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 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.</li>
	<li>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).</li>
</ol>
2. Isolation methods for entomopathogenic fungi
<ol>
	<li>Isolation using CTC selective artificial medium.
	<ol>
		<li>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.</li>
		<li>Weigh 0.35 &#177; 0.05 g of each soil sample (with or without roots) and place it in a 1.5 mL microtube.</li>
		<li>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.</li>
		<li>Remove 50 &#181;L 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.</li>
		<li>Incubate the plates in climate chambers (25 &#177; 1 &#176;C, relative humidity &#8805;80%) in the dark and observe the growth of fungal colonies after 7, 14, and 21 days of incubation.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Isolation using insect baits
	<ol>
		<li>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.</li>
		<li>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 &#177; 1 &#176;C and relative humidity &#8805; 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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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 &#8805; 80%) at 25 &#177; 1 &#176;C for 7 days to favor the exteriorization of entomopathogenic fungi (mycosis).</li>
		<li>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 &#177; 1 &#176;C and relative humidity &#8805; 80%.</li>
		<li>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. 
		&#8203;NOTE: Use the description keys presented below in step 3 for the identification of EPF colonies.</li>
	</ol>
	</li>
</ol>
3. Identification of EPF (Metarhizium spp. and Beauveria spp.)
<ol>
	<li>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 &#177; 1 &#176;C and relative humidity &#8805; 80%.</li>
	<li>Transfer the aerial conidia to slide cultures (microculture technique)16 for 3 days at 25 &#177; 1 &#176;C and relative humidity &#8805; 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.</li>
	<li>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 Humber17,18,19,20. 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.</li>
	<li>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.</li>
	<li>After confirming the isolates to be EPF, deposit the isolates in a collection of&#160;fungal cultures. In the present study, the isolates were deposited in the entomopathogenic fungal cultures collection from&#160;the Laboratory of Microbial Control (LCM) at the Federal Rural University of Rio de Janeiro.</li>
</ol>

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The authors have no conflicts of interest.

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.

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			<td>Petri plates</td>
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			<td>K30-6015</td>
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			<td>LMP 200 / LMP 1000</td>
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			<td>Prafesta descart&#225;veis</td>
			<td>8314</td>
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			<td>Extrusa</td>
			<td>38034273/5561</td>
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			<td>Potato dextrose agar</td>
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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 ...

comparison-methods-for-isolating-entomopathogenic-fungi-from-soil

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JoVE Journal

Biology

4.2K Views.  Universidade Federal Rural do Rio de Janeiro. 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).

Video: Comparison of Methods for Isolating Entomopathogenic Fungi from Soil Samples

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical proteins, such as antibodies, vaccine candidates and enzymes. These must be purified from the plant biomass but chromatography steps are hindered by the high concentrations of host cell proteins (HCPs) in plant extracts. However, most HCPs irreversibly aggregate at temperatures above 60 &#176;C facilitating subsequent purification of the target protein. Here, three methods are presented to achieve the heat precipitation of tobacco HCPs in either intact leaves or extracts. The blanching of intact leaves can easily be incorporated into existing processes but may have a negative impact on subsequent filtration steps. The opposite is true for heat precipitation of leaf extracts in a stirred vessel, which can improve the performance of downstream operations albeit with major changes in process equipment design, such as homogenizer geometry. Finally, a heat exchanger setup is well characterized in terms of heat transfer conditions and easy to scale, but cleaning can be difficult and there may be a negative impact on filter capacity. The design-of-experiments approach can be used to identify the most relevant process parameters affecting HCP removal and product recovery. This facilitates the application of each method in other expression platforms and the identification of the most suitable method for a given purification strategy.

Three heat precipitation methods are presented that effectively remove more than 90% of host cell proteins (HCPs) from tobacco extracts prior to any other purification step. The plant HCPs irreversibly aggregate at temperatures above 60 &#176;C.

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical ...

Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility with high-throughput formatting. This protocol describes the use of three different methods for measuring cell proliferation in vitro including conventional hemocytometer counting chamber, a luminescence-based assay that utilizes the change in the metabolic activity of viable cells as a measure of the relative number of cells, and a multi-mode cell imager that measures cell number using a counting algorithm. Each method presents its own advantages and disadvantages for the measurement of cell proliferation, including time, cost and high-throughput compatibility. This protocol demonstrates that each method could accurately measure cell proliferation over time, and was sensitive to detect growth at differing cellular densities. Additionally, measurement of cell proliferation using a cell imager was able to provide further information such as morphology, confluence and allowed for a continual monitoring of cell proliferation over time. In conclusion, each method is capable of measuring cell proliferation, but the chosen method is user-dependent.

This protocol describes the use of three different methods for analyzing cell proliferation in breast cancer cell lines. This includes the use of conventional cell counting, luminescence-based cell viability, and cell counting through the use of a cell imager. Each offers advantages for the reproducible measurement of cell proliferation.

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility ...

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4&#8211;24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.&#160;

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, ...

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

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.

Here we present a protocol based on the&#160;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.

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important ...

Mass Production of Entomopathogenic Fungi, Metarhizium robertsii and Metarhizium pinghaense, for Commercial Application Against Insect Pests

Entomopathogenic fungi of the Metarhizium anisopliae species complex have gained importance as the biological control agents of agricultural insect pests. The increase in pest resistance to chemical insecticides, the growing concerns regarding the negative effects of insecticides on human health, and the environmental pollution from pesticides have led to a global drive to find novel sustainable strategies for crop protection and pest control. Previously, attempts to mass culture such entomopathogenic fungi (EPF) species as Beauveria bassiana have been conducted. However, only limited attempts have been conducted to mass culture Metarhizium robertsii and M. pinghaense for use against insect pests. This study aimed to mass-produce a sufficient number of resilient infective propagules of South African isolates of M. robertsii and M. pinghaense for commercial application. Three agricultural grain products, flaked oats, flaked barley, and rice, were used as the EPF solid fermentation substrates. Two inoculation methods, conidial suspensions and the liquid fungal culture of blastospores were used to inoculate the solid substrates. Inoculation using conidial suspensions was observed to be relatively less effective, as increased levels of contamination were observed on the solid substrates relative to when using the blastospore inoculation method. Flaked oats were found not to be a suitable growth substrate for both M. robertsii and M. pinghaense, as no dry conidia were harvested from the substrate. Flaked barley was found to favor the production of M. robertsii conidia over that of M. pinghaense, and an average of 1.83 g &plusmn; 1.47 g of dry M. robertsii conidia and zero grams of M. pinghaense conidia was harvested from the substrate. Rice grains were found to favor the conidial mass production of both M. pinghaense and M. robertsii isolates, with an average of 8.2 g &plusmn; 4.38 g and 6 g &plusmn; 2 g harvested from the substrate, respectively.

Mass Production of Entomopathogenic Fungi, Metarhizium robertsii and Metarhizium pinghaense, for Commercial Application Against Insect Pests

Entomopathogenic fungi have gained importance as the biological control agents of agricultural insect pests. In this study, the mass production of a sufficient number of resilient infective propagules of South African isolates of both Metarhizium robertsii and M. pinghaense for commercial application against insect pests was successfully conducted using agricultural grain products.

Entomopathogenic fungi of the Metarhizium anisopliae species complex have gained importance as the biological control agents of agricultural insect pests. ...

Isolation and Screening from Soil Biodiversity for Fungi Involved in the Degradation of Recalcitrant Materials

Environmental pollution is an increasing problem, and identifying fungi involved in the bioremediation process is an essential task. Soil hosts an incredible diversity of microbial life and can be a good source of these bioremediative fungi. This work aims to search for soil fungi with bioremediation potential by using different screening tests. Mineral culture media supplemented with recalcitrant substances as the sole carbon source were used as growth tests. First, soil dilutions were plated on Petri dishes with mineral medium amended with humic acids or lignocellulose. The growing fungal colonies were isolated and tested on different substrates, such as complex mixtures of hydrocarbons (petrolatum and used motor oil) and powders of different plastic polymers (PET, PP, PS, PUR, PVC). Qualitative enzymatic tests were associated with the growth tests to investigate the production of esterases, laccases, peroxidases, and proteases. These enzymes are involved in the main degradation processes of recalcitrant material, and their constitutive secretion by the examined fungal strains could have the potential to be exploited for bioremediation. More than 100 strains were isolated and tested, and several isolates with good bioremediation potential were found. In conclusion, the described screening tests are an easy and low-cost method to identify fungal strains with bioremediation potential from the soil. In addition, it is possible to tailor the screening tests for different pollutants, according to requirements, by adding other recalcitrant substances to minimal culture media.

Here, we present a protocol for screening soil biodiversity to look for fungal strains involved in the degradation of recalcitrant materials. First, fungal strains able to grow on humic acids or lignocellulose are isolated. Their activity is then tested both in enzymatic assays and on pollutants such as hydrocarbons and plastics.

Environmental pollution is an increasing problem, and identifying fungi involved in the bioremediation process is an essential task. Soil hosts an ...

Optimized Bioassay for Entomopathogenic Fungi Against Mustard Aphid

Assessment of Aphidicidal Effect of Entomopathogenic Fungi against Parthenogenetic Insect, Mustard Aphid, Lipaphis erysimi (Kalt.)

The mustard aphid (L. erysimi) is a pest that infests various cruciferous crops and transmits plant viruses. To achieve eco-friendly pest management, entomopathogenic fungi (EPF) are potential microbial control agents for controlling this pest. Therefore, virulence screening of EPF isolates under Petri dish conditions is necessary before field application. However, the mustard aphid is a parthenogenetic insect, making it difficult to record data during Petri dish experiments. A modified system for detached-leaf bioassays was developed to address this issue, using a micro-sprayer to inoculate conidia onto aphids and prevent drowning by facilitating air-drying after spore suspension. The system maintained high relative humidity throughout the observation period, and the leaf disc remained fresh for over ten days, allowing parthenogenetic reproduction of the aphids. To prevent offspring buildup, a process of daily removal using a painting brush was implemented. This protocol demonstrates a stable system for evaluating the virulence of EPF isolates against mustard aphids or other aphids, enabling the selection of potential isolates for aphid control.

Author Spotlight: Eco-Friendly Pest Management Using Entomopathogenic Fungi Against Mustard Aphids 

This protocol presents an optimized detached-leaf bioassay system for evaluating the effectiveness of entomopathogenic fungi (EPF) against the mustard aphid (Lipaphis erysimi (Kalt.)), a parthenogenetic insect. The method outlines the data collection process during Petri dish experiments, enabling researchers to consistently measure the virulence of EPF against mustard aphids and other parthenogenetic insects.

The mustard aphid (L. erysimi) is a pest that infests various cruciferous crops and transmits plant viruses. To achieve eco-friendly pest management, ...

Exploring Entomopathogenic Fungi Resources from Forest Wood Borers

Isolation, Behavioral Identification, and Pathogenicity Assessment of Entomopathogenic Fungi from a Forest Wood Borer

Forest wood borers (FWB) cause severe tree damage and economic losses worldwide. The release of entomopathogenic fungi (EPF) during the FWB emergence period is considered an acceptable alternative to chemical control. However, EPF resources have been significantly less explored for FWBs, in contrast to agricultural insect pests. This paper presents a protocol for exploring EPF resources from FWBs using wild Monochamus alternatus populations as an example. In this protocol, the assignment of traps baited with M. alternatus attractants to different populations guaranteed the collection of adequate samples with natural infection symptoms, during the emergence periods of the beetle. Following finely dissecting integuments and placing them onto a selective medium, fungal species were isolated from each part of beetle bodies and identified based on both molecular and morphological traits.
Several fungal species were certified as parasitic EPFs via re-infection of healthy M. alternatus with spore suspensions. Their behavioral phenotypes on M. alternatus were observed using scanning electron microscopy and further compared with those on the Coleopteran model insect Tribolium castaneum. For EPFs that present consistent parasitism phenotypes on both beetle species, evaluation of their activities on T. castaneum provided valuable information on lethality for future study on M. alternatus. This protocol helped the discovery of EPF newly reported on M. alternatus populations in China, which could be applied as an efficient approach to explore more EPF resources from other FWBs.

Author Spotlight: Evaluation of Entomopathogenic Fungi in Wild Monochamus alternatus Populations for Biocontrol Applications in Forest Wood Borers

Here we present a protocol for obtaining entomopathogenic fungi from a forest wood borer and a substitutive way to evaluate their entomopathogenic activities using a Coleopteran model insect. This method is efficient and convenient for exploring entomopathogenic fungal resources from wood-boring insect pests in natural forests.

Forest wood borers (FWB) cause severe tree damage and economic losses worldwide. The release of entomopathogenic fungi (EPF) during the FWB emergence ...

Characterizing Surface Antigens and Proliferation Ability in hiPSC-Driving MSCs

Comparison of Two Representative Methods for Differentiation of Human Induced Pluripotent Stem Cells into Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are restricted by limited donation, quality variations, and biosafety, the past 10 years have seen a great rise in efforts to generate MSCs from human induced pluripotent stem cells (hiPSCs). Past and recent efforts in the differentiation of hiPSCs into MSCs have been centered around two culture methodologies: (1) the formation of embryoid bodies (EBs) and (2) the use of monolayer culture. This protocol describes these two representative methods in deriving MSC from hiPSCs. Each method presents its advantages and disadvantages, including time, cost, cell proliferation ability, the expression of MSC markers, and their capability of differentiation in vitro. This protocol demonstrates that both methods can derive mature and functional MSCs from hiPSCs. The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation, while the EB method is characterized by lower time consumption.

Author Spotlight: Advancements in iPSCs and Genetic Disease Research 

This protocol describes and compares two representative methods for differentiating hiPSCs into mesenchymal stromal cells (MSCs). The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation. The embryoid bodies (EBs) method is characterized by lower time consumption.

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are ...