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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</ol>

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.

<table><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&amp;aacute;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>&lt;em&gt;Tenebrio mollitor&lt;/em&gt;</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 ...

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Research

JoVE Journal

Biology

4.3K Views.  Universidade Federal Rural do Rio de Janeiro. De isolatie van entomopathogene schimmels is belangrijk voor de ontwikkeling van een biopesticide en kan nuttig zijn om de diversiteit, verspreiding en ecologie van deze specifieke groep te bestuderen. Het gebruik van insectenaas om entomopathogene schimmels te isoleren wordt beschouwd als een goedkope en zeer efficiënte methode. chlooramfenicol, thiabendazool, cycloheximide, kunstmatige media gebruiken geen dodine en vereisen minder ruimte in het bodemmonsterproces.Kritieke stappen o...

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

Soil Sampling and Isolation of Entomopathogenic Nematodes (Steinernematidae, Heterorhabditidae)

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to two families: Steinernematidae and Heterorhabditidae. Until now, more than 70 species have been described in the Steinernematidae and there are about 20 species in the Heterorhabditidae. The nematodes have a mutualistic partnership with Enterobacteriaceae bacteria and together they act as a potent insecticidal complex that kills a wide range of insect species.
Herein, we focus on the most common techniques considered for collecting EPN from soil. The second part of this presentation focuses on the insect-baiting technique, a widely used approach for the isolation of EPN from soil samples, and the modified White trap technique which is used for the recovery of these nematodes from infected insects. These methods and techniques are key steps for the successful establishment of EPN cultures in the laboratory and also form the basis for other bioassays that consider these nematodes as model organisms for research in other biological disciplines. The techniques shown in this presentation correspond to those performed and/or designed by members of S. P. Stock laboratory as well as those described by various authors.

Soil Sampling and Isolation of Entomopathogenic Nematodes Steinernematidae, Heterorhabditidae

Entomopathogenic nematodes are soil-inhabiting roundworms that parasitize a wide range of insects. We demonstrate sampling methods used for the isolation of these nematodes from the soil using two techniques: the insect baiting and the modified White trap, for the recovery of nematodes from soil samples and infected insect cadavers, respectively.

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to ...

Environment

Methods for Patch Clamp Capacitance Recordings from the Calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal.

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. ...

Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance

A major concern when developing commercial mycoinsecticides is the kill speed compared to that of chemical insecticides. Therefore, isolation and screening for the selection of a fast-acting, highly virulent entomopathogenic fungus are important steps. Entomopathogenic fungi, such as Metarhizium, Beauveria, and Nomurea, which act by contact, are better suited than Bacillus thuringiensis or nucleopolyhedrosis virus (NPV), which must be ingested by the insect pest. In the present work, we isolated 68 Metarhizium strains from infected insects using a soil dilution and bait method. The isolates were identified by the amplification and sequencing of the ITS1-5.8S-ITS2 and 26S rDNA region. The most virulent strain of Metarhizium anisopliae was selected based on the median lethal concentration (LC50) and time (LT50) obtained in insect bioassays against III-instar larvae of Helicoverpa armigera. The mass production of spores by the selected strain was carried out with solid-state fermentation (SSF) using rice as a substrate for 14 days. Spores were extracted from the sporulated biomass using 0.1% tween-80, and different formulations of the spores were prepared. Field trials of the formulations for the control of an H. armigera infestation in pigeon peas were carried out by randomized block design. The infestation control levels obtained with oil and aqueous formulations (78.0% and 70.9%, respectively) were better than the 63.4% obtained with chemical pesticide.

Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance

Here, we report the different stages involved in the knowledge-based development of an effective mycoinsecticide, including the isolation, identification, screening, and selection of the "best-fit" entomopathogenic fungus, Metarhizium anisopliae, for the control of insect pests in agriculture.

A major concern when developing commercial mycoinsecticides is the kill speed compared to that of chemical insecticides. Therefore, isolation and ...

Establishing Fungal Entomopathogens as Endophytes: Towards Endophytic Biological Control

Beauveria bassiana is a fungal entomopathogen with the ability to colonize plants endophytically. As an endophyte, B. bassiana may play a role in protecting plants from herbivory and disease. This protocol demonstrates two inoculation methods to establish B. bassiana endophytically in the common bean (Phaseolus vulgaris), in preparation for subsequent evaluations of endophytic biological control. Plants are grown from surface-sterilized seeds for two weeks before receiving a B. bassiana treatment of 108 conidia/ml (or water) applied either as a foliar spray or a soil drench. Two weeks later, the plants are harvested and their leaves, stems and roots are sampled to evaluate endophytic fungal colonization. For this, samples are individually surface sterilized, cut into multiple sections, and incubated in potato dextrose agar media for 20 days. The media is inspected every 2-3 days to observe fungal growth associated with plant sections and record the occurrence of B. bassiana to estimate the extent of its endophytic colonization. Analyses of inoculation success compare the occurrence of B. bassiana within a given plant part (i.e. leaves, stems or roots) across treatments and controls. In addition to the inoculation method, the specific outcome of the experiment may depend on the target crop species or variety, the fungal entomopathogen species strain or isolate used, and the plant's growing conditions.

This protocol demonstrates two inoculation methods to introduce the fungal entomopathogen Beauveria bassiana as an endophyte in the common bean (Phaseolus vulgaris), in preparation for subsequent evaluations of endophytic biological control.

 Beauveria bassiana is a fungal entomopathogen with the ability to colonize plants endophytically. As an endophyte, B. bassiana may play a role in ...

Bioengineering

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 ...

Isolating Stem Cells from Soft Musculoskeletal Tissues

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of excitement for treating injured and diseased tissues due to their impressive capabilities to undergo multi-lineage cell differentiation and their self-renewal ability. Most importantly, these qualities have made them advantageous for use in autologous cell transplantation therapies. The current protocol will introduce the readers to the modified preplate technique where soft tissues of the musculoskeletal system, e.g. tendon and muscle, are 1st enzymatically dissociated and then placed in collagen coated flasks with medium. The supernatant, which is composed of medium and the remaining floating cells, is serially transferred daily to new flasks. The stem cells are the slowest to adhere to the flasks which is usually takes 5-7 days (serial transfers or preplates) . By using this technique, adult stem cells present in these tissues can be easily harvested through fairly non-invasive procedures.

Isolating adult stem cells from musculoskeletal soft tissues based on the cell's adherence speed to flask.

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of ...

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 ...

Isolation of Culturable Yeasts and Molds from Soils to Investigate Fungal Population Structure

Soil is host to an incredible amount of microbial life, with each gram containing up to billions of bacterial, archaeal, and fungal cells. Multicellular fungi such as molds and unicellular fungi, broadly defined as yeasts, fulfill essential roles in soil ecosystems as decomposers of organic material and as food sources for other soil dwellers. Fungal species diversity in soil is dependent on a multitude of climatic factors such as rainfall and temperature, as well as soil properties including organic matter, pH, and moisture. Lack of adequate environmental sampling, especially in regions of Asia, Africa, South America, and Central America, hinders the characterization of soil fungal communities and the discovery of novel species. 
We characterized soil fungal communities in nine countries across six continents using ~4,000 soil samples and a protocol developed in the laboratory for the isolation of yeasts and molds. This protocol begins with separate selective enrichment for yeasts and the medically relevant mold Aspergillus fumigatus, in liquid media while inhibiting bacterial growth. Resulting colonies are then transferred to solid media and further processed to obtain pure cultures, followed by downstream genetic characterization. Yeast species identity is established via sequencing of their internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene cluster, while global population structure of A. fumigatus is explored via microsatellite marker analysis. 
The protocol was successfully applied to isolate and characterize soil yeast and A. fumigatus populations in Cameroon, Canada, China, Costa Rica, Iceland, Peru, New Zealand, and Saudi Arabia. These findings revealed much-needed insights on global patterns in soil yeast diversity, as well as global population structure and antifungal resistance profiles of A. fumigatus. This paper presents the method of isolating both yeasts and A. fumigatus from international soil samples.

This protocol is an effective, speedy method of culturing yeasts and the mold Aspergillus fumigatus from large sets of soil samples in as little as 7 days. The methods can be easily modified to accommodate a range of incubation media and temperatures as needed for experiments.

Soil is host to an incredible amount of microbial life, with each gram containing up to billions of bacterial, archaeal, and fungal cells. Multicellular ...

Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as any other vital resource, the fungi with which these plants intimately associate are essential for them. Hence, some of the most relevant techniques in studying mycoheterotrophic species are the ones that enable the investigation of associated fungi, especially those inhabiting roots and subterranean organs. In this context, techniques for identifying culture-dependent and culture-independent endophytic fungi are commonly applied. Isolating fungal endophytes provides a means for morphologically identifying them, analyzing their diversity, and maintaining inocula for applications in the symbiotic germination of orchid seeds. However, it is known that there is a large variety of non-culturable fungi inhabiting plant tissues. Thus, culture-independent molecular identification techniques offer a broader cover of species diversity and abundance. This article aims to provide the methodological support necessary for starting two investigation procedures: a culture-dependent and an independent one. Regarding the culture-dependent protocol, the processes of collecting and maintaining plant samples from collection sites to laboratory facilities are detailed, along with isolating filamentous fungi from subterranean and aerial organs of mycoheterotrophic plants, keeping a collection of isolates, morphologically characterizing hyphae by slide culture methodology, and molecular identification of fungi by total DNA extraction. Encompassing culture-independent methodologies, the detailed procedures include collecting plant samples for metagenomic analyses and total DNA extraction from achlorophyllous plant organs using a commercial kit. Finally, continuity protocols (e.g., polymerase chain reaction [PCR], sequencing) are also suggested for analyses, and techniques are presented here.

The current article aims to provide detailed and adequate protocols for the isolation of plant-associated endophytic fungi, long-term preservation of isolates, morphological characterization, and total DNA extraction for subsequent molecular identification and metagenomic analyses.

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as ...

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 ...

Comparison of Methods for Isolating Entomopathogenic Fungi from Soil Samples

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