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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 management^1,2. 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 hosts^3,4,5. 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 resource^3,6. 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 soil^3,4,7,8,9,10.

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 bacteria^2,3,9,11. However, it has been reported that this method may distort the strain'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 method^3,4,7,6. 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 Coleoptera^6,12,13,14. Among these insect baits, Galleria mellonella (order Lepidoptera) and Tenebrio molitor (order Coleoptera) 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.

Protokół

NOTE: The whole flowchart is shown in Figure 1.

1. Isolation and selection of potential Entomopathogenic fungi (EPF)

1. Collect the soil sample
   1. 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.
   2. 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.
   3. 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 °C for 1 week until the start of the protocol^18,19.
2. Bait and isolate the EPF with mealworm (Tenebrio molitor)
   1. 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 (14^th instar) of T. molitor larvae helps in fungal isolate screens²⁰.
   2. 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.
   3. 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 (¼ SDA) plate (55 mm) in the laboratory²¹. Incubate the culture plate at 25 °C for 7 days to obtain the primary culture of fungi.
      NOTE: The ¼ 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 ¼ SDA into each 55 mm Petri dish before solidification. The solidified ¼ SDA plates are stored at 4 °C until use.
   4. Re-streak each primary culture fungi on one 55 mm ¼ SDA plate in a laminar flow and incubate the culture plate at 25 °C for 7 days to obtain single colonies of fungi.
   5. 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.
3. Store the EPF isolates
   1. 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' storage after 2^nd virulence test. Molecular identification is also recommended if storage space is limited.
   2. Add 250 µL of 0.03% surfactant solution (Table of Materials) and 250 µL of 60% glycerol into the 1.5 mL micro-centrifuge tube using a micropipette; then, vortex for 10 s.
   3. Seal the 1.5 mL micro-centrifuge tube with paraffin film and precool it in a -20 °C refrigerator for 24 h. Then, transfer the precooled fungal stocks to a -80 °C refrigerator for cryopreservation.
      NOTE: Extra pure fungal culture plates (aside from the cryo-preserved samples) were used in section 1.4.
4. 1^st pathogenicity screen for fungal isolates
   1. Place five T. molitor larvae directly on the surface of each pure fungal culture plate at 25 °C.
   2. 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 2^nd virulence test to confirm their virulence to T. molitor larvae. Alternatively, the researcher can adjust the criteria as per their own study.
5. 2^nd virulence test of fungal isolates
   NOTE: Based on the 1^st pathogenicity screen, recover the selected fungal isolates from -80 °C for the 2^nd virulence test. The purpose of the 2^nd virulence test is to quantify the pathogenicity of the selected fungal isolates after the 1^st round of screening.
   1. Harvest conidia of each fungal isolate by vortexing for 1 min and count the number of conidia using a hemocytometer.
   2. Adjust the conidia suspension to a concentration of 1 x 10⁷ conidia/mL in a 0.03% surfactant solution (Table of Materials).
   3. Spread 10 µL of the fungal suspension onto 55 mm ¼ SDA plates and grow for 7 days at 25 °C in the dark.
   4. 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 °C in the dark.
   5. Observe and record the mycosis and mortality for 10 days.
   6. 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 3^rd virulence test to confirm their virulence to target the pest.
6. 3^rd virulence test of fungal isolates for target pest (Spodoptera litura as an example)
   1. Repeat steps 1.5.2 to 1.5.6 with selected isolates from 2^nd virulence to test virulence against target pest.
   2. Calculate the LT₅₀ of each fungal isolate²².
      ​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.

2. Molecular identification of EPF

1. Extraction of fungal genomic DNA
   1. Collect c.a. 1 cm² EPF from the 7-day ¼ SDA plate.
   2. Extract the fungal genomic DNA using a fungal genomic DNA extraction kit according to the manufacturer's instructions²³ (Table of Materials).
2. PCR amplification and DNA sequencing
   1. 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 °C for 1 min, and then 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, followed by a 7 min final extension at 72 °C.
      NOTE: The ITS1F/ITS4R primer set is for the genus level identification.
   2. Sequence the PCR by commercial sequencing service.
   3. 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' portion of translation elongation factor 1 alpha (TEF)^25,26.
3. Phylogenetic analysis
   1. 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.
   2. 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 1^st pathogenicity screen are used for molecular identification at the genus level. The fungal isolates screened by the 2^nd virulence test are used for the species level molecular and morphological identification.

3. Morphological identification of EPF

1. Observation of the fungal colony morphology
   1. Use a camera to capture the fungal culture colony growth for 7 days, and record the growth, form (fluffy, firm), and color of the colonies.
2. Observation of conidia and conidiophores
   1. 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.
   2. 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.
   3. 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.
   4. Measure and record the width and length of the conidia and conidiophores to compare the differences between different fungal isolates.
   5. Use Welch'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 3^rd virulence test are used for the physiological characterization and ECN ranking in sections 4 and 5.

4. Investigation of conidial productivity and thermotolerance

1. Conidial production assay
   1. Culture the selected fungal isolate on ¼ SDA medium at 25 ± 1 °C in dark for 10 days.
   2. 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.
   3. Drop three droplets of 10 µL of conidial suspension on¼ SDA and incubate at 25 °C in the dark for 7, 10, and 14 days to count the sporulation of fungi.
      NOTE: 10 µL is the best volume to collect the 5 mm block with even fungal sporulation after fungal growth for 7-14 days.
   4. 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.
   5. 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 ÷ 80 × (4 × 10⁶)
   6. Repeat three times for each isolate.
2. Thermotolerance assay
   1. Culture the selected fungal isolate on ¼ SDA medium at 25 ± 1 °C in dark for 10 days.
   2. 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.
   3. Vortex the conidial suspension and heat it in a 45 °C dry bath for 0, 30, 60, 90, and 120 min. Drop three droplets of 5 µL of the conidial suspension on 55 mm ¼ SDA medium at each time point post heat-exposure and incubate at 25 ± 1 °C for 18 h.
      NOTE: Avoid spreading the fungal droplets to be able to better focus on the area.
   4. Count the number of germinated conidia spores with five randomly selected fields under 200x light microscopy to determine germination rate.
   5. Perform three replicates for each isolate.

5. Effective conidia number (ECN) ranking

1. ECN calculation
   NOTE: Obtain the conidial production and thermotolerance data of each potential fungal strain before calculating the total ECN²¹.
   1. Calculate the fold-change (FC) of conidial production at each time point:
      
      where, x = time point for data collection; n_cp = number of conidia after each day of growth; and I = initial number of seeded conidia.
   2. Calculate the conidia number under the stress treatment at each time point using the following formula:
      
      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); TT_z = stress coefficient is the conidia germination rate at different times of heat treatment (z).
   3. Calculate the total ECN using the following formula:
      
   4. Compare the ECN of each fungal strain.
2. 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.
3. Use R software to create PCA by coding:
   #Input PCA data file
   ​a = read.table("PCA.csv",sep=',',header=T)
   1. # Processing sample data
      row.names(a) <- c("NCHU-9","NCHU-11", "NCHU-64", "NCHU-69", "NCHU-95", "NCHU-113")
      X=row.names(a)
      df<- a[2:11]
   2. #PCA calculation
      pca <- prcomp(df, center = TRUE, scale = TRUE)
      vars <- (pca$sdev)^2
      pc1_percent = vars[1] / sum(vars)
      pc2_percent = vars[2] / sum(vars)
      value = pca$x
   3. #Output PCA visualization file
      png(file = 'pca.png', 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.
4. Select the best-performing fungal strains based on ECN or PCA and perform the virulence test of target pests for further research.

Wyniki

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...

Dyskusje

Entomopathogenic fungi (EPF) have been used for insect control. There are several methods to isolate, select, and identify EPF^30,31,32. Comparing the different types of insect bait methods, Beauveria bassiana and Metarhizium anisopliae were commonly found in insect baits^6,12,13,14. Am...

Materiały

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