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 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.
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 rhizosphere4,5.
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 medium7,8,9. 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 interactions10,11. 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 cost7,12.
Culture-independent methods such as using a variety of PCR techniques can be applied to detect and quantify EPF on their substrates, including soil13,14. 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.
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
2. Isolation methods for entomopathogenic fungi
3. Identification of EPF (Metarhizium spp. and Beauveria spp.)
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 of isolation (Galleria-bait, Tenebrio-bait, and the selective CTC culture medium), so the relative efficiency of these methods could be evaluated. A total of 51 EPF strains were isolated from 524 samples (41 Metarhizium spp. and 10 Beauveria spp.) (Figure 1). Micromorphological characteristics of some isolates are shown in Figure 2. All fungal strains were isolated from grassland or cropland (Supplementary Table 1). The results revealed that Metarhizium spp. is more prevalent than Beauveria spp. (Supplementary Table 1). Nine of the Metarhizium isolates (LCM S01 to LCM S09) were molecularly identified using the ef1-a (eukaryotic translation elongation factor 1-alpha) gene21. Of these, seven isolates (LCM S01-LCM S06 and LCM S08) were identified as Metarhizium anisopliae sensu stricto while two isolates (LCM S07 and LCM S09) were identified as Metarhizium pingshaense21.
The occurrence of EPF (% of positive EPF samples) in the 24 soil samples studied using the three different methods of isolation is shown in Table 1. Recovery rates of EPF were analyzed by chi-square test. As shown in Table 1, Galleria bait proved to be more efficient in the isolation of EPF (91.7% (22/24) of positive samples) followed by T. molitor bait (62.5% (15/24) of EPF positive samples) and CTC medium (41.7% (14/24) of EPF positive samples). These 24 soil samples showed no recovery of Beauveria spp., but only Metarhizium.
Figure 1: Entomopathogenic fungal colonies of strains isolated from soil samples. Colonies were cultivated on CTC artificial medium. (1) Petri plate exhibiting fungal colonies from soil samples 14 days after incubation on CTC selective medium before pure cultures are obtained; (2-42) Pure Metarhizium spp. colonies; (43-52) Pure Beauveria spp. colonies. Please click here to view a larger version of this figure.
Figure 2: Micromorphological characteristics of entomopathogenic fungi isolated from soil samples. Colonies were incubated for 3 days on potato dextrose agar at 25 ± 1 °C and relative humidity ≥ 80%. The microscope slide was stained with lactophenol blue solution. Images show conidiophores and conidia of (A) Metarhizium anisopliae sensu stricto (s.s) isolate LCM S01; (B) Metarhizium anisopliae s.s. isolate LCM S03; (C) Metarhizium sp. isolate LCM S27; (D–F) Beauveria spp. isolates LCM S23, LCM S24, and LCM S20, respectively. All strains represented here were isolated using the CTC medium. LCM S27 was also recovered from soil using insect baits. * Conidiophores and conidia. ** Conidial chains show the characteristic side-by-side placement of Metarhizium spores in adjacent chains. Black arrows indicate Metarhizium cylindrical to ellipsoid conidia. Red arrows indicate Beauveria globe-shaped conidia. Please click here to view a larger version of this figure.
Method of isolation | Entomopathogenic fungi* | χ2** | |
Positive | Negative | ||
Galleria-bait | 91.7% (22/24) | 8.3% (2/24) | 13.4 |
Tenebrio-bait | 62.5% (15/24) | 37.2% (9/24) | |
CTC selective medium | 41.7% (10/24) | 58.3% (14/24) | |
* Only Metarhizium spp. were isolated | |||
** Chi-square analysis, DF2. P = 0.0013 |
Table 1: Occurrence of entomopathogenic fungi (% of positive samples) in 24 soil samples using different isolation methods.
Supplementary Table 1: Geographical coordinates, isolation method, code, year of collection, and land-use types of samples positive for entomopathogenic fungi. Please click here to download this Table.
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 subjects23,24. After collection, it is recommended that the samples are processed as soon as possible (preferably within 7 days) because the viability of conidia in these soil samples can eventually decrease. Critical steps in the EPF isolation using CTC include: a) investigation of CTC plates 1 and 2 weeks after incubation (the first weeks are critical because, at later stages, other fungal colonies can narrow EPF development), and b) accurately identifying EPF colonies based on their macromorphology and micromorphology. For isolation using insect baits, it is essential to keep the soil sample humid but not soak it in water.
The results reported by several studies have led to an interpretation that M. anisopliae is more common in cultivated soils than natural ecosystems8,25,26. Differences in the distribution and occurrence of these fungi can occur. In the present study, all strains were isolated either from cultivated soil (crops) or grasslands, and there was a predominance of Metarhizium spp. over Beauveria spp. It is suggested that cultivation practices and the high content of organic matter favor the presence of saprophytic fungi in the soil27. Accordingly, effective isolation techniques seeking EPF should consider reducing fungal contaminants.
Selective artificial media are commonly used for isolation because they are easy to use and have proven efficient in isolating entomopathogenic fungi, mainly Metarhizium spp. and Beauveria spp.28. These selective media use specific chemicals to reduce the growth of contaminants. In the 1980s and 1990s, the fungicide dodine became a widely used selective medium to isolate Metarhizium spp. and Beauveria spp.29,30. Although these artificial media are effective, some EPF species such as Metarhizium acridum can be susceptible to dodine31. That is why the dodine-free CTC medium was chosen in the present study. According to Fernandes et al.9, CTC was developed to maximize the isolation of naturally occurring entomopathogenic fungi, including M. acridum. Using a selective medium rather than insect baits in the isolation of EPF is convenient because the former requires less space in the sample processing. The main disadvantage in CTC use relies on the fact that some of its components (i.e., cycloheximide and chloramphenicol) are toxic, so the use of personal protection equipment is mandatory.
As observed in the present study, a higher percentage of positive samples has been reported with insect baits as compared to artificial selective media for isolation of EPF15,32,33,34,35. The use of insect baits is considered a low-cost and high-efficiency alternative in the search for new EPF. Despite this, there are disadvantages associated with the use of insect baits over selective media. As the amount of soil to analyze using insects is higher, it is also necessary to have more physical space to store the samples and incubate the pots. The acquisition of insects can also be a limitation. In Brazil, for example, G. mellonella is not commercially available, so it is necessary to establish a colony in the lab to use this insect as bait. It is essential to keep the salubrity of the insects' colonies, avoiding natural infection by EPF. An EPF infection in the colony can make the isolation results unreliable. Therefore, one has to observe the remaining larvae in the colony seeking invertebrate pathological signs. As an alternative, control pots with sterile soil can be included in the study to check the health status of the insect larvae.
Seeking new fungal isolates with outstanding biocontrol traits is crucial to increase the effectiveness of fungi in arthropod-pest control. Fungi isolated from soil can be well adapted to growing in this environment22, and they are likely to have high field persistence, which is an essential characteristic of successful EPF in pest control21. Accordingly, locally isolated EPF can improve the biological control of local pests because of their geographic and temporal congruence, increasing the chances of success and reducing the environmental impacts otherwise caused by the application of synthetic insecticides.
The authors have nothing to disclose.
This study was financed in part by the Coordenacão de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) from Brazil, finance code 001, Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) (project number E-26/010.001993/2015), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) from Brazil.
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 |