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 fungi used for arthropod-pest control³. EPF have been successfully isolated from soil, arthropods with visible mycosis, colonized plants, and plant rhizosphere^4,5.

Isolation of EPF can also be useful to study the diversity, distribution, and ecology of this particular group. Recent literature reported that the use of EPF is underestimated, citing several unconventional applications of EPF such as their capacity to improve plant growth⁴, to remove toxic contaminants from the soil, and to be used in medicine⁶. The present study aims to compare the efficiency of isolating EPF from soil using insect baits versus artificial culture medium^7,8,9. The use of Galleria mellonella L. (Lepidoptera: Phyralidae) as an insect bait in the context of EPF isolation has been well accepted. These larvae are used worldwide by the scientific community as an experimental model to study host-pathogen interactions^10,11. Tenebrio molitor L. (Coleoptera: Tenebrionidae) larva is considered another insect model for studies involving virulence and for isolation of EPF since this insect is easy to rare in the laboratory at a low cost^7,12.

Culture-independent methods such as using a variety of PCR techniques can be applied to detect and quantify EPF on their substrates, including soil^13,14. Nevertheless, to properly isolate these fungal colonies, their substrate should be cultured onto a selective artificial medium⁹, or the fungi present in the samples can be baited using sensitive insects¹⁵. On one hand, CTC is a dodine-free artificial medium that consists of potato dextrose agar enriched with yeast extract supplemented with chloramphenicol, thiabendazole, and cycloheximide. This medium was developed by Fernandes et al.⁹ to maximize the recovery of naturally occurring Beauveria spp. and Metarhizium spp. from the soil. On the other hand, G. mellonella and T. molitor larvae can also to be successfully used as baits to obtain EPF isolates from the soil. Nevertheless, according to Sharma et al.¹⁵, fewer studies reported the concomitant use and comparison of these two bait insects. Portuguese vineyards soils exhibited significant recoveries of Metarhizium robertsii (Metscn.) Sorokin using T. molitor larvae in comparison to G. mellonella larvae; in contrast, Beauveria bassiana (Bals. -Criv.) Vuill isolation was linked to the use of G. mellonella baits¹⁵. Therefore, the decision on which EPF isolation method to use (i.e., G. mellonella-bait, T. molitor-bait or CTC medium) should be considered according to the study's goal and the laboratory infrastructure. The goal of the present study is to compare the effectiveness of using insect baits versus artificial selective medium for isolating EPF from soil samples.

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 spores may be less viable over time. In the present study, samples were analyzed no more than 7 days after the collection.
2. Use a GPS to identify the location of the collected samples in latitude and longitude and classify the collected area according to the type of soil (for example, grasslands, native rainforest, lakeshores, or croplands).

2. Isolation methods for entomopathogenic fungi

1. Isolation using CTC selective artificial medium.
   1. To prepare the CTC medium [potato dextrose agar plus yeast extract (PDAY) supplemented with 0.5 g/L of chloramphenicol, 0.001 g/L of thiabendazole, and 0.25 g/L of cycloheximide⁹], weigh all the reagents individually, mix them in distilled water, and sterilize the medium in autoclave. In a biosafety cabinet, plate 23 mL of the medium into 60 mm x 15 mm Petri plates.
      CAUTION: While weighing CTC reagents, use a lab coat, mask, gloves, and goggles because cycloheximide and chloramphenicol are toxic.
   2. Weigh 0.35 ± 0.05 g of each soil sample (with or without roots) and place it in a 1.5 mL microtube.
   3. In a biosafety cabinet, add 1 mL of sterile 0.01% (vol/vol) polyoxyethylene sorbitan monooleate aqueous suspension to the microtube containing soil and vortex for 30 s.
   4. Remove 50 µL of the supernatant and pipette it onto the center of Petri plates with CTC medium. Homogenously disperse the suspensions onto the surface of the medium using a sterile Drigalski spatula (6 mm in diameter).
      NOTE: At least three replicates for each soil sample should be prepared.
   5. Incubate the plates in climate chambers (25 ± 1 °C, relative humidity ≥80%) in the dark and observe the growth of fungal colonies after 7, 14, and 21 days of incubation.
   6. Observe the macromorphology and micromorphology of the fungal colonies seeking EPF. Transfer the EPF cultures to potato dextrose agar medium plus 0.05% chloramphenicol (PDAC) until pure cultures are obtained.
      NOTE: Use the description keys presented below in step 3 for the identification of EPF colonies.
2. Isolation using insect baits
   1. Use surface-disinfected G. mellonella and T. molitor late-stage larvae. Immerse the larvae into 0.5% sodium hypochlorite for 1 min for sterilization. Wash the larvae twice using sterile water.
      NOTE: G. mellonella larvae from the fourth stage were used in the present study. T. molitor larval stages were not standardized.
   2. Use plastic pots to assemble the baits. Add 250 g of collected soil to each plastic pot (98 mm width x 47 mm height x 142 mm length). Separate 15 larvae of each species (T. molitor and G. mellonella) and deposit five larvae per plastic pot. Store the pots at 25 ± 1 °C and relative humidity ≥ 80% in the dark.
      NOTE: Drill 10 small holes (2 mm in diameter) in the pot lids to allow ventilation. A sharp heated iron device can be used to drill the holes.
   3. Homogenize the soil every other day to allow maximum contact of larvae with soil.
      NOTE: Moisture is important to support the fungal infection of larvae. To maintain moisture in the soil, spray sterile distilled water on the soil surface whenever necessary. Do not soak the soil sample in water.
   4. Analyze the pots daily seeking dead insects.
      NOTE: Observe the remaining larvae in the colony daily for invertebrate pathological signs to make sure the insects are not infected. As an alternative, control pots with sterile soil can be included in the study to check the health status of the insect larvae.
   5. Remove dead insects and superficially sterilize them with 0.5% sodium hypochlorite for 1 min. Place the sterile insects in a humid chamber (relative humidity ≥ 80%) at 25 ± 1 °C for 7 days to favor the exteriorization of entomopathogenic fungi (mycosis).
   6. Upon mycosis, harvest the conidia from the insect surface. Use a microbiological loop to place the conidia on PDAC medium under a stereoscopic microscope. As an alternative, place the whole infected larvae on the PDAC medium. Incubate the culture plates in a climate chamber at 25 ± 1 °C and relative humidity ≥ 80%.
   7. Observe the macromorphology and micromorphology of the fungal colonies on the plates to confirm the identity of EPF. Repeat culturing on PDAC until pure fungal colonies are obtained.
      ​NOTE: Use the description keys presented below in step 3 for the identification of EPF colonies.

3. Identification of EPF (Metarhizium spp. and Beauveria spp.)

1. Analyze the macromorphological characteristics of the fungal cultures on the plates (i.e., surface and reverse of colonies, their shape, edge, growth rate, color, texture, diffusible pigment, exudates, and aerial conidia) after 14 days at 25 ± 1 °C and relative humidity ≥ 80%.
2. Transfer the aerial conidia to slide cultures (microculture technique)¹⁶ for 3 days at 25 ± 1 °C and relative humidity ≥ 80% and stain with lactophenol blue to observe the microscopic features (i.e., arrangement of conidia, conidiophores, shape, and size of conidia)^17,18,19,20.
3. Observe the microscopic fungal structures at 400x using an optical microscope to confirm the EPF identification.
   NOTE: Morphological keys for EPF are described in the reports by Bischoff et al., Rehner et al., Seifert et al., and Humber^17,18,19,20. The macro and micromorphology of fungal colonies are the most frequent criteria used to identify filamentous fungi at the genus level. Depending on the genus of the EPF, these morphological characteristics will change. Humber²⁰ presents an identification key to major genera of fungal entomopathogens. Metarhizium spp. colonies, for example, are usually circular, powdery, exhibiting varying shades of green, and can present exudate. Microscopically, these colonies have conidiogenous cells apical on broadly branched, densely intertwined conidiophores forming a compact hymenium, and cylindrical to ellipsoid conidia in parallel chains forming columns or plate-like masses. Beauveria spp. colonies are usually white, powdery, or cotton-like. They exhibit conidiogenous cells with a dilated basal portion extending apically in a zigzag direction. Beauveria conidiophores form dense clusters of globe-shaped conidia. Molecular analyses are needed for the identification of EPF at the species level.
4. Perform molecular analyses on the isolates for taxonomic identification at the species level. For the EPF strains isolated in this study, namely, Metarhizium spp. and Beauveria spp., perform molecular analyses based on the reports of Bischoff et al.¹⁷ and Rehner et al.¹⁸.
5. After confirming the isolates to be EPF, deposit the isolates in a collection of fungal cultures. In the present study, the isolates were deposited in the entomopathogenic fungal cultures collection from the Laboratory of Microbial Control (LCM) at the Federal Rural University of Rio de Janeiro.

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

List of materials used in this article

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