Comparison of Methods for Isolating Entomopathogenic Fungi from Soil Samples

Summary

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).

Abstract

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.

Introduction

Soil is the source of several microorganisms, including entomopathogenic fungi (EPF). This particular group of fungi is recognized by their ability to colonize and often kill arthropod hosts, especially insects¹. After isolation, characterization, selection of virulent strains, and registration, EPF are mass-produced for arthropod-pest control, which supports their economical relevance². Accordingly, the isolation of EPF is considered the first step to the development of a biopesticide. Beauveria spp. (Hypocreales: Cordycipitaceae) and Metarhizium spp. (Hypocreales: Clavicipitaceae) are the most common ....

Protocol

As the present study accessed Brazilian genetic heritage, the research was registered at the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (Sisgen) under the code AA47CB6.

1. Soil sampling

1. Collect 800 g of soil (with or without incident secondary plant roots) to a depth of 10 cm using a small shovel. Store them in polypropylene bags at room temperature until the start of the experiment.
   ​NOTE: Small roots can also be collected as EPF are reported to have rhizosphere competence. The faster the processing of the samples, the better because the fungal spore....

Results

A total of 524 soil samples were collected from grassland: livestock pasture (165 samples), native tropical forest (90 samples), lakeside (42 samples), and cultivated/cropland (227 samples) between 2015 and 2018 in the Rio de Janeiro State, Brazil. Details of geographic coordinates of samples positive for EPF are given in Supplementary Table 1.

Of the 524 soil samples, 500 samples were analyzed only using CTC medium, and 24 samples were concomitantly analyzed using three forms.......

Discussion

Natural and agricultural soil habitats are typical environments for EPF²² and an excellent natural reservoir. In the present study, two methods of EPF isolation using insect baits versus selective medium were addressed. The first step for isolation is the collection of the soil samples. Proper storage and identification of soil samples are crucial. Information on the latitude, longitude, soil type, and biome is essential for studies involving epidemiological, modeling, and geospatial subjects

Materials

Name	Company	Catalog Number	Comments
Autoclave	Phoenix Luferco	9451
Biosafety cabinet	Airstream ESCO	AC2-4E3
Chloramphenicol	Sigma-Aldrich	C0378
Climate chambers	Eletrolab	EL212/3
Coverslip	RBR	3871
Cycloheximide	Sigma-Aldrich	C7698
Drigalski spatula	Marienfeld	1800024
GPS app	Geolocation app	2.1.2005
Lactophenol blue solution	Sigma-Aldrich	61335
Microscope	Zeiss Axio star plus	1169 149
Microscope camera	Zeiss Axiocam 105 color	426555-0000-000
Microscope softwere	Zen lite Zeiss 3.0
Microscope slide	Olen	k5-7105-1
Microtube	BRAND	Z336769-1PAK
Petri plates	Kasvi	K30-6015
Pipette tip	Vatten	VT-230-200C/VT-230-1000C
Pippette	HTL - Labmatepro	LMP 200 / LMP 1000
Plastic pots	Prafesta descartáveis	8314
Polypropylene bags	Extrusa	38034273/5561
Potato dextrose agar	Kasvi	K25-1022
Prism software 9.1.2	Graph Pad
Shovel	Tramontina	77907009
Tenebrio mollitor	Safari	QP98DLZ36
Thiabendazole	Sigma-Aldrich	T8904
Tween 80	Vetec	60REAVET003662
Vortex	Biomixer	QL-901
Yeast extract	Kasvi	K25-1702