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

Summary

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.

Abstract

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.

Introduction

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 distribu....

Protocol

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.......

Discussion

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.......

Materials

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