Name: isolation and screening from soil biodiversity for fungi involved in the degradation of recalcitrant materials
Uploaded: 2022-05-16T14:00:00.000+00:00
Duration: 8 min 21 s
Description: Watch this Scientific Journal Video about Isolation and Screening from Soil Biodiversity for Fungi Involved in the Degradation of Recalcitrant Materials at JoVE.com

We acknowledge Scuola di Alta Formazione Dottorale (SAFD) of the University of Pavia and Professor Solveig Tosi for providing the opportunity for this work.

1. Selection of fungal strains able to degrade recalcitrant materials from soil
<ol>
	<li>Preparation of antibiotic solution.
	<ol>
		<li>Put penicillin (50 mg/L), streptomycin (40 mg/L), chlortetracycline (40 mg/L), neomycin (100 mg/L), and chloramphenicol (100 mg/L) into 250 mL of deionized sterile water.</li>
		<li>Before adding chloramphenicol to the antibiotic solution, dissolve it into 3 mL of &#8805;99% ethanol.</li>
		<li>Place the antibiotic solution on a magnetic stirrer (no heat) with a magnetic stirring bar inside the solution for 10 min. Store it at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Preparation of growth media.
	<ol>
		<li>Put 1 g of commercial humic acid, 3.26 g of Bushnell-Haas broth (BH), and 15 g of agar into 1 L of deionized water and autoclave it for 20 min at 121 &#176;C (humic acid agar).</li>
		<li>Plate the humic acid agar into Petri dishes under a laminar flow cabinet.</li>
		<li>Put 4 g of lignocellulose, 3.26 g of BH, and 15 g of agar into 1 L of deionized water and autoclave it for 20 min at 121 &#176;C (lignocellulose agar).</li>
		<li>Plate the lignocellulose agar into Petri dishes under a laminar flow cabinet.</li>
		<li>Under a laminar flow cabinet, after the media have solidified in the Petri dishes, transfer 1 mL of the antibiotic solution onto the prepared plates using a sterile syringe with a 0.2 &#181;m filter to sterilize it.</li>
		<li>Distribute the antibiotic solution evenly on the plate surfaces using a sterile disposable L-shaped cell spreader and let it air-dry under the laminar flow cabinet.</li>
		<li>Put 20 g of malt extract broth and 15 g of agar into 1 L of deionized water and autoclave it for 20 min at 121 &#176;C (malt extract agar - MEA).</li>
		<li>Plate the MEA into Petri dishes under a laminar flow cabinet.</li>
	</ol>
	</li>
	<li>Soil sampling and soil dilutions.
	<ol>
		<li>Sample the soil of interest at a depth of 10 cm, removing any plants, grass, and dead leaves from the top layer, and put it into sterile polyethylene bags.</li>
		<li>After reaching the laboratory, sieve the soil using a 2 mm mesh, removing any roots and plant debris.</li>
		<li>If it is not possible to proceed with the downstream analysis right away, store the sieved soil samples at 4 &#176;C.</li>
		<li>Working under a laminar flow cabinet, put 1 g of the sieved soil into a sterile 15 mL tube with 10 mL of sterile deionized water (1 x 10-1 dilution). Shake the tube horizontally for 30 min.</li>
		<li>Under a laminar flow cabinet, before the suspension settles, take 1 mL of the 1 x 10-1 dilution with a sterile pipette and transfer it to a 9 mL of deionized water blank (1 x 10-2 dilution). Vortex it thoroughly.</li>
		<li>Before the suspension settles, take 1 mL of the 1 x 10-2 dilution with a sterile pipette and transfer it to a 9 mL of deionized water blank (1 x 10-3 dilution). Vortex it thoroughly.</li>
	</ol>
	</li>
	<li>Plating soil dilutions on selective media.
	<ol>
		<li>Working under a laminar flow cabinet, collect 100 &#181;L of the 1 x 10-3 dilution using a micropipette with a sterile point and transfer it onto a humic agar Petri dish. Do this for at least 4 or 5 Petri dishes.</li>
		<li>Distribute the dilution evenly on the Petri dish surface using a sterile disposable L-shaped cell spreader.</li>
		<li>Let it air-dry for 10-15 min under the laminar flow cabinet.</li>
		<li>Repeat Steps 1.4.1.-1.4.3. using lignocellulose Petri dishes.</li>
		<li>Incubate at 25 &#176;C in the dark for a maximum of 15 days.</li>
	</ol>
	</li>
	<li>Isolation of fungal colonies grown from selective media to growth media.
	<ol>
		<li>Starting from 3 days after preparation, check the prepared selective media Petri dishes daily for any fungal colony growth for a maximum of 15 days.</li>
		<li>Check all the fungal colonies present for similarities. If necessary, prepare a slide to be observed under a light microscope. 
		NOTE: The aim is to isolate the highest number of fungal colonies, but it is necessary to avoid always isolating the same fungal strain from different plates, so this check is very important. Look for colonies with different macro and micromorphology.</li>
		<li>Under a laminar flow cabinet or at a Bunsen burner, isolate each chosen fungal strain by gently removing a small part of the mycelium from the colony with a sterile inoculation needle and transferring it to a new MEA Petri dish. 
		NOTE: In this phase, it is very important to be delicate and precise because there is a high risk of contamination from the other fungal colonies present in the original Petri dish. If more than one fungal strain grows on the inoculated MEA plate, repeat Step 1.5.3.</li>
		<li>Incubate at 25 &#176;C in the dark for 7 days. These are the fungal strains to be tested further on specific recalcitrant substances.</li>
	</ol>
	</li>
</ol>
2. Growth tests on recalcitrant substances
<ol>
	<li>Growth test on petrolatum.
	<ol>
		<li>Transfer 20 mL of liquid BH (3.26 g/L of BH) into 50 mL vials and sterilize them by autoclaving at 121 &#176;C for 20 min.</li>
		<li>Transfer 7 mL of deionized water into 15 mL glass vials and add some pieces of broken glass coverslips into each vial.</li>
		<li>Sterilize them by autoclaving at 121 &#176;C for 20 min.</li>
		<li>Under the laminar flow cabinet, add 1 mL of petrolatum to each 50 mL BH vial using a sterile pipette. Keep some vials with only BH as the negative control.</li>
		<li>Under the laminar flow cabinet, remove the mycelium on the surface of the medium from the MEA plates with the chosen fungal strains (prepared at Step 1.5.3.) using a sterile needle and transfer it to 15 mL glass vials with the broken coverslips.</li>
		<li>Agitate each vial on a vortex mixer for 2 min, allowing the broken coverslips to cut the cube of the fungal colony, making a fungal suspension.</li>
		<li>Inoculate each petrolatum vial with 200 &#181;L of fungal suspension. Make two replicates for each fungal strain.</li>
		<li>For the negative control, put 200 &#181;L of fungal suspension into vials with only liquid BH. Moreover, keep a couple of vials with petrolatum without any fungal inoculation to check for contamination in the medium.</li>
		<li>Incubate the vials at 25 &#176;C in the dark and check them after 15 days and 30 days from the inoculum.</li>
	</ol>
	</li>
	<li>Growth test on used engine oil.
	<ol>
		<li>Put 3.26 g of BH and 15 g of agar into 1 L of deionized water and autoclave it for 20 min at 121 &#176;C (BHA).</li>
		<li>Plate the BHA into Petri dishes under a laminar flow cabinet.</li>
		<li>When it has solidified, transfer 500 &#181;L of used engine oil onto the BHA Petri dishes and distribute it evenly on the plate surfaces using a sterile disposable L-shaped cell spreader.</li>
		<li>Inoculate each used engine oil BHA plate with each fungal strain, collecting the mycelium from the plates prepared at Step 1.5.3. Work at a Bunsen burner or under a laminar flow hood to avoid contamination. Make two replicates for each fungal strain.</li>
		<li>For the negative control, inoculate the Petri dishes with BHA only. Moreover, keep a couple of used engine oil BHA Petri dishes without any fungal inoculation to check for contamination in the medium.</li>
		<li>Incubate the Petri dishes at 25 &#176;C in the dark and check them after 15 days and 30 days from the inoculum.</li>
	</ol>
	</li>
	<li>Growth test on plastics.
	<ol>
		<li>Transfer 200 &#181;L of fungal suspension (Steps 2.1.5.-2.1.6.) into a 96-microwell plate. Make three replicates of the fungal strain-plastic combination.</li>
		<li>To each well with the fungal suspension, add 10 mg of a different kind of plastic powder (polyethylene terephthalate - PET, polyvinyl chloride - PVC, high-density polyethylene - HDPE, polystyrene - PS, polyurethane - PUR).</li>
		<li>For the negative control, instead of adding the plastic powder, add 200 &#181;L of sterilized BH solution to the well with the fungal suspension. Moreover, for each type of plastic powder, fill a well with 200 &#181;L of sterilized BH solution and 10 mg of each plastic dust to check for contamination of the medium.</li>
		<li>Incubate the microwell plates at 25 &#176;C in the dark and check them after 15 days and 30 days from the inoculum.</li>
	</ol>
	</li>
</ol>
3. Qualitative enzymatic tests
<ol>
	<li>Qualitative ligninolytic enzymes colorimetric test.
	<ol>
		<li>Put 27 g of potato dextrose broth (PD) and 15 g of agar into 1 L of deionized water and autoclave it for 20 min at 121 &#176;C (PDA).</li>
		<li>When the medium has cooled down a little but is still liquid, add gallic acid (5 g/L) under a laminar flow hood and plate the medium into Petri dishes.</li>
		<li>Inoculate the PDA + gallic acid plates with each fungal strain, collecting the mycelium from the plates prepared at Step 1.5.3. Work at a Bunsen burner or under a laminar flow hood to avoid contamination.</li>
		<li>As negative controls, prepare Petri dishes with only PDA (no gallic acid) and inoculate them with the fungal strains (one for each fungal strain), as well as one Petri dish with PDA + gallic acid and no fungal inoculation.</li>
		<li>Incubate the Petri dishes at 25 &#176;C in the dark and check them after 7 days from the inoculum.</li>
	</ol>
	</li>
	<li>Qualitative laccases colorimetric test.
	<ol>
		<li>Put 27 g of PD and 15 g of agar into 1 L of deionized water and autoclave it for 20 min at 121 &#176;C (PDA).</li>
		<li>When the medium has cooled down a little but is still liquid, add guaiacol (400 &#181;L/L) under a laminar flow hood and plate it into Petri dishes.</li>
		<li>Inoculate the PDA + guaiacol plates with each fungal strain, collecting the mycelium from the plates prepared at Step 1.5.3. Work at a Bunsen burner or under a laminar flow hood to avoid any contamination.</li>
		<li>As negative controls, prepare Petri dishes with only PDA (no guaiacol) And inoculate them with the fungal strains (one for each fungal strain), as well as one Petri dish with PDA + guaiacol and no fungal inoculation.</li>
		<li>Incubate the Petri dishes at 25 &#176;C in the dark and check them after 7 days from the inoculum.</li>
	</ol>
	</li>
	<li>Qualitative proteases test.
	<ol>
		<li>Prepare the YES medium by adding K2HPO4 (1 g/L) , KH2PO4 (0.5 g/L), MgSO4&#183;7H2O (0.5 g/L), MnCl2&#183;4H2O (0.001 g/L), CuCl2&#183;2H2O (1.4 x 10-5 g/L), ZnCl2 (1.1 x 10-5 g/L), CoCl2&#183;6H2O (2 x 10-5 g/L), Na2MoO4&#183;2H2O (1.3 x 10-5 g/L), FeCl3&#183;6H2O (7.5 x 10-5 g/L), and gelatin/peptone (0.02 g/L) to deionized water.</li>
		<li>Place the YES medium on a magnetic stirrer (no heat) with a magnetic stirring bar inside the medium for 10 min.</li>
		<li>When all the salts have dissolved into the medium, transfer 5 mL of the solution into 20 mL sterile glass vials and autoclave them for 20 min at 121 &#176;C.</li>
		<li>For the negative control, prepare some 20 mL sterile glass vials with 5 mL of BH solution and autoclave them for 20 min at 121 &#176;C.</li>
		<li>Under a laminar flow cabinet, transfer 200 &#181;L of fungal suspension (Steps 2.1.5.-2.1.6.) for each strain to the YES medium sterilized vials (make at least 2 replicates per fungal strain). For each fungal strain, add 200 &#181;L of the same fungal suspension to BH sterilized vials to have a negative control.</li>
		<li>Incubate the vials at 25 &#176;C in the dark for 21 days and check them every 7 days.</li>
	</ol>
	</li>
	<li>Qualitative esterases test.
	<ol>
		<li>Prepare the Tween 80 medium by adding peptone (10 g/L), NaCl (5 g/L), CaCl2 (0.1 g/L), and 10 mL of Tween 80 to 1 L of deionized water.</li>
		<li>Place the Tween 80 medium on a magnetic stirrer (no heat) with a magnetic stirring bar inside the medium for 10 min.</li>
		<li>When everything is melted inside the medium, transfer 5 mL of the solution into 20 mL sterile glass vials and autoclave them for 20 min at 121 &#176;C.</li>
		<li>For the negative control, prepare some 20 mL sterile glass vials with 5 mL of BH solution and autoclave them for 20 min at 121 &#176;C.</li>
		<li>Under a laminar flow cabinet, transfer 200 &#181;L of fungal suspension (Steps 2.1.5.-2.1.6.) for each strain to the Tween 80 medium sterilized vials (make at least 2 replicates per fungal strain). For each fungal strain, add 200 &#181;L of the same fungal suspension to BH sterilized vials for negative control.</li>
		<li>Incubate the vials at 25 &#176;C in the dark for 21 days and check them every 7 days.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

The rich biodiversity of soil is an abundant source of fungi that possess numerous metabolic abilities, some of which could be potential candidates for bioremediation. Selective media tests (Section 1 of the protocol) are easy-to-perform and effective methods for isolating fungi able to grow on natural complex polymers as their sole carbon source. Fungi can produce extracellular, non-specific hydrolases and oxidoreductases30 such as the ligninolytic enzymes laccases and peroxidases31. These enzymes can degrade lignocellulose and humic acids, but also many different xenobiotics, including hydrocarbons, aromatic compounds, and chlorinated organic compounds32. 
The described methods produce results that are easy to interpret and are low-cost to carry out. These characteristics allow them to be used by non-experts. However, certain aspects, such as sterility and morphological identification, require special attention. For example, the 30 min agitation of the soil suspension (Step 1.3.4.) is necessary so that the fungal spores and propagules are freed from the soil microstructure and can be suspended in the solution. In this way, by plating dilutions of this soil suspension, it is possible to isolate the maximum number of fungal strains. Macro and microscopic morphological identification of fungal strains are important in order to differentiate between strains and avoid isolating the same fungal strain multiple times. Moreover, sterility and avoidance of microbial contaminations are crucial because the presence of other active fungal strains could misrepresent the activity of the studied colonies.
After the selective tests, an array of growth tests were performed on different recalcitrant substances, such as petrolatum, used engine oil, and different plastic polymers. This stage can be customized according to the recalcitrant substances of interest. The important step is to add the selected substance to BH or BHA as the sole carbon source and to check the fungal growth against a control without the added recalcitrant substance.
The aim of the growth tests on plastics and hydrocarbons was to assess whether the fungus could use these substances as its sole carbon source by qualitatively observing its growth on the material. Unfortunately, due to the extreme recalcitrance of these materials, it is very difficult to quantify the actual decrease in the weight of the substrate and the increase in the weight of the fungal biomass, especially after a relatively short amount of time. If a fungal strain performs very well in these screening growth tests, further studies should be undertaken to quantify the degradation of the recalcitrant substance.
The used enzymatic tests (Section 3 of the protocol) were chosen because they proved to be efficient and fast to perform, but they produce only qualitative results. They highlight the degradation potential of a fungal strain, but they cannot precisely quantify it. If a strain of particular interest is isolated, further tests, such as spectrophotometric quantitative enzyme essays, could be carried out to describe the metabolic activity more precisely. The only enzymatic test that showed suboptimal efficacy was the qualitative proteases test, with a low number of strains able to grow on the YES medium. Indeed, the high percentage of strains able to grow on PUR suggests that the fungi produced proteases involved in breaking the amide and urethane bonds, with esterase targeting the ester bonds40. The almost complete absence of protease-producing fungi suggests there may have been a problem in the method used. Other tests could be adopted for protease activity, such as casein41, skimmed milk42, or milk powder43 agar plate tests. The gallic acid test proved to be very useful for detecting the production of ligninolytic enzymes, but, unfortunately, it was not very specific and could not distinguish the type of ligninolytic enzyme produced (either peroxidases or laccases).
The results of this work suggest that the use of lignocellulose or humic acids as selective media allows fungal strains with bioremediation potential to be isolated from the rich soil biodiversity. Indeed, more than 70% of the fungal strains isolated by the selective methods were able to grow on petrolatum or used engine oil, and 60% were able to grow on different plastic polymers as the sole carbon source. Moreover, the qualitative enzymatic tests described the metabolic activity of these fungi linked to biodegradation processes. The selection of fungal strains showing +++ activity in one or more tests is recommended because it increases the chances of finding a fungal strain that is very active in bioremediation. Tests using gas mass-chromatography (GC-MS) on the recalcitrant substances after prolonged contact with the fungal strains selected with our screening tests have shown actual biodegradation of the materials9,44. The use of these simple screening tests in different ecosystems will allow the investigation of fungal biodiversity in different soils. Searching for fungi that can degrade recalcitrant materials will increase the number of identified strains that have the potential for bioremediation and help tackle the growing problem of environmental pollution.

Soil is a fundamental component of life on Earth and is the basis of many ecosystems. The minerals, organic matter, and microorganisms in the soil can be considered as one system, with close associations and interactions occurring between them. The interactions of these compounds have an important impact on terrestrial processes, environmental quality, and ecosystem health1. Soil pollution poses serious environmental problems worldwide. The indiscriminate, long-term, and excessive application of recalcitrant and toxic substances, such as pesticides, petroleum products, plastics, and other chemicals, has serious effects on soil ecology and, as a result, can alter soil microbiota. Microbial communities in soils are composed of a wide range of organisms in different physiological states, with the majority being bacteria and fungi. Many of the contaminants in soils have medium- to long-term stability, and their persistence can lead to the development of adaptive mechanisms that allow the microorganisms to utilize recalcitrant substances as nutrients2,3. These microorganisms can, therefore, be considered for bioremediation techniques.
Bioremediation tries to mitigate the effects of pollution by using microorganisms and their enzymes for the degradation or transformation of waste into less toxic or non-toxic compounds. Various species of archaea, bacteria, algae, and fungi possess this bioremediation ability4. As a result of their particular biodegradative actions, fungi are especially promising organisms for bioremediation. They can attack different substrates using their hyphal network, enabling them to penetrate the soil matrix more efficiently than other microorganisms. Additionally, they can reach inaccessible interstices where contaminants are difficult to remove5, and they can also survive low moisture levels6. Moreover, fungi synthesize different cassettes of unspecific enzymes, usually to degrade natural recalcitrant substances such as cellulose, lignin, and humic acids. Those that lack the target substrate can be involved in the degradation of a wide range of recalcitrant pollutants, such as hydrocarbons, plastics, and pesticides7,8,9,10. Therefore, although many fungal species have already been reported as bioremediation agents, there is increasing interest in exploring species that have not yet been studied to select candidates for the bioremediation of recalcitrant contaminating substances. The species already known to have bioremediation properties belong to the phyla Ascomycota11,12,13, Basidiomycota14,15,&#160;and Mucoromycota. For example, the genera Penicillium and Aspergillus are well known to be involved in the degradation of aliphatic hydrocarbons13, different plastic polymers16,17,18, heavy metals19, and dyes20. Similarly, studies carried out on basidiomycetes fungi, such as Phanerochaete chrysosporium and Trametes versicolor, have revealed their involvement in the oxidation of recalcitrant materials such as aromatic hydrocarbons13 and plastics21. Another example of fungi involved in the biodegradation processes are the zygomycetes Rhizopus spp., Mucor spp., and Cunninghamella spp.22,23. In particular, Cunninghamella is able to oxidase aromatic hydrocarbons and is considered a model organism for studying the detoxification of products from a wide range of xenobiotics13.
There are several fungal enzymes involved in the major degradative processes of recalcitrant materials24,25, such as esterase, laccase, peroxidase, and protease. Laccases are copper-containing oxidases produced in the cell and subsequently secreted, that allow the oxidation of a variety of phenolic and aromatic compounds. They can degrade ortho and para diphenols, the amino group-containing phenols, lignin, and the aryl group-containing diamines26. Peroxidases use hydrogen peroxide as a mediator to degrade lignin and other aromatic compounds. There are many different peroxidases, but the ones with the greatest potential to degrade toxic substances are lignin peroxidase and manganese peroxidase27.
Esterases and proteases belong to the group of extra- or ecto-cellular enzymes, which act outside their cells of origin but are still bound to them. These enzymes can catalyze the hydrolysis of large recalcitrant molecules into smaller ones. Due to their low substrate specificity, these enzymes can play a key role in the bioremediation of various pollutants, such as textile dyes, effluents released from the pulp and paper industries and leather tanning, petroleum products, plastics, and pesticides28,29,30.
A number of screening methods to select for bioremediative fungal strains have already been published. For example, straw-based agar medium has been used to screen for white-rot fungi with high potential in the polycyclic aromatic hydrocarbons (PAH) degradation31; and small pieces of rotting wood have been placed onto malt extract agar (MEA) to isolate wood-rotting fungi32. However, most of the methods that have already been proposed select very specific fungi for their activity of interest. This research proposes a wider approach for selecting soil fungi with a broader range of action. The method relies on the initial plating of serial dilutions of soil samples onto a medium amended with humic acids or lignocellulose mixed with antibiotics to select fungi with the ability to degrade these natural recalcitrant substances. Humic acids and lignocellulose, in fact, are substances that are extremely resistant to biodegradation since they have very complex molecular structures, and this allows them to be excellent indicators of the degradative ability of the tested fungi33,34. Subsequently, the fungi selected in the first tests are screened to identify those with the potential to degrade specific pollutants such as petrolatum, used engine oil, and plastics. Finally, qualitative enzymatic tests are performed to detect fungal strains able to produce enzymes involved in the biodegradation processes of recalcitrant substances. For this purpose, protease and esterase tests are conducted, while gallic acid and guaiacol are used as indicators of laccase and other ligninolytic enzyme production35,36. These substrates are used because a strong correlation has been found between the ability of fungi to oxidize them to their brown-colored form and the possession of ligninolytic ability37,38,39.
Through these protocols, it is possible to isolate fungal strains with high degradative potential and a broad spectrum of action directly from soil samples. The isolation of these fungal strains could help find new candidates for bioremediation purposes.

<table><tbody><tr><td>96 microwell plate</td><td>Greiner bio-one</td><td>650185</td><td></td></tr><tr><td>Agar</td><td>VWR</td><td>84609.05</td><td></td></tr><tr><td>Bushnell-Haas Broth</td><td>Fluka</td><td>B5051</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C5670</td><td></td></tr><tr><td>Chloroamphenicol</td><td>Eurobio</td><td>GABCRL006Z</td><td></td></tr><tr><td>Chlortetracycline</td><td>Sigma-Aldrich</td><td>Y0001451</td><td></td></tr><tr><td>CoCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>C8661</td><td></td></tr><tr><td>CuCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>C3279</td><td></td></tr><tr><td>Ethanol</td><td>VWR Chemicals</td><td>20821.296</td><td></td></tr><tr><td>FeCl&lt;sub&gt;3&lt;/sub&gt;&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>236489</td><td></td></tr><tr><td>Filter 0.2 &amp;micro;m</td><td>Whatman</td><td>10462200</td><td></td></tr><tr><td>gallic acid</td><td>Sigma-Aldrich</td><td>G7384</td><td></td></tr><tr><td>Glass cover slips</td><td>Biosigma</td><td>VBS634</td><td></td></tr><tr><td>Glass vials 15 mL</td><td>SciLabware</td><td>P35467</td><td></td></tr><tr><td>guaiacol</td><td>Sigma-Aldrich</td><td>G5502</td><td></td></tr><tr><td>High-density polyethylene (HDPE)</td><td>Sigma-Aldrich</td><td>434272</td><td></td></tr><tr><td>Humic acids</td><td>Aldrich Chemistry</td><td>53680</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P8281</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P5655</td><td></td></tr><tr><td>Lignocellulose</td><td>/</td><td>/</td><td>Sterilized bioethanol production waste</td></tr><tr><td>L-shaped cell spreader</td><td>Laboindustria S.p.a</td><td>21133</td><td></td></tr><tr><td>magnetic stirrer</td><td>A.C.E.F</td><td>8235</td><td></td></tr><tr><td>Malt Extract Broth</td><td>Sigma-Aldrich</td><td>70146</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>M2643</td><td></td></tr><tr><td>Micropipette 1000 &amp;mu;L</td><td>Gilson</td><td>FA10006M</td><td></td></tr><tr><td>Micropipette 200&amp;nbsp; &amp;mu;L</td><td>Gilson</td><td>FA10005M</td><td></td></tr><tr><td>MnCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>M5005</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;MoO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>M1651</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S5886</td><td></td></tr><tr><td>Neomycin</td><td>Sigma-Aldrich</td><td>N0401000</td><td></td></tr><tr><td>Penicillin</td><td>Sigma-Aldrich</td><td>1504489</td><td></td></tr><tr><td>peptone</td><td>Sigma-Aldrich</td><td>83059</td><td></td></tr><tr><td>Polyethylene terephthalate (PET)</td><td>Goodfellow</td><td>ES306031</td><td></td></tr><tr><td>Petri dishes</td><td>Laboindustria S.p.a</td><td>21050</td><td></td></tr><tr><td>Petrolatum (Paraffin liquid)</td><td>A.C.E.F</td><td>009661</td><td></td></tr><tr><td>Potato Dextrose Broth</td><td>Sigma-Aldrich</td><td>P6685</td><td></td></tr><tr><td>Polystyrene (PS)</td><td>Sigma-Aldrich</td><td>331651</td><td></td></tr><tr><td>Polyurethane (PUR)</td><td>Sigma-Aldrich</td><td>GF20677923</td><td></td></tr><tr><td>Polyvinyl chloride (PVC)</td><td>Sigma-Aldrich</td><td>81388</td><td></td></tr><tr><td>Sterile falcon tube</td><td>Greiner bio-one</td><td>227 261</td><td></td></tr><tr><td>Sterile glass vials 20 mL</td><td>Sigma-Aldrich</td><td>SU860051</td><td></td></tr><tr><td>Sterile point 1000&amp;nbsp; &amp;mu;L</td><td>Gilson</td><td>F172511</td><td></td></tr><tr><td>Sterile point 200&amp;nbsp; &amp;mu;L</td><td>Gilson</td><td>F172311</td><td></td></tr><tr><td>Sterile polyethylene bags</td><td>WHIRL-PAK</td><td>B01018</td><td></td></tr><tr><td>sterile syringe</td><td>Rays</td><td>5523CM25</td><td></td></tr><tr><td>Streptomycin</td><td>Sigma-Aldrich</td><td>S-6501</td><td></td></tr><tr><td>Tween 80</td><td>Sigma-Aldrich</td><td>P1754</td><td></td></tr><tr><td>Used engine oil</td><td>/</td><td>/</td><td>complex mixture of hydrocarbons, engine additives, and metals, provided by an Italian private company</td></tr><tr><td>Vials 50 mL</td><td>Sigma-Aldrich</td><td>33108-U</td><td></td></tr><tr><td>ZnCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>Z0152</td><td></td></tr></tbody></table>

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.

The selective media methods (Section 1 of the protocol) allowed the rich biodiversity of soil to be screened and the fungi with high bioremediation potential to be selected. With the humic acid and lignocellulose media, more than 100 fungal strains were isolated. These fungi produced enzymes involved in the biodegradation of natural recalcitrant materials, which have a chemical structure resembling many pollutants. However, the fungal strains isolated with the selective media needed further screening. Specifically, the selective tests isolated the strains able to degrade humic acids and lignocellulose, the growth tests screened the isolated fungi for those able to use a specific pollutant as their sole carbon source, and the qualitative enzymatic tests described their metabolic activities.
The growth tests were focused on the fungal ability to degrade hydrocarbons (petrolatum and used engine oil) and plastics (PET, PVC, HDPE, PS, PUR). Each of these substances was used in a test as the sole carbon source for the selected fungal strains. The fungal ability to exploit these substances was evaluated as the difference in colony growth between the samples with the added substance and the control samples with only minimal medium (BH or BHA, depending on the test). A qualitative scale was used to describe the results (Figure 1), ranging from the absence of growth difference with the control (0) to low (+), medium (++), and high (+++) levels of difference in growth.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63445/63445fig01.jpg" /> 
Figure 1: Rate of growth success of fungal strains in the growth tests. The percentage of fungal strains able to grow on petrolatum, used engine oil, and plastic powders (polyethylene terephthalate, PET; polyvinyl chloride, PVC; high-density polyethylene, HDPE; polystyrene, PS; polyurethane, PUR) as their sole carbon source with different degrees of success (no growth, 0; low growth, +; medium growth, ++; high growth, +++). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63445/63445fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The petrolatum and used engine oil tests evaluated the hydrocarbon biodegradation potential of the selected fungal strains. Petrolatum, a mixture of long-chain alkanes (&#62;C25), was used as the sole carbon source. The fungal ability to exploit this substance was evaluated as the difference in colony growth between vials with BH + petrolatum and control vials with only BH (Figure 2). Almost 75% of the tested fungal strains were able to use petrolatum as their sole carbon source, and 21% of the strains exhibited maximum growth (+++, Figure 1).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63445/63445fig02.jpg" /> 
Figure 2: Pictures of the qualitative scale of growth on petrolatum as the sole carbon source. The qualitative scale is based on the growth difference between the treated samples and BH control. It ranges from the absence of growth difference (0), to low (+), medium (++), and high (+++) levels of growth difference. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63445/63445fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Used engine oil is a complex mixture of hydrocarbons, engine additives, and metals. It was used in the growth tests to evaluate the fungal ability to utilize a complex and toxic hydrocarbon blend as a carbon source. Like the other growth tests, growth was evaluated by comparing the fungal growth in plates with the engine oil with growth in the control plates containing just BHA (Figure 3). The growth results confirmed the efficacy of the selective media (Section 1 of protocol). Indeed, almost 90% of the isolated fungal strains were able to grow on used engine oil, with 39% showing maximum growth (+++, Figure 1).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63445/63445fig03.jpg" /> 
Figure 3: Pictures of the qualitative scale of growth on used engine oil as the sole carbon source. The qualitative scale is based on the growth difference between the treated samples and BHA control. It ranges from absence of growth difference (0), to low (+), medium (++), and high (+++) levels of growth difference. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63445/63445fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Growth tests on plastic powders were used to select the fungal strains that could use specific plastic polymers as their primary carbon source; hence, these strains have a high potential for plastic bioremediation. The growth in multiwell plates was observed by stereomicroscope and evaluated using a qualitative scale, ranging from the absence of growth (0) to high production of hyphae (+++). PUR was the plastic powder that had the highest percentage of fungal strains showing growth (92%), with 31% of strains showing maximum growth (+++). Approximately 70% of the strains isolated from humic acid and lignocellulose media grew on PS powder, while almost 60%-65% of them were able to use HDPE, PVC, and PET as their sole carbon source. Therefore, a large percentage of fungi were able to grow on the different plastic powders in multiwell plates, but a very low number of them showed high colonization of plastics. Indeed, only 10% of the strains were able to thrive on PET and HDPE, and 13% were able to thrive on PS. The only plastic where high fungal growth was regularly observed was PUR, on which approximately 30% of fungal strains managed to grow very abundantly.
Qualitative enzymatic tests were performed to detect fungal strains that can produce enzymes involved in the biodegradation processes of recalcitrant substances. Peroxidases and laccases are ligninolytic enzymes whose production is indicated by a brownish halo on the underside of the medium in gallic acid and guaiacol tests (Step 3.1. and Step 3.2. of the protocol). In both tests, the increase in the intensity of the color indicated a higher production of these enzymes (Figure 4).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63445/63445fig04.jpg" /> 
Figure 4: Pictures of the qualitative scale of qualitative ligninolytic enzymes. The qualitative scale is based on the production of a red/brownish halo around the colony. 0 = no activity, + = some activity, ++ = high activity, +++ very high activity. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63445/63445fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Approximately 60% of the fungi tested were able to produce ligninolytic enzymes, indicating the success of the selective media. Moreover, 12% of the isolated fungal strains produced an intense dark halo around the colony (+++) in gallic acid medium, with 17% doing so in guaiacol medium (laccases), indicating high fungal secretion of these enzymes (Figure 5).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63445/63445fig05.jpg" /> 
Figure 5: Rate of enzyme activity by fungal strains during qualitative enzymatic tests. The percentage of fungal strains able to produce ligninolytic enzymes, laccases, proteases, and esterases with different levels of success (0 = no activity, + = activity, ++ = high activity, +++ very high activity) during their corresponding qualitative enzymatic tests. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63445/63445fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Another screening test used to analyze fungal activity was the proteases test, which was based on the difference in fungal growth between YES medium vials and control vials with BH. However, approximately 70% of the fungal strains in the YES medium showed similar growth to controls, or even decreased growth. No fungal strain reached the maximum level of difference (+++). These results suggest that this test may be unsuitable for screening for protease activity.
Finally, esterases activity was evaluated by the formation of a white precipitate in Tween 80 medium (Figure 5). Almost 60% of the selected fungal strains showed esterase activity, and 13% displayed a high formation of the precipitate (+++), suggesting that the strains in the 13% should be selected to scrutinize their biodegradation potential (Figure 5).
Regarding all the growth and enzymatic tests, the most successful strains (i.e., those that obtained +++ in at least one test) were the most interesting. These fungal strains were able to efficiently use recalcitrant substances as their sole carbon source, or they produced a high level of enzymes involved in the biodegradation of recalcitrant substances. Indeed, 39 of the 115 fungal strains tested were able to thrive (+++) on hydrocarbon sources (petrolatum or used engine oil), and 58 had high growth on at least one plastic polymer. Only 32 showed very high enzymatic activity (+++) in at least one of the enzymatic tests performed. These results show the high efficacy of all the reported screening tests, apart from the proteases test, which did not select any high-performing strains.

Watch this Scientific Journal Video about Isolation and Screening from Soil Biodiversity for Fungi Involved in the Degradation of Recalcitrant Materials at JoVE.com

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

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 ...

isolation-screening-from-soil-biodiversity-for-fungi-involved

Universidad San Sebastian

Research

JoVE Journal

Biology

4.6K Views.  University of Pavia. 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. 

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

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 ...

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see1,2), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.

A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic matter, as well as regulation of nutrient levels. There they also find numerous opportunities to interact with a variety of other microbes such as bacteria or other fungi. Studying fungal interactions at the cellular level, however, can be challenging owing to the black box-like nature of soil. New microfluidic tools are being developed for the study of fungal interactions; two platforms designed to study bacterial-fungal and fungal-fungal interactions are highlighted. Within these microchannels, fungal-microbial interactions can be monitored in controlled physico-chemical environments at higher temporal and spatial resolution than previously possible. Application of these tools have yielded numerous novel biological insights, such as the observation of bacterial polar attachment to hyphae or revealing uncharacterised fungal-fungal antagonisms. A key feature of these methodologies regards the ease of use of this tool by non-experts, yielding highly translatable technologies for use in microbiology labs.

Owing to the opacity of soil, interactions between its constituent microbes cannot easily be visualised with cellular resolution. Here, two microfluidic tools are presented, which offer new opportunities for investigating fungal-microbial interactions. The devices are versatile and simple to use, enabling high spatiotemporal control and high-resolution imaging at the cellular level.

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic ...

Bioengineering

Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture

Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to degrade plastics. Typically, when formulations of plastics are known and a source of the feedstock is available, powdered plastic can be suspended in agar-based media and degradation determined by visualization of clearing zones. However, this approach poorly mimics in situ degradation of BDMs. First, BDMs are not dispersed as small particles throughout the soil matrix. Secondly, BDMs are not sold commercially as pure polymers, but rather as films containing additives (e.g. fillers, plasticizers and dyes) that may affect microbial growth. The procedures described herein were used for isolates acquired from soil-buried mulch films. Fungal isolates acquired from excavated BDMs were tested individually for growth on pieces of new, disinfested BDMs laid atop defined medium containing no carbon source except agar. Isolates that grew on BDMs were further tested in liquid medium where BDMs were the sole added carbon source. After approximately ten weeks, fungal colonization and BDM degradation were assessed by scanning electron microscopy. Isolates were identified via analysis of ribosomal RNA gene sequences. This report describes methods for fungal isolation, but bacteria also were isolated using these methods by substituting media appropriate for bacteria. Our methodology should prove useful for studies investigating breakdown of intact plastic films or products for which plastic feedstocks are either unknown or not available. However our approach does not provide a quantitative method for comparing rates of BDM degradation.

Plastic films labeled "biodegradable" are commercially available for agricultural use as mulches. Tillage represents an attractive disposal method, but degradation under field conditions is poorly understood. The purpose of this study was to develop methods for isolating native soil fungi and bacteria that colonize plastic mulch films after field burial.

Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to ...

Assays for the Degradation of Misfolded Proteins in Cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...

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 ...

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.

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 ...

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 ...

Filamentous Fungi

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona 
Demonstrating Author: Bradley Schmitz
Fungi are heterotrophic eukaryotic organisms, and with the exception of yeasts, are aerobic. They are abundant in surface soils and are important for their role in nutrient cycling and the decomposition of organic matter and organic contaminants. White rot fungi (phanerochaete chryosporium) for example, (Figure 1) are known to degrade aromatics.

<img alt="Figure 1" src="/files/ftp_upload/10030/10030fig1.jpg" /> 
Figure 1. White rot on birch.

Culture, Enumeration and Identification of Filamentous Fungi from Soil

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona
Demonstrating Author: Bradley Schmitz
Fungi are heterotrophic ...

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

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

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

Filamentous Fungi