observe and record the larvae daily for mortality and mycosis; keep the dead larvae in the cup until 2 weeks for fungal isolation. 
		NOTE: The fungal conidia spore in the soil samples will attach to the mealworm larvae during the above process. The fungal mycosis will be observed as the hyphae grow from the intersegmental membrane, and then the whole body will be covered with the mycelium. Sporulation will start after 7 days and the color of the fungal infection will change to the color of the conidia mass.</li>
		<li>Transfer the dead insects to a clean bench and use a sterile toothpick to collect the conidia. Streak them on a quarter strength Sabouraud dextrose agar medium (&#188; SDA) plate (55 mm) in the laboratory21. Incubate the culture plate at 25 &#176;C for 7 days to obtain the primary culture of fungi. 
		NOTE: The &#188; SDA plate is prepared as follows: Mix 1.5 g of Sabouraud dextrose broth and 3 g of agar in 200 mL of H2O, and then sterilize for 20 min. Aliquot &#188; SDA into each 55 mm Petri dish before solidification. The solidified &#188; SDA plates are stored at 4 &#176;C until use.</li>
		<li>Re-streak each primary culture fungi on one 55 mm &#188; SDA plate in a laminar flow and incubate the culture plate at 25 &#176;C for 7 days to obtain single colonies of fungi.</li>
		<li>Repeat this re-isolation ~2-3 times and observe under light microscopy to obtain single and pure morphological fungal colonies. 
		NOTE: Isolate all EPF using T. molitor-bait and store as described in the following section.The separation, preservation, pure culture, and streaking must be performed in a laminar flow in the subsequent sections.</li>
	</ol>
	</li>
	<li>Store the EPF isolates
	<ol>
		<li>Cut 3 of the 5 mm agar blocks at the edge of each isolated pure fungal cultured plate with a cork borer and place it into a 1.5 mL micro-centrifuge tube as one replicate. 
		NOTE: Three replicates for each fungal isolate are recommended for the EPF strains&#39; storage after 2nd virulence test. Molecular identification is also recommended if storage space is limited.</li>
		<li>Add 250 &#181;L of 0.03% surfactant solution (Table of Materials) and 250 &#181;L of 60% glycerol into the 1.5 mL micro-centrifuge tube using a micropipette; then, vortex for 10 s.</li>
		<li>Seal the 1.5 mL micro-centrifuge tube with paraffin film and precool it in a -20 &#176;C refrigerator for 24 h. Then, transfer the precooled fungal stocks to a -80 &#176;C refrigerator for cryopreservation. 
		NOTE: Extra pure fungal culture plates (aside from the cryo-preserved samples) were used in section 1.4.</li>
	</ol>
	</li>
	<li>1st pathogenicity screen for fungal isolates
	<ol>
		<li>Place five T. molitor larvae directly on the surface of each pure fungal culture plate at 25 &#176;C.</li>
		<li>Observe and record the mycosis and mortality for 10 days. Select the fungal isolate for further analysis. 
		NOTE: Fungal isolates causing 100% mortality are selected for 2nd virulence test to confirm their virulence to T. molitor larvae. Alternatively, the researcher can adjust the criteria as per their own study.</li>
	</ol>
	</li>
	<li>2nd virulence test of fungal isolates 
	NOTE: Based on the 1st pathogenicity screen, recover the selected fungal isolates from -80 &#176;C for the 2nd virulence test. The purpose of the 2nd virulence test is to quantify the pathogenicity of the selected fungal isolates after the 1st round of screening.
	<ol>
		<li>Harvest conidia of each fungal isolate by vortexing for 1 min and count the number of conidia using a hemocytometer.</li>
		<li>Adjust the conidia suspension to a concentration of 1 x 107 conidia/mL in a 0.03% surfactant solution (Table of Materials).</li>
		<li>Spread 10 &#181;L of the fungal suspension onto 55 mm &#188; SDA plates and grow for 7 days at 25 &#176;C in the dark.</li>
		<li>Place five T. molitor larvae directly on the surface of each pure fungal culture plate (c.a. 6 x 107 conidia). Seal the plates with paraffin film and incubate at 25 &#176;C in the dark.</li>
		<li>Observe and record the mycosis and mortality for 10 days.</li>
		<li>Repeat the test (from step 1.51 to 1.5.5) in triplicate for each fungal isolate. 
		NOTE: Fungal isolates causing 100% mortality are selected for 3rd virulence test to confirm their virulence to target the pest.</li>
	</ol>
	</li>
	<li>3rd virulence test of fungal isolates for target pest (Spodoptera litura as an example)
	<ol>
		<li>Repeat steps 1.5.2 to 1.5.6 with selected isolates from 2nd virulence to test virulence against target pest.</li>
		<li>Calculate the LT50 of each fungal isolate22. 
		&#8203;NOTE: The LT50 of each fungal isolate was calculated through generalized linear models (GLMs) using R studio (version 3.4.1); the quasibinomial error distribution and a log link function can be used to account for overdispersion.</li>
	</ol>
	</li>
</ol>
2. Molecular identification of EPF
<ol>
	<li>Extraction of fungal genomic DNA
	<ol>
		<li>Collect c.a. 1 cm2 EPF from the 7-day &#188; SDA plate.</li>
		<li>Extract the fungal genomic DNA&#160;using a fungal genomic DNA extraction kit according to the manufacturer&#39;s instructions23 (Table of Materials).</li>
	</ol>
	</li>
	<li>PCR amplification and DNA sequencing
	<ol>
		<li>Amplify Fungal ITS region by PCR of the DNA sample21 using the PCR Master Mix (2x), ITS1F/ITS4R primer set24 (Table 1) with the following PCR program: 94 &#176;C for 1 min, and then 35 cycles of 94 &#176;C for 30 s, 55 &#176;C for 30 s, and 72 &#176;C for 1 min, followed by a 7 min final extension at 72 &#176;C. 
		NOTE: The ITS1F/ITS4R primer set is for the genus level identification.</li>
		<li>Sequence the PCR by commercial sequencing service.</li>
		<li>Use NCBI BLAST search for similar fungi in the NCBI database and select the relative fungal type species for phylogenetic analysis. 
		NOTE: The fungal species belonging to the genus of Metarhizium or Beauveria should be further identified to the species level with tef-983F/tef-2218R primer set25 (repeat steps 2.1.1 to 2.2.3). For fungi that do not belong to the genera Metarhizium or Beauveria, other molecular markers can be used to identify the species, including DNA lyase (APN2), beta tubulin (BTUB), RNA polymerase II largest subunit (RPB1), RNA polymerase II second largest subunit (RPB2), and 3&#39; portion of translation elongation factor 1 alpha (TEF)25,26.</li>
	</ol>
	</li>
	<li>Phylogenetic analysis
	<ol>
		<li>Use ClustalX 2.1 software27 to align the multiple sequences from steps 2.2.2 and 2.2.3. Trim the conserved sequences region manually with GeneDoc28.</li>
		<li>Perform the phylogenetic analysis by MEGA7 software29 based on the minimum evolution (ME), Neighbor-Joining (NJ), and maximum likelihood (ML) methods. 
		NOTE: Performing all three methods can help to confirm and accurately conclude the classification status. The fungal isolates screened by the 1st pathogenicity screen are used for molecular identification at the genus level. The fungal isolates screened by the 2nd virulence test are used for the species level molecular and morphological identification.</li>
	</ol>
	</li>
</ol>
3. Morphological identification of EPF
<ol>
	<li>Observation of the fungal colony morphology
	<ol>
		<li>Use a camera to capture the&#160;fungal culture colony growth for 7 days, and record the growth, form (fluffy, firm), and color of the colonies.</li>
	</ol>
	</li>
	<li>Observation of conidia and conidiophores
	<ol>
		<li>Scrape conidia from the pure culture fungal colony with an inoculation loop and transfer the spores to a glass slide with 0.1% Tween 80 solution. Then, cover with a coverslip for light microscopic observation of conidia.</li>
		<li>Use a scalpel to cut a 5 mm2 agar block of the hyphal strand at the edge of the fungal colony, and then transfer the agar block to a glass slide.</li>
		<li>Perform the cleaning as follows: Add the 0.1% Tween 80 solution on the agar block with a plastic dropper and wash off most of the excess conidia using tweezers. Then, cover it with a coverslip for lightmicroscopic observation. 
		NOTE: 0.1% Tween 80 can be substituted with another more potent surfactant (Table of Materials) depending on fungal species and hydrophobicity.</li>
		<li>Measure and record the width and length of the conidia and conidiophores to compare the differences between different fungal isolates.</li>
		<li>Use Welch&#39;s ANOVA test and Games-Howell test (post-hoc test) to analyze the conidial width and length of each strain using R studio (version 3.4.1). 
		NOTE: Data analysis of morphological characters can be adjusted by cases. The fungal isolates screened with the 3rd virulence test are used for the physiological characterization and ECN ranking in sections 4 and 5.</li>
	</ol>
	</li>
</ol>
4. Investigation of conidial productivity and thermotolerance
<ol>
	<li>Conidial production assay
	<ol>
		<li>Culture the selected fungal isolate on &#188; SDA medium at 25 &#177; 1 &#176;C in dark for 10 days.</li>
		<li>Prepare 1 mL of the conidial suspension of the fungal isolate in 0.03% surfactant solution and adjust to 1 x 107 conidia/mL as described above.</li>
		<li>Drop three droplets of 10 &#181;L of conidial suspension on&#188; SDA and incubate at 25 &#176;C in the dark for 7, 10, and 14 days to count the sporulation of fungi. 
		NOTE: 10 &#181;L is the best volume to collect the 5 mm block with even fungal sporulation after fungal growth for 7-14 days.</li>
		<li>Use the cork borer to detach 5 mm agar block from the center of the colony and transfer into 1 mL of 0.03% surfactant solution (Table of Materials) in a 1.5 mL micro-centrifuge tube at each time point.</li>
		<li>Vortex the tube at 3,000 rpm at room temperature for 15 min and use a hemocytometer to count the number of conidia. 
		NOTE: The formula used for counting is the number of conidia per 25 squares of the smallest cell (size = 0.025 mm2; chamber depth = 0.1 mm): 
		Total No of conidia in 5 squares&#160;&#247; 80&#160;&#215; (4 &#215; 106)</li>
		<li>Repeat three times for each isolate.</li>
	</ol>
	</li>
	<li>Thermotolerance assay
	<ol>
		<li>Culture the selected fungal isolate on &#188; SDA medium at 25 &#177; 1 &#176;C in dark for 10 days.</li>
		<li>Prepare 1 mL of the conidial suspension of fungal isolate in 0.03% surfactant solution and adjust to 1 x 107 conidia/mL as described above.</li>
		<li>Vortex the conidial suspension and heat it in a 45 &#176;C dry bath for 0, 30, 60, 90, and 120 min. Drop three droplets of 5 &#181;L of the conidial suspension on 55 mm &#188; SDA medium at each time point post heat-exposure and incubate at 25 &#177; 1 &#176;C for 18 h. 
		NOTE: Avoid spreading the fungal droplets to be able to better focus on the area.</li>
		<li>Count the number of germinated conidia spores with five randomly selected fields under 200x light microscopy to determine germination rate.</li>
		<li>Perform three replicates for each isolate.</li>
	</ol>
	</li>
</ol>
5. Effective conidia number (ECN) ranking
<ol>
	<li>ECN calculation 
	NOTE: Obtain the conidial production and thermotolerance data of each potential fungal strain before calculating the total ECN21.
	<ol>
		<li>Calculate the fold-change (FC) of conidial production at each time point: 
		<img alt="Equation 1" src="/files/ftp_upload/62882/62882eq01.jpg" /> 
		where, x = time point for data collection; ncp&#160;= number of conidia after each day of growth; and I = initial number of seeded conidia.</li>
		<li>Calculate the conidia number under the stress treatment at each time point using the following formula: 
		<img alt="Equation 2" src="/files/ftp_upload/62882/62882eq02.jpg" /> 
		Where, y = the ECN of the time point under treatment; TT0 = the germination rate of conidia not undergoing heat stress (= germination rate of 0 min heat treatment); TTz = stress coefficient is the conidia germination rate at different times of heat treatment (z).</li>
		<li>Calculate the total ECN using the following formula: 
		<img alt="Equation 3" src="/files/ftp_upload/62882/62882eq03.jpg" /></li>
		<li>Compare the ECN of each fungal strain.</li>
	</ol>
	</li>
	<li>Principal component analysis (PCA) of fungal strains 
	NOTE:The PCA analysis confirms the ranking of ECN and helps in understanding the correlation between the physiological character values. Compare the ECN values and select EPF isolates having higher ECN values.</li>
	<li>Use R software to create PCA by coding: 
	#Input PCA data file 
	&#8203;a = read.table(&#34;PCA.csv&#34;,sep=&#39;,&#39;,header=T)
	<ol>
		<li># Processing sample data 
		row.names(a) &#60;- c(&#34;NCHU-9&#34;,&#34;NCHU-11&#34;, &#34;NCHU-64&#34;, &#34;NCHU-69&#34;, &#34;NCHU-95&#34;, &#34;NCHU-113&#34;) 
		X=row.names(a) 
		df&#60;- a[2:11]</li>
		<li>#PCA calculation 
		pca &#60;- prcomp(df, center = TRUE, scale = TRUE) 
		vars &#60;- (pca$sdev)^2 
		pc1_percent = vars[1] / sum(vars) 
		pc2_percent = vars[2] / sum(vars) 
		value = pca$x</li>
		<li>#Output PCA visualization file 
		png(file = &#39;pca.png&#39;, height = 2000, width = 2000, res = 300) 
		NOTE: Use 7 to 14 days conidial production and all thermotolerance data to execute principal component analysis (PCA) for confirming the ECN ranking.</li>
	</ol>
	</li>
	<li>Select the best-performing fungal strains based on ECN or PCA and perform the virulence test of target pests for further research.</li>
</ol>

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

Entomopathogenic fungi (EPF) have been used for insect control. There are several methods to isolate, select, and identify EPF30,31,32. Comparing the different types of insect bait methods, Beauveria bassiana and Metarhizium anisopliae were commonly found in insect baits6,12,13,14. Am...

Currently, entomopathogenic fungi (EPF) are widely used in the microbial control of agricultural, forest, and horticultural pests. The advantages of EPF are its wide host ranges, good environmental adaptability, ecofriendly nature, and that it can be used with other chemicals to show the synergistic effect for integrated pest management1,2. For the application as a pest control agent, it is necessary to isolate a large number of EPF from either diseased insects or the natural environment.
The sampling of these organisms from their hosts helps in understanding the geographic distribution and prevalence rate of EPF in natural hosts3,4,5. However, the collection of fungal infected insects are usually limited by environmental factors and insect populations in the field4. Considering that insect hosts will die after EPF infection and then fall into the soil, isolation of EPF from soil samples might be a stable resource3,6. For example, saprophytes are known to use the dead host as their resource for growth. The soil bait and selective medium systems have been widely used to detect and isolate EPF from the soil3,4,7,8,9,10.
In the selective medium method, the diluted soil solution is plated onto a medium containing broad-spectrum antibiotics (e.g., chloramphenicol, tetracycline, or streptomycin) to inhibit the growth of bacteria2,3,9,11. However, it has been reported that this method may distort the strain&#39;s diversity and density and can cause an over- or under-estimation of many microbial communities6. Moreover, the isolated strains are less pathogenic and compete with saprophytes during isolation. It is difficult to isolate EPF from the diluted soil solution3. Instead of using a selective medium, the soil bait method isolates EPF from the infected dead insects, which can be stored for 2-3 weeks, thereby providing a more efficient and standard EPF separation method3,4,7,6. Because the method is easy to operate, one can isolate a variety of pathogenic strains at a low cost4. Therefore, it is widely used by many researchers.
Upon comparing the different types of insect bait systems, Beauveria bassiana and Metarhizium anisopliae are the most common EPF species that are found in insects belonging to the Hemiptera, Lepidoptera, Blattella, and Coleoptera6,12,13,14. Among these insect baits, Galleria mellonella (order Lepidoptera) and Tenebrio molitor (order Coleoptera)&#160;show higher recovery rates of Beauveria and Metarhizium spp., when compared with other insects. Therefore, G. mellonella and T. molitor are commonly used for insect baiting. Over the years, the United States Department of Agriculture (USDA) has established an EPF Library (Agricultural Research Service Collection of EPF cultures, ARSEF) that contains a wide variety of species, including 4081 species of Beauveria spp., 18 species of Clonostachys spp., 878 species of Cordyceps spp., 2473 species of Metarhizium spp., 226 species of Purpureocillium spp., and 13 species of Pochonia spp. among others15. Another EPF Library was constructed by the Entomology Research Laboratory (ERL) from the University of Vermont in the United States for c.a. 30 years. It includes 1345 strains of EPF from the United States, Europe, Asia, Africa, and the Middle East16.
To control local or invasion pests in Taiwan, isolation and selection of indigenous EPF is required. Therefore, in this protocol, we have modified and described the procedure of the soil bait method and combined it with the insect bait (mealworm, Tenebrio molitor) system17. Based on this protocol, an EPF library was established. Two rounds of screening (quantification of inoculation) were performed for the preliminary EPF isolates. EPF isolates showed pathogenicity to insects. The potential strains were subjected to morphological and molecular identifications and further analyzed by the thermotolerance and conidial production assay. Further, a concept of effective conidia number (ECN) was also proposed. Using ECN formula and principal component analysis (PCA), the potential strains were analyzed under simulated environmental pressure to complete the process of establishing and screening the EPF library. Subsequently, pathogenicity of promising EPF strains were tested for the target pest (e.g., Spodoptera litura). The current protocol integrates thermotolerance and conidial production data into the ECN formula and PCA analysis, which can be used as a standard ranking system for EPF related research.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agar Bacteriological grade</td>
			<td>BIOMAN SCIENTIFIC Co., Ltd.</td>
			<td>AGR001</td>
			<td>Suitable in most cell culture/molecular, biology applications.</td>
		</tr>
		<tr>
			<td>AGAROSE, Biotechnology Grade</td>
			<td>BIOMAN SCIENTIFIC Co., Ltd.</td>
			<td>AGA001</td>
			<td>For DNA electrophoresis.</td>
		</tr>
		<tr>
			<td>BioGreen Safe DNA Gel Buffer</td>
			<td>BIOMAN</td>
			<td>SDB001T</td>
			<td></td>
		</tr>
		<tr>
			<td>Brass cork borer</td>
			<td>Dogger</td>
			<td>D89A-44001</td>
			<td></td>
		</tr>
		<tr>
			<td>Canon kiss x2</td>
			<td>Canon</td>
			<td>EOS 450D</td>
			<td>For record strain colony morphology</td>
		</tr>
		<tr>
			<td>Constant temperature incubator</td>
			<td>Yihder Co., Ltd.</td>
			<td>LE-509RD</td>
			<td>Fungal keeping.</td>
		</tr>
		<tr>
			<td>cubee Mini-Centrifuge</td>
			<td>GeneReach</td>
			<td>MC-CUBEE</td>
			<td></td>
		</tr>
		<tr>
			<td>DigiGel 10 Digital Gel Image System</td>
			<td>TOPBIO</td>
			<td>DGIS-12S</td>
			<td></td>
		</tr>
		<tr>
			<td>Finnpipette F2 0.2 to 2 &#181;L Pipette</td>
			<td>Thermo Scientific</td>
			<td>4642010</td>
			<td></td>
		</tr>
		<tr>
			<td>Finnpipette F2 1 to 10 &#181;L Pipette</td>
			<td>Thermo Scientific</td>
			<td>4642030</td>
			<td></td>
		</tr>
		<tr>
			<td>Finnpipette F2 10 to 100 &#181;L Pipette</td>
			<td>Thermo Scientific</td>
			<td>4642070</td>
			<td></td>
		</tr>
		<tr>
			<td>Finnpipette F2 100 to 1000 &#181;L Pipette</td>
			<td>Thermo Scientific</td>
			<td>4642090</td>
			<td></td>
		</tr>
		<tr>
			<td>Finnpipette F2 2 to 20 &#181;L Pipette</td>
			<td>Thermo Scientific</td>
			<td>4642060</td>
			<td></td>
		</tr>
		<tr>
			<td>Finnpipette F2 20 to 200 &#181;L Pipette</td>
			<td>Thermo Scientific</td>
			<td>4642080</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneAmp PCR System 9700</td>
			<td>Applied Biosystems</td>
			<td>4342718</td>
			<td></td>
		</tr>
		<tr>
			<td>GenepHlow Gel/PCR Kit</td>
			<td>Geneaid</td>
			<td>DFH100</td>
			<td></td>
		</tr>
		<tr>
			<td>Genius Dry Bath Incubator</td>
			<td>Major Science</td>
			<td>MD-01N</td>
			<td></td>
		</tr>
		<tr>
			<td>Graduated Cylinder Custom A 100mL</td>
			<td>SIBATA</td>
			<td>SABP-1195906</td>
			<td>Measure the volume of reagents.</td>
		</tr>
		<tr>
			<td>Hand tally counter</td>
			<td>SDI</td>
			<td>NO.1055</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>bioman</td>
			<td>AP-0650010</td>
			<td>Calculate the number of spore</td>
		</tr>
		<tr>
			<td>Inoculating loop</td>
			<td>Dogger</td>
			<td>D8GA-23000</td>
			<td></td>
		</tr>
		<tr>
			<td>lid</td>
			<td>IDEAHOUSE</td>
			<td>RS92004</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro cover glass</td>
			<td>MUTO PURE CHEMICALS CO.,LTD</td>
			<td>24241</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope imaging system</td>
			<td>SAGE VISION CO.,LTD</td>
			<td>SGHD-3.6C</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>DOGGER</td>
			<td>DG75001-07105</td>
			<td></td>
		</tr>
		<tr>
			<td>Mupid-2plus DNA Gel Electrophoresis</td>
			<td>ADVANCE</td>
			<td>AD110</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon optical microscope</td>
			<td>SAGE VISION CO.,LTD</td>
			<td>Eclipse CI-L</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic cup</td>
			<td>IDEAHOUSE</td>
			<td>CS60016</td>
			<td></td>
		</tr>
		<tr>
			<td>Presto Mini gDNA Yeast Kit</td>
			<td>Geneaid</td>
			<td>GYBY300</td>
			<td>Fungal genomic DNA extraction kit</td>
		</tr>
		<tr>
			<td>Sabouraud Dextrose Broth (Sabouraud Liquid Medium)</td>
			<td>HiMedia Leading BioSciences Company</td>
			<td>M033</td>
			<td>Used for cultivation of yeasts, moulds and aciduric microorganisms.</td>
		</tr>
		<tr>
			<td>Scalpel Blade No.23</td>
			<td>Swann-Morton</td>
			<td>310</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Handle No.4</td>
			<td>AGARWAL SURGICALS</td>
			<td>SSS -FOR-01-91</td>
			<td></td>
		</tr>
		<tr>
			<td>Shovel</td>
			<td>Save &#38; Safe</td>
			<td>A&#160;-1580242&#160;-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Silwet L-77</td>
			<td>bioman(phytotech)</td>
			<td>S7777</td>
			<td>Surfactant</td>
		</tr>
		<tr>
			<td>Sorvall Legend Micro 17 Microcentrifuge</td>
			<td>Thermo Scientific</td>
			<td>75002403</td>
			<td></td>
		</tr>
		<tr>
			<td>Steel Tweezers</td>
			<td>SIPEL ELECTRONIC SA</td>
			<td>GG-SA</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Petri Dish</td>
			<td>BIOMAN SCIENTIFIC Co., Ltd.</td>
			<td>1621</td>
			<td>Shallow cylindrical containers with fitted lids, specifically for microbiology or cell culture use.</td>
		</tr>
		<tr>
			<td>ThermoCell MixingBlock</td>
			<td>BIOER</td>
			<td>MB-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 80</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>164-21775</td>
			<td></td>
		</tr>
		<tr>
			<td>TwinGuard ULT Freezer</td>
			<td>Panasonic Healthcare Holdings Co., Ltd.</td>
			<td>MDF-DU302VX</td>
			<td>-80&#176;C sample stored.</td>
		</tr>
		<tr>
			<td>Vertical floor type cabinet</td>
			<td>Chih Chin</td>
			<td>BSC-3</td>
			<td>Fungal operating culturing.</td>
		</tr>
		<tr>
			<td>Vortex Genie II</td>
			<td>Scientific</td>
			<td>SIG560</td>
			<td></td>
		</tr>
		<tr>
			<td>Zipper storage bags</td>
			<td>Save &#38; Safe</td>
			<td>A&#160;-1248915&#160;-00</td>
			<td></td>
		</tr>
		<tr>
			<td>100 bp DNA Ladder</td>
			<td>Geneaid</td>
			<td>DL007</td>
			<td></td>
		</tr>
		<tr>
			<td>-20&#176;C Freezer</td>
			<td>FRIGIDAIRE</td>
			<td>Frigidaire FFFU21M1QW</td>
			<td>-20&#176;C sample and experimental reagents stored.</td>
		</tr>
		<tr>
			<td>2X SuperRed PCR Master Mix</td>
			<td>TOOLS</td>
			<td>TE-SR01</td>
			<td></td>
		</tr>
		<tr>
			<td>50X TAE Buffer</td>
			<td>BIOMAN</td>
			<td>TAE501000</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Isolation and selectionof potential Entomopathogenic fungi (EPF) 
By using the Tenebrio molitor-mediated Entomopathogenic fungi (EPF) library construction method, the number of fungi without insect-killing activity would be excluded; thus, the isolation efficiency and selection of EPF could be largely increased. During the application of this method, the information of sampling sites, soil samples, and the fungal germination rates were recorded (Tab...

Watch this Scientific Journal Video about Isolation and Selection of Entomopathogenic Fungi from Soil Samples and Evaluation of Fungal Virulence against Insect Pests at JoVE.com

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 field.

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

isolation-selection-entomopathogenic-fungi-from-soil-samples

Research

JoVE Journal

Biology

8.9K Views.  National Chung Hsing University. Here we present a protocol based on the 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.

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

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

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 ...

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 ...

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

Entomopathogenic fungi of the Metarhizium anisopliae species complex have gained importance as the biological control agents of agricultural insect pests. The increase in pest resistance to chemical insecticides, the growing concerns regarding the negative effects of insecticides on human health, and the environmental pollution from pesticides have led to a global drive to find novel sustainable strategies for crop protection and pest control. Previously, attempts to mass culture such entomopathogenic fungi (EPF) species as Beauveria bassiana have been conducted. However, only limited attempts have been conducted to mass culture Metarhizium robertsii and M. pinghaense for use against insect pests. This study aimed to mass-produce a sufficient number of resilient infective propagules of South African isolates of M. robertsii and M. pinghaense for commercial application. Three agricultural grain products, flaked oats, flaked barley, and rice, were used as the EPF solid fermentation substrates. Two inoculation methods, conidial suspensions and the liquid fungal culture of blastospores were used to inoculate the solid substrates. Inoculation using conidial suspensions was observed to be relatively less effective, as increased levels of contamination were observed on the solid substrates relative to when using the blastospore inoculation method. Flaked oats were found not to be a suitable growth substrate for both M. robertsii and M. pinghaense, as no dry conidia were harvested from the substrate. Flaked barley was found to favor the production of M. robertsii conidia over that of M. pinghaense, and an average of 1.83 g &plusmn; 1.47 g of dry M. robertsii conidia and zero grams of M. pinghaense conidia was harvested from the substrate. Rice grains were found to favor the conidial mass production of both M. pinghaense and M. robertsii isolates, with an average of 8.2 g &plusmn; 4.38 g and 6 g &plusmn; 2 g harvested from the substrate, respectively.

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 products.

Entomopathogenic fungi of the Metarhizium anisopliae species complex have gained importance as the biological control agents of agricultural insect pests. ...

Comparison of Methods for Isolating Entomopathogenic Fungi from Soil Samples

The goal of the present study is to compare the effectiveness of using insect baits versus artificial selective medium for isolating entomopathogenic fungi (EPF) from soil samples. The soil is a rich habitat for microorganisms, including EPF particularly belonging to the genera Metarhizium and Beauveria, which can regulate arthropod pests. Biological products based on fungi are available in the market mainly for agricultural arthropod pest control. Nevertheless, despite the high endemic biodiversity, only a few strains are used in commercial bioproducts worldwide. In the present study, 524 soil samples were cultured on potato dextrose agar enriched with yeast extract supplemented with chloramphenicol, thiabendazole, and cycloheximide (CTC medium). The growth of fungal colonies was observed for 3 weeks. All Metarhizium and Beauveria EPF were morphologically identified at the genus level. Additionally, some isolates were molecularly identified at the species level. Twenty-four out of these 524 soil samples were also surveyed for EPF occurrence using the insect bait method (Galleria mellonella and Tenebrio molitor). A total of 51 EPF strains were isolated (41 Metarhizium spp. and 10 Beauveria spp.) from the 524 soil samples. All fungal strains were isolated either from croplands or grasslands. Of the 24 samples selected for comparison, 91.7% were positive for EPF using Galleria bait, 62.5% using Tenebrio bait, and 41.7% using CTC. Our results suggested that using insect baits to isolate the EPF from the soil is more efficient than using the CTC medium. The comparison of isolation methods in addition to the identification and conservation of EPF has a positive impact on the knowledge about biodiversity. The improvement of EPF collection supports scientific development and technological innovation.

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 medium).

The goal of the present study is to compare the effectiveness of using insect baits versus artificial selective medium for isolating entomopathogenic ...

Isolation of Culturable Yeasts and Molds from Soils to Investigate Fungal Population Structure

Soil is host to an incredible amount of microbial life, with each gram containing up to billions of bacterial, archaeal, and fungal cells. Multicellular fungi such as molds and unicellular fungi, broadly defined as yeasts, fulfill essential roles in soil ecosystems as decomposers of organic material and as food sources for other soil dwellers. Fungal species diversity in soil is dependent on a multitude of climatic factors such as rainfall and temperature, as well as soil properties including organic matter, pH, and moisture. Lack of adequate environmental sampling, especially in regions of Asia, Africa, South America, and Central America, hinders the characterization of soil fungal communities and the discovery of novel species. 
We characterized soil fungal communities in nine countries across six continents using ~4,000 soil samples and a protocol developed in the laboratory for the isolation of yeasts and molds. This protocol begins with separate selective enrichment for yeasts and the medically relevant mold Aspergillus fumigatus, in liquid media while inhibiting bacterial growth. Resulting colonies are then transferred to solid media and further processed to obtain pure cultures, followed by downstream genetic characterization. Yeast species identity is established via sequencing of their internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene cluster, while global population structure of A. fumigatus is explored via microsatellite marker analysis. 
The protocol was successfully applied to isolate and characterize soil yeast and A. fumigatus populations in Cameroon, Canada, China, Costa Rica, Iceland, Peru, New Zealand, and Saudi Arabia. These findings revealed much-needed insights on global patterns in soil yeast diversity, as well as global population structure and antifungal resistance profiles of A. fumigatus. This paper presents the method of isolating both yeasts and A. fumigatus from international soil samples.

This protocol is an effective, speedy method of culturing yeasts and the mold Aspergillus fumigatus from large sets of soil samples in as little as 7 days. The methods can be easily modified to accommodate a range of incubation media and temperatures as needed for experiments.

Soil is host to an incredible amount of microbial life, with each gram containing up to billions of bacterial, archaeal, and fungal cells. Multicellular ...

Isolation and Screening from Soil Biodiversity for Fungi Involved in the Degradation of Recalcitrant Materials

Environmental pollution is an increasing problem, and identifying fungi involved in the bioremediation process is an essential task. Soil hosts an incredible diversity of microbial life and can be a good source of these bioremediative fungi. This work aims to search for soil fungi with bioremediation potential by using different screening tests. Mineral culture media supplemented with recalcitrant substances as the sole carbon source were used as growth tests. First, soil dilutions were plated on Petri dishes with mineral medium amended with humic acids or lignocellulose. The growing fungal colonies were isolated and tested on different substrates, such as complex mixtures of hydrocarbons (petrolatum and used motor oil) and powders of different plastic polymers (PET, PP, PS, PUR, PVC). Qualitative enzymatic tests were associated with the growth tests to investigate the production of esterases, laccases, peroxidases, and proteases. These enzymes are involved in the main degradation processes of recalcitrant material, and their constitutive secretion by the examined fungal strains could have the potential to be exploited for bioremediation. More than 100 strains were isolated and tested, and several isolates with good bioremediation potential were found. In conclusion, the described screening tests are an easy and low-cost method to identify fungal strains with bioremediation potential from the soil. In addition, it is possible to tailor the screening tests for different pollutants, according to requirements, by adding other recalcitrant substances to minimal culture media.

Here, we present a protocol for screening soil biodiversity to look for fungal strains involved in the degradation of recalcitrant materials. First, fungal strains able to grow on humic acids or lignocellulose are isolated. Their activity is then tested both in enzymatic assays and on pollutants such as hydrocarbons and plastics.

Environmental pollution is an increasing problem, and identifying fungi involved in the bioremediation process is an essential task. Soil hosts an ...

Optimized Bioassay for Entomopathogenic Fungi Against Mustard Aphid

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 

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, ...

Exploring Entomopathogenic Fungi Resources from Forest Wood Borers

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

Forest wood borers (FWB) cause severe tree damage and economic losses worldwide. The release of entomopathogenic fungi (EPF) during the FWB emergence period is considered an acceptable alternative to chemical control. However, EPF resources have been significantly less explored for FWBs, in contrast to agricultural insect pests. This paper presents a protocol for exploring EPF resources from FWBs using wild Monochamus alternatus populations as an example. In this protocol, the assignment of traps baited with M. alternatus attractants to different populations guaranteed the collection of adequate samples with natural infection symptoms, during the emergence periods of the beetle. Following finely dissecting integuments and placing them onto a selective medium, fungal species were isolated from each part of beetle bodies and identified based on both molecular and morphological traits.
Several fungal species were certified as parasitic EPFs via re-infection of healthy M. alternatus with spore suspensions. Their behavioral phenotypes on M. alternatus were observed using scanning electron microscopy and further compared with those on the Coleopteran model insect Tribolium castaneum. For EPFs that present consistent parasitism phenotypes on both beetle species, evaluation of their activities on T. castaneum provided valuable information on lethality for future study on M. alternatus. This protocol helped the discovery of EPF newly reported on M. alternatus populations in China, which could be applied as an efficient approach to explore more EPF resources from other FWBs.

Author Spotlight: Evaluation of Entomopathogenic Fungi in Wild Monochamus alternatus 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 forests.

Forest wood borers (FWB) cause severe tree damage and economic losses worldwide. The release of entomopathogenic fungi (EPF) during the FWB emergence ...