Name: comparison of methods for isolating entomopathogenic fungi from soil samples
Uploaded: 2022-01-06T14:00:00
Description: Watch this Scientific Journal Video about Comparison of Methods for Isolating Entomopathogenic Fungi from Soil Samples at JoVE.com

This study was financed in part by the Coordenac&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior (CAPES) from Brazil, finance code 001, Funda&#231;&#227;o Carlos Chagas Filho de Amparo &#224; Pesquisa do Estado do Rio de Janeiro (FAPERJ) (project number E-26/010.001993/2015), and Conselho Nacional de Desenvolvimento Cient&#237;fico e Tecnol&#243;gico (CNPq) from Brazil.

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
<ol>
	<li>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. 
	&#8203;NOTE: Small roots c...

<ol>
	<li>Roberts, D. W., St. Leger, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metarhizium+spp.,+cosmopolitan+insect-pathogenic+fungi:+Mycological+aspects.">Metarhizium spp., cosmopolitan insect-pathogenic fungi: Mycological aspects.</a> Advances in Applied Microbiology. 54, 1-70 (2004).</li><li>do Nascimento Silva, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+cost-effective+bioconversion+process+of+palm+kernel+cake+into+bioinsecticides+based+on+Beauveria+bassiana+and+Isaria+javanica.">New cost-effective bioconversion process of palm kernel cake into bioinsecticides based on Beauveria bassiana and Isaria javanica.</a> Applied Microbiology and Biotechnology. 102 (6), 2595-2606 (2018).</li><li>Faria, M. R., Wraight, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycoinsecticides+and+Mycoacaricides:+A+comprehensive+list+with+worldwide+coverage+and+international+classification+of+formulation+types.">Mycoinsecticides and Mycoacaricides: A comprehensive list with worldwide coverage and international classification of formulation types.</a> Biological Control. 43 (3), 237-256 (2007).</li><li>Vega, F. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+fungal+entomopathogens+as+endophytes+in+biological+control:+a+review.">The use of fungal entomopathogens as endophytes in biological control: a review.</a> Applied Mycology. 110 (1), 4-30 (2018).</li><li>Sharma, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+entomopathogen+isolation:+A+case+of+bacteria+and+fungi.">Advances in entomopathogen isolation: A case of bacteria and fungi.</a> Microorganisms. 9 (1), 1-28 (2021).</li><li>Litwin, A., Nowak, M., R&#243;&#380;alska, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entomopathogenic+fungi:+unconventional+applications.">Entomopathogenic fungi: unconventional applications.</a> Reviews in Environmental Science and Bio/Technology. 19, 23-42 (2020).</li><li>Kim, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tenebrio+molitor-mediated+entomopathogenic+fungal+library+construction+for+pest+management.">Tenebrio molitor-mediated entomopathogenic fungal library construction for pest management.</a> Journal of Asia-Pacific Entomology. 21 (1), 196-204 (2018).</li><li>Meyling, N., Eilenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocurrence+and+distribution+of+soil+borne+entomopathogenic+fungi+within+a+single+organic+agroecosystem.">Ocurrence and distribution of soil borne entomopathogenic fungi within a single organic agroecosystem.</a> Agriculture, Ecosystems and Environment. 113 (1), 336-341 (2006).</li><li>Fernandes, E. K. K., Keyser, C. A., Rangel, D. E. N., Foster, R. N., Roberts, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CTC+medium:+A+novel+dodine-free+selective+medium+for+isolating+entomopathogenic+fungi,+especially+Metarhizium+acridum,+from+soil.">CTC medium: A novel dodine-free selective medium for isolating entomopathogenic fungi, especially Metarhizium acridum, from soil.</a> Biological Control. 54 (3), 197-205 (2010).</li><li>Ortiz-Urquiza, A., Keyhani, N. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+genetics+of+Beuveria+bassiana+infection+of+insects.">Molecular genetics of Beuveria bassiana infection of insects.</a> Advantages in Genetics. 94, 165-249 (2016).</li><li>Pereira, M. F., Rossi, C. C., Silva, G. C., Rosa, J. N., Bazzolli, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galleria+mellonella+as+infection+model:+an+in+depth+look+at+why+it+works+and+practical+considerations+for+successful+application.">Galleria mellonella as infection model: an in depth look at why it works and practical considerations for successful application.</a> Pathogens and Disease. 78 (8), (2020).</li><li>Souza, P. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tenebrio+molitor+(Coleoptera:+Tenebrionidae)+as+an+alternative+host+to+study+fungal+infections.">Tenebrio molitor (Coleoptera: Tenebrionidae) as an alternative host to study fungal infections.</a> Journal of Microbiological Methods. 118, 182-186 (2015).</li><li>Canfora, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+method+for+detection+and+quantification+of+B.+brongniartii+and+B.+bassiana+in+soil.">Development of a method for detection and quantification of B. brongniartii and B. bassiana in soil.</a> Scientific Reports. 6, 22933 (2016).</li><li>Garrido-Jurado, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+endophytic+colonization+of+melon+plants+by+entomopathogenic+fungi+after+foliar+application+for+the+control+of+Bemisia+tabaci+Gennadius+(Hemiptera:+Aleyrodidae).">Transient endophytic colonization of melon plants by entomopathogenic fungi after foliar application for the control of Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae).</a> Journal of Pest Science. 90, 319-330 (2016).</li><li>Sharma, L., Oliveira, I., Torres, L., Marques, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entomopathogenic+fungi+in+Portuguese+vineyards+soils:+suggesting+a+'Galleria-Tenebrio-bait+method'+as+bait-insects+Galleria+and+Tenebrio+significantly+underestimate+the+respective+recoveries+of+Metarhizium+(robertsii)+and+Beauveria+(bassiana).">Entomopathogenic fungi in Portuguese vineyards soils: suggesting a 'Galleria-Tenebrio-bait method' as bait-insects Galleria and Tenebrio significantly underestimate the respective recoveries of Metarhizium (robertsii) and Beauveria (bassiana).</a> MycoKeys. 38, 1-23 (2018).</li><li>Riddell, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permanent+stained+mycological+preparations+obtained+by+slide+culture.">Permanent stained mycological preparations obtained by slide culture.</a> Mycologia. 42 (2), 265-270 (1950).</li><li>Bischoff, J., Rehner, S. A., Humber, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multilocus+phylogeny+of+the+Metarhizium+anisopliae+lineage.">A multilocus phylogeny of the Metarhizium anisopliae lineage.</a> Mycologia. 101 (4), 512-530 (2009).</li><li>Rehner, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogeny+and+systematics+of+the+anamorphic,+entomopathogenic+genus+Beauveria.">Phylogeny and systematics of the anamorphic, entomopathogenic genus Beauveria.</a> Mycologia. 103 (5), 1055-1073 (2011).</li><li>Seifert, K. A., Gams, W., Seifert, K. A., Morgan-Jones, G., Gams, W., Kendrick, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anamorphs+of+Clavicipitaceae,+Cordycipitaceae+and+Ophiocordycipitaceae.">Anamorphs of Clavicipitaceae, Cordycipitaceae and Ophiocordycipitaceae.</a> The Genera of Hyphomycetes. CBS Biodiversity Series. CBS-KNAW Fungal Biodiversity Centre. 9, 903-906 (2011).</li><li>Humber, R. A., Lacey, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+entomopathogenic+fungi.">Identification of entomopathogenic fungi.</a> Manual of Techniques in Invertebrate Pathology., 2nd ed. , 151-187 (2012).</li><li>Mesquita, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+a+native+isolate+of+the+entomopathogenic+fungus+Metarhizium+anisopliae+against+larval+tick+outbreaks+under+semifield+conditions.">Efficacy of a native isolate of the entomopathogenic fungus Metarhizium anisopliae against larval tick outbreaks under semifield conditions.</a> BioControl. 65 (3), 353-362 (2020).</li><li>St Leger, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+adaptations+of+Metarhizium+anisopliae+to+life+in+the+soil.">Studies on adaptations of Metarhizium anisopliae to life in the soil.</a> Journal of Invertebrate Pathology. 98 (3), 271-276 (2008).</li><li>Mar, T. T., Suwannarach, N., Lumyong, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+entomopathogenic+fungi+from+Nortern+Thailand+and+their+production+in+cereal+grains.">Isolation of entomopathogenic fungi from Nortern Thailand and their production in cereal grains.</a> World Journal of Microbiology and Biotechnology. 28 (12), 3281-3291 (2012).</li><li>Rocha, L. F. N., Inglis, P. W., Humber, R. A., Kipnis, A., Luz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Occurrence+of+Metarhizium+spp.+in+central+Brazilian+soils.">Occurrence of Metarhizium spp. in central Brazilian soils.</a> Journal of Basic Microbiology. 53 (3), 251-259 (2013).</li><li>Quesada-Moraga, E., Navas-Cort&#233;s, J. A., Maranhao, E. A. A., Ortiz-Urquiza, A., Santiago-&#193;lvarez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+the+occurrence+and+distribution+of+entomopathogenic+fungi+in+natural+and+cultivated+soils.">Factors affecting the occurrence and distribution of entomopathogenic fungi in natural and cultivated soils.</a> Mycological Research. 111 (8), 947-966 (2007).</li><li>Mora, M. A. E., Rouws, J. R. C., Fraga, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Occurrence+of+entomopathogenic+fungi+in+atlantic+forest+soils.">Occurrence of entomopathogenic fungi in atlantic forest soils.</a> Microbiology Discovery. 4 (1), 1-7 (2016).</li><li>Goble, T. A., Dames, J. F., Hill, M. P., Moore, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=D.The+effects+of+farming+system,+habitat+type+and+bait+type+on+the+isolation+of+entomopathogenic+fungi+from+citrus+soils+in+the+Eastern+Cape+Province,+South+Africa.">D.The effects of farming system, habitat type and bait type on the isolation of entomopathogenic fungi from citrus soils in the Eastern Cape Province, South Africa.</a> BioControl. 55 (3), 399-412 (2010).</li><li>Medo, J., Cag&#225;&#328;, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+the+occurrence+of+entomopathogenic+fungi+in+soils+of+Slovakia+as+revealed+using+two+methods.">Factors affecting the occurrence of entomopathogenic fungi in soils of Slovakia as revealed using two methods.</a> Biological Control. 59 (2), 200-208 (2011).</li><li>Chase, A. R., Osborne, L. S., Ferguson, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+isolation+of+the+entomopathogenic+fungi+Beauveria+bassiana+and+Metarhizium+anisopliae+from+an+artificial+potting+medium.">Selective isolation of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae from an artificial potting medium.</a> Florida Entomologist. 69, 285-292 (1986).</li><li>Liu, Z. Y., Milner, R. J., McRae, C. F., Lutton, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+dodine+in+selective+media+for+the+isolation+of+Metarhizium+spp.+from+soil.">The use of dodine in selective media for the isolation of Metarhizium spp. from soil.</a> Journal of Invertebrate Pathology. 62, 248-251 (1993).</li><li>Rangel, D. E. N., Dettenmaier, S. J., Fernandes, E. K. K., Roberts, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Susceptibility+of+Metarhizium+spp.+and+other+entomopathogenic+fungi+to+dodine-based+selective+media.">Susceptibility of Metarhizium spp. and other entomopathogenic fungi to dodine-based selective media.</a> Biocontrol Science and Technology. 20 (4), 375-389 (2010).</li><li>Keller, S., Kessler, P., Schweizer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+insect+pathogenic+soil+fungi+in+Switzerland+with+special+reference+to+Beauveria+brongniartii+and+Metharhizium+anisopliae.">Distribution of insect pathogenic soil fungi in Switzerland with special reference to Beauveria brongniartii and Metharhizium anisopliae.</a> BioControl. 48 (3), 307-319 (2003).</li><li>Enkerli, J., Widmer, F., Keller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+field+persistence+of+Beauveria+brongniartii+strains+applied+as+biocontrol+agents+against+European+cockchafer+larvae+in+Switzerland.">Long-term field persistence of Beauveria brongniartii strains applied as biocontrol agents against European cockchafer larvae in Switzerland.</a> Biological Control. 29 (1), 115-123 (2004).</li><li>Imoulan, A., Alaoui, A., El Meziane, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+occurrence+of+soil-borne+entomopathogenic+fungi+in+the+Moroccan+endemic+forest+of+Argania+spinosa+and+their+pathogenicity+to+Ceratitis+capitata.">Natural occurrence of soil-borne entomopathogenic fungi in the Moroccan endemic forest of Argania spinosa and their pathogenicity to Ceratitis capitata.</a> World Journal of Microbiology and Biotechnology. 27 (11), 2619-2628 (2011).</li><li>Keyser, C. 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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<sup ...

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)&#160;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&#160;using T. molitor&#160;larvae in comparison to G. mellonella larvae; in contrast, Beauveria bassiana (Bals. -Criv.) Vuill&#160;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&#160;or CTC medium) should be considered according to the study&#39;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Autoclave</td>
			<td>Phoenix Luferco</td>
			<td>9451</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety cabinet</td>
			<td>Airstream ESCO</td>
			<td>AC2-4E3</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Sigma-Aldrich</td>
			<td>C0378</td>
			<td></td>
		</tr>
		<tr>
			<td>Climate chambers</td>
			<td>Eletrolab</td>
			<td>EL212/3</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip</td>
			<td>RBR</td>
			<td>3871</td>
			<td></td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma-Aldrich</td>
			<td>C7698</td>
			<td></td>
		</tr>
		<tr>
			<td>Drigalski spatula</td>
			<td>Marienfeld</td>
			<td>1800024</td>
			<td></td>
		</tr>
		<tr>
			<td>GPS app</td>
			<td>Geolocation app</td>
			<td>2.1.2005</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactophenol blue solution</td>
			<td>Sigma-Aldrich</td>
			<td>61335</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Zeiss Axio star plus</td>
			<td>1169 149</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope camera</td>
			<td>Zeiss Axiocam 105 color</td>
			<td>426555-0000-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope softwere</td>
			<td>Zen lite Zeiss 3.0</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>Olen</td>
			<td>k5-7105-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtube</td>
			<td>BRAND</td>
			<td>Z336769-1PAK</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri plates</td>
			<td>Kasvi</td>
			<td>K30-6015</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tip</td>
			<td>Vatten</td>
			<td>VT-230-200C/VT-230-1000C</td>
			<td></td>
		</tr>
		<tr>
			<td>Pippette</td>
			<td>HTL - Labmatepro</td>
			<td>LMP 200 / LMP 1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic pots</td>
			<td>Prafesta descart&#225;veis</td>
			<td>8314</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene bags</td>
			<td>Extrusa</td>
			<td>38034273/5561</td>
			<td></td>
		</tr>
		<tr>
			<td>Potato dextrose agar</td>
			<td>Kasvi</td>
			<td>K25-1022</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism software 9.1.2</td>
			<td>Graph Pad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Shovel</td>
			<td>Tramontina</td>
			<td>77907009</td>
			<td></td>
		</tr>
		<tr>
			<td>Tenebrio mollitor</td>
			<td>Safari</td>
			<td>QP98DLZ36</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiabendazole</td>
			<td>Sigma-Aldrich</td>
			<td>T8904</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 80</td>
			<td>Vetec</td>
			<td>60REAVET003662</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Biomixer</td>
			<td>QL-901</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Kasvi</td>
			<td>K25-1702</td>
			<td></td>
		</tr>
	</tbody>
</table>

comparison of methods for isolating entomopathogenic fungi from soil samples

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.

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...

Watch this Scientific Journal Video about Comparison of Methods for Isolating Entomopathogenic Fungi from Soil Samples at JoVE.com

Comparison of Methods for Isolating Entomopathogenic Fungi from Soil Samples

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 ...

The isolation of entomopathogenic fungi is significant for developing a biopesticide and can be helpful to study the diversity, distribution, and the ecology of this particular group. Using insect baits to isolate entomopathogenic fungi is considered a low cost and highly efficient method. chloramphenicol, thiabendazole, cycloheximide, artificial media does not use dodine and requires less space in the soil sample process.Critical steps include investigating CTC plates after one and two weeks to prevent other fungi from narrowing and entomopathogenic fungi in development, and then identifying entomopathogenic fungi colonies based on their macro morphology and micro morphology. Begin by collecting 800 grams of soil to a depth of 10 centimeters using a small shovel. Store the samples in polypropylene bags at room temperature until the start of the experiment.Small roots can also be collected since entomopathogenic fungi are known to have rhizosphere competence. For isolation of fungi using chloramphenicol, thiabendazole, cycloheximide, or CTC selective artificial medium, weigh 0.35 grams of the soil sample with or without roots. Place this sample in a 1.5 milliliter microtube.While working in a biosafety cabinet, add one milliliter of sterile 0.01 volume percent polyoxyethylene sorbitan monooleate aqueous suspension to the microtube containing soil and vortex the tube for 30 seconds. When done remove and pipette 50 microliters of the supernatant onto the center of Petri plates with CTC medium. Then use a sterile Drigalski spatula of a six millimeter diameter to homogeneously disperse the suspensions onto the surface of the medium.Incubate the plates in climate chambers at 25 degrees Celsius and a minimum of 80%relative humidity in the dark. After seven, 14, and 21 days of incubation, observe the plates for the growth of fungal colonies. To isolate the fungi using surface disinfected Galleria mellonella and Tenebrio molitor late stage larvae as insect baits.Immerse the larva into 0.5%sodium hypochlorite for one minute for sterilization. Then wash the larvae twice with sterile water. Assemble the baits in plastic pots with 10 small holes of 10 millimeter diameter in the lid containing 250 grams of the collected soil.Homogenize the soil every other day to allow maximum contact of larvae with soil and analyze the pots daily, seeking dead insects. After removing dead insects from the pot, superficially sterilize insects with 0.5%sodium hypochlorite for one minute. To favor the mycosis, place the sterile insects in a humid chamber at 80%relative humidity and 25 degrees Celsius for seven days.On mycosis, harvest the conidia from the insect surface and with the help of the microbiologic loop, place the conidia on potato dextrose agar medium containing 0.05%chloramphenicol or PDAC medium under a stereoscopic microscope. As an alternative, place the whole infected larvae on the PDAC medium. And incubate the culture plates in a climate chamber as explained earlier.After 14 days of incubation, identify the EPF species by analyzing the macro morphological characteristics of the fungal cultures on the plates as described in the manuscript. Next transfer the aerial conidia to slide cultures for three days at 80%relative humidity and 25 degrees Celsius. After three days stain the slide with lactophenol blue to observe the microscopic features.Under an optical microscope, observe the fungal structures such as arrangement, conidiophores, shape, and size of conidia at 400 times and confirm the EPF identification. In the study, 51 EPF strains of Metarhizium and Beaveria species were isolated from 524 soil samples. The representative analysis shows the micromorphological characteristics of a few isolates of Metarhizium species and Beaveria species.The occurrence of EPF in the 24 soil samples was studied using three different methods of isolation. Galleria mellonella bait proved to be more efficient in the isolation, followed by Tenebrio molitor bait and CTC medium. The same 24 soil samples showed no recovery of Beaveria species, but only Metarhizium.The most important things to remember are transferring the aerial conidia to slide coat observing the microscope features and confirming the entomopathogenic fungi identification using morphological keys. Additional methods include the use of other selective artificial media with a specific chemicals to reduce the growth of contaminants such as dodine. Seeking new fungal isolates with outstanding bio control traits is crucial to increasing fungi effectiveness in arthropod-pest control and exploring further questions in the invertebrate pathology field.

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