Name: isolation, characterization, and total dna extraction to identify endophytic fungi in mycoheterotrophic plants
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Description: Watch this Scientific Journal Video about Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants at JoVE.com

We thank funding from FAPESP (2015/26479-6) and CNPq (447453/2014-9). JLSM thanks CNPq for productivity grants (303664/2020-7). MPP thanks Capes (master&#39;s degree scholarship, process 88887.600591/2021-00) and CNPq.

1. Plant sample collection
<ol>
	<li>Sample collection for culture-dependent methods
	<ol>
		<li>Carefully dig the subterranean organs; these can be roots, stems, rhizomes, or storage organs of the plant to be collected. Apart from highly compact soils, collect these samples by hand. 
		NOTE: The use of tools such as trowels or scoops in this step is ill-advised, as it can damage the fragile structures of mycoheterotrophic plants and may cause tissue contamination by non-endophytic fungi.</li>
		<...

<ol>
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L., Kasuya, M. C. M., Borges, A. C., Ara&#250;jo, E. F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+and+molecular+characterization+of+mycorrhizal+fungi+isolated+from+neotropical+orchids+in+Brazil.">Morphological and molecular characterization of mycorrhizal fungi isolated from neotropical orchids in Brazil.</a> Canadian Journal of Botany. 83 (1), 54-65 (2005).</li><li>Riddell, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permanent+stained+mycological+preparations+obtained+by+slide+culture.">Permanent stained mycological preparations obtained by slide culture.</a> Mycologia. 42 (2), 265-270 (1950).</li><li>Walsh, T. J., Hayden, R. T., Larone, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Larone's Medically Important Fungi: A Guide to Identification. , (2018).</li><li>Microscopy: Chemical Reagents. British Mycological Society Available from: <a target="_blank" href="https://www.britmycolsoc.org.uk/field_mycology/microscopy/reagents">https://www.britmycolsoc.org.uk/field_mycology/microscopy/reagents</a> (2022)</li><li>Senanayake, I. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+approaches+in+studying+fungi:+Collection,+examination,+isolation,+sporulation+and+preservation.">Morphological approaches in studying fungi: Collection, examination, isolation, sporulation and preservation.</a> Mycosphere. 11 (1), 2678-2754 (2020).</li><li>Slifkin, M., Cumbie, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Congo+red+as+a+fluorochrome+for+the+rapid+detection+of+fungi.">Congo red as a fluorochrome for the rapid detection of fungi.</a> Journal of Clinical Microbiology. 26 (5), 827-830 (1988).</li><li>Raeder, U., Broda, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+preparation+of+DNA+from+filamentous+fungi.">Rapid preparation of DNA from filamentous fungi.</a> Letters in Applied Microbiology. 1 (1), 17-20 (1985).</li><li>Martins, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+endophytic+microorganisms.">Molecular characterization of endophytic microorganisms.</a> Endophytic microorganisms: theoretical and practical aspects of isolation and characterization. 1st edition. , 189-211 (2014).</li><li>Rayner, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Mycological+Colour+Chart.">A Mycological Colour Chart.</a> Commonwealth Mycological Institute. , (1970).</li><li>Kornerup, A., Wanscher, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methuen Handbook of Colour. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toluidine+blue:+A+review+of+its+chemistry+and+clinical+utility.">Toluidine blue: A review of its chemistry and clinical utility.</a> Journal of Oral and Maxillofacial Pathology. 16 (2), 251-255 (2012).</li><li>Smith, D., Onions, A. H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+some+preservation+techniques+for+fungi.">A comparison of some preservation techniques for fungi.</a> Transactions of the British Mycological Society. 81 (3), 535-540 (1983).</li><li>Ryan, M. J., Smith, D., Jeffries, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+decision-based+key+to+determine+the+most+appropriate+protocol+for+the+preservation+of+fungi.">A decision-based key to determine the most appropriate protocol for the preservation of fungi.</a> World Journal of Microbiology and Biotechnology. 16 (2), 183-186 (2000).</li><li>Lalaymia, I., Cranenbrouck, S., Declerck, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+and+preservation+of+ectomycorrhizal+and+arbuscular+mycorrhizal+fungi.">Maintenance and preservation of ectomycorrhizal and arbuscular mycorrhizal fungi.</a> Mycorrhiza. 24 (5), 323-337 (2014).</li><li>Zettler, L. W., Corey, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orchid+mycorrhizal+fungi:+isolation+and+identification+techniques.">Orchid mycorrhizal fungi: isolation and identification techniques.</a> Orchid Propagation: From Laboratories to Greenhouses-Methods and Protocols. , 27-59 (2018).</li><li>Yu, S., Wang, Y., Li, X., Yu, F., Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+factors+affecting+the+reproducibility+of+micro-volume+DNA+mass+quantification+in+Nanodrop+2000+spectrophotometer.">The factors affecting the reproducibility of micro-volume DNA mass quantification in Nanodrop 2000 spectrophotometer.</a> Optik. 145, 555-560 (2017).</li><li>Martos, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independent+recruitment+of+saprotrophic+fungi+as+mycorrhizal+partners+by+tropical+achlorophyllous+orchids.">Independent recruitment of saprotrophic fungi as mycorrhizal partners by tropical achlorophyllous orchids.</a> New Phytologist. 184 (3), 668-681 (2009).</li><li>Schoch, C. 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The superficial disinfestation of plant samples is one of the most critical stages in the presented protocol. No contamination in the PDA dishes with drops from the last wash are highly desirable. Bacteria are frequently observed as contaminants in the isolation dishes, usually more than airborne sporulating fungi, considering endophytic bacteria are also common within plant tissues3,11. Thus, the addition of antibiotics in the culture medium when installing the ...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65135">Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants</a>. The Authors section was updated from:
Juliana Lishcka Sampaio Mayer
to:
Juliana Lischka Sampaio Mayer

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65135">Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants</a>. The Authors section was updated.

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

Endophytic fungi are, by definition, those that inhabit the interior of plant organs and tissues in inconspicuous infections (i.e., without causing harm to their host)1,2. These fungi can neutrally or beneficially interact with host plants, may confer resistance to pathogens and unfavorable environmental conditions, and may contribute to the synthesis of beneficial compounds for the plant (e.g., growth factors and other phytohormones)1,3. Mycorrhizal endophytes are fungi that establish mycorrhizal associations with the plant, taking part in nutrient transfer4. In Orchidaceae, the interaction with mycorrhizal endophytes is fundamental for seed germination in the vast majority of species, and seedling establishment in all the plants in the family5. In such contexts, mycoheterotrophic orchids represent a case of total dependence regarding their mycorrhizal partners, as they depend on mineral nutrients and carbon compounds transference by these fungi during their whole life cycle6. Therefore, the isolation and identification of associating fungi is a fundamental base when investigating mycoheterotrophic life strategies. Moreover, little is known about the roles of fungal endophytes in mycoheterotrophic plants or even the real diversity of these fungi7,8.
The investigation of endophytic fungi may be conducted via different techniques, traditionally described as culture-independent or -dependent, for instance: (a) direct observation, (b) fungal isolation and morphological and/or molecular identification, and (c) total DNA extraction of plant tissues and molecular identification9. In direct observation (a), endophytic fungi may be investigated while still in the interior of plant cells and tissues by light or electron microscopy9, as different microscopy protocols are detailed by Pena-Passos et al.10. By isolation methods (b), fungal endophytes can be characterized according to their colonies, hyphae, and reproductive or resistance structure morphology. Also, via isolation techniques, it is possible to conduct the molecular identification of isolates through DNA extraction, amplification of molecular identification sequences (barcodes or fingerprints), and sequencing11. The latter technique (c) enables the molecular identification of endophytic fungi per DNA extraction while in the interior of plant tissues (metabarcoding), followed by library preparation and sequencing12.
Moreover, fungal isolates may be applied in symbiotic germination trials, using seeds from autotrophic or mycoheterotrophic orchids. An example of such an application is the investigation conducted by Sisti et al.13, describing the germination and initial stages of protocorm development in Pogoniopsis schenckii, a mycoheterotrophic orchid, in association with some of its isolates, comprising non-mycorrhizal endophytic fungi. The applied symbiotic germination protocol is detailed and presented in a video by Pena-Passos et al.10. Isolating fungi in association with different plant organs allows diverse investigation focuses regarding the nature of plant-fungal interactions (e.g., to comprehend either ecological or physiological aspects of the association, as well as inquiries into the nutrient transference from fungi to the plant)9.
The methodologies presented in section 1 are based on a collection of subterranean organ samples, as these organs present the most difficulties in collection, and they are of major interest since mycorrhizal endophytes colonize them. However, both included protocols (steps 1.1 and 1.2) may be applied to other mycoheterotrophic plant organs (e.g., rhizomes, floral stems, and fruits). The collection methodology described in step 1.1 is designated for isolating endophytic fungi (section 2) for morphological characterization (sections 4 and 5) and/or total DNA extraction for isolate identification (section 6). On the other hand, the collection methodology described in step 1.2 is exclusively assigned to total DNA extraction of plant tissues for metabarcoding techniques (section 7). In section 3, four methods for filamentous fungi storage and preservation are presented, two for short-term storage (3-6 months) and the other two adequate for long-term storage (&#62;1 year). The morphological characterization (sections 4 and 5) may be associated with molecular identification to reinforce it and provide important information on fungal macro- and micromorphology. Figure 1 summarizes the collective methodologies described thereafter.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65135/65135fig01.jpg" /> 
Figure 1: Schematic summarization of the presented methods. Plant collection and fungal isolation, preservation, and molecular identification by culture-dependent and -independent methodologies. <a href="https://www.jove.com/files/ftp_upload/65135/65135fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adhesive tape</td>
			<td></td>
			<td></td>
			<td>(from any company, for adhesive tape mount in micromorphological analyses)</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Sigma-Aldrich</td>
			<td>A5354</td>
			<td>(for installation of plant fragments; other antibiotics may be used - check step 2.2.1)</td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td></td>
			<td></td>
			<td>(from any company, for materials sterilization in many steps)</td>
		</tr>
		<tr>
			<td>Bacteriological agar</td>
			<td>Sigma-Aldrich</td>
			<td>A1296</td>
			<td>(for many steps)</td>
		</tr>
		<tr>
			<td>C1, C2, C3, C4, C5, and C6 solutions</td>
			<td>Qiagen</td>
			<td>12888-50</td>
			<td>(purchased with DNeasy PowerSoil kit)</td>
		</tr>
		<tr>
			<td>Centrifuge&#160;&#160;</td>
			<td>Merck/Eppendorf</td>
			<td>5810 G</td>
			<td>(for total DNA extraction from fungal isolates)</td>
		</tr>
		<tr>
			<td>Centrifuge tubes</td>
			<td>Merck</td>
			<td>CLS430828</td>
			<td>(for samples collection)</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>C2432</td>
			<td>(for total DNA extraction from fungal isolates)</td>
		</tr>
		<tr>
			<td>Congo red</td>
			<td>Supelco</td>
			<td>75768</td>
			<td>(for hyphae staining)</td>
		</tr>
		<tr>
			<td>Cryotubes</td>
			<td>Merck</td>
			<td>BR114831</td>
			<td>(for many steps)</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Supelco</td>
			<td>100983</td>
			<td>It will be necessary to carry out the appropriate dilutions (for many steps)</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>3609</td>
			<td>(for total DNA extraction from fungal isolates)</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Merck</td>
			<td>WHA10010155</td>
			<td>(for many steps)</td>
		</tr>
		<tr>
			<td>Glass test tubes</td>
			<td>Merck</td>
			<td>CLS7082516</td>
			<td>(for cryopreservation in unhulled rice grains)</td>
		</tr>
		<tr>
			<td>Glass wool</td>
			<td>Supelco</td>
			<td>20411</td>
			<td>(for cryopreservation in unhulled rice grains)</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td>Or dextrose (for cryopreservation in vermiculite)</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td>Or glycerin (for cryopreservation in vermiculite, for preparing LPCB)</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma-Aldrich</td>
			<td>563935</td>
			<td>(for total DNA extraction from fungal isolates)</td>
		</tr>
		<tr>
			<td>Lactic acid</td>
			<td>Sigma-Aldrich</td>
			<td>252476</td>
			<td>(for preparing LPCB - hyphae staining)</td>
		</tr>
		<tr>
			<td>Lactophenol blue solution (LPCB)</td>
			<td>Sigma-Aldrich</td>
			<td>61335</td>
			<td>(for hyphae staining)</td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td></td>
			<td></td>
			<td>(class I, from any company, for many steps)</td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td></td>
			<td></td>
			<td>(from any company, for hyphae observation)</td>
		</tr>
		<tr>
			<td>MB Spin Columns</td>
			<td>Qiagen</td>
			<td>12888-50</td>
			<td>(purchased with DNeasy PowerSoil kit)</td>
		</tr>
		<tr>
			<td>Methyl blue (cotton blue)</td>
			<td>Sigma-Aldrich</td>
			<td>M5528</td>
			<td>(for preparing LPCB - hyphae staining)</td>
		</tr>
		<tr>
			<td>Microcentrifuge tube (1.5 mL)</td>
			<td>Merck</td>
			<td>HS4323</td>
			<td>(for total DNA extraction from fungal isolates)</td>
		</tr>
		<tr>
			<td>Microcentrifuge tube (2 mL)</td>
			<td>Merck</td>
			<td>BR780546</td>
			<td>(for many steps)</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td></td>
			<td></td>
			<td>(for preservation of fungal isolates)</td>
		</tr>
		<tr>
			<td>Paper bags</td>
			<td></td>
			<td></td>
			<td>Average size 150 mm x 200 mm (for samples collection)</td>
		</tr>
		<tr>
			<td>Petri dish (Glass, 120 mm x 20 mm)</td>
			<td>Merck/Pyrex</td>
			<td>SLW1480/10D</td>
			<td>(autoclavable, for fungi slide culture, prefer higher ones)</td>
		</tr>
		<tr>
			<td>Petri dish (Glass, 50 mm x 17 mm)</td>
			<td>Merck/Aldrich</td>
			<td>Z740618</td>
			<td>(for purification of fungal isolates); alternatively: polystyrene petri dishes (sterile, &#947;-irradiated, non-autoclavable)</td>
		</tr>
		<tr>
			<td>Petri dish (Glass, 80 mm x 15 mm)</td>
			<td>Merck/Brand</td>
			<td>BR455732</td>
			<td>(for installation of plant fragments); alternatively: polystyrene petri dishes (sterile, &#947;-irradiated, non-autoclavable)</td>
		</tr>
		<tr>
			<td>Phenol</td>
			<td>Sigma-Aldrich</td>
			<td>P1037</td>
			<td>(for total DNA extraction from fungal isolates, for preparing LPCB)</td>
		</tr>
		<tr>
			<td>Porcelain mortar</td>
			<td>Sigma-Aldrich</td>
			<td>Z247464</td>
			<td>(for total DNA extraction from fungal isolates)</td>
		</tr>
		<tr>
			<td>Porcelain pestle</td>
			<td>Sigma-Aldrich</td>
			<td>Z247502</td>
			<td>(for total DNA extraction from fungal isolates)</td>
		</tr>
		<tr>
			<td>Potato dextrose agar (PDA)</td>
			<td>Millipore</td>
			<td>P2182</td>
			<td>(for many steps)</td>
		</tr>
		<tr>
			<td>PowerBead tubes</td>
			<td>Qiagen</td>
			<td>12888-50</td>
			<td>(purchased with DNeasy PowerSoil kit)</td>
		</tr>
		<tr>
			<td>Rapid mounting medium (Entellan)</td>
			<td>Sigma-Aldrich</td>
			<td>1.0796</td>
			<td>(for fungi slide culture)</td>
		</tr>
		<tr>
			<td>Silica gel</td>
			<td>Supelco</td>
			<td>717185</td>
			<td>(for cryopreservation in unhulled rice grains)</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td>(for total DNA extraction from fungal isolates)</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma-Aldrich</td>
			<td>L3771</td>
			<td>Lauryl sulfate sodium salt (for total DNA extraction from fungal isolates)</td>
		</tr>
		<tr>
			<td>Sodium hypochlorite (w/ 2% active chlorine)</td>
			<td></td>
			<td></td>
			<td>(commercial product, for superficial desinfestation)</td>
		</tr>
		<tr>
			<td>Soil DNA extraction kit (DNeasy PowerSoil kit)</td>
			<td>Qiagen</td>
			<td>12888-50</td>
			<td>(for total DNA extraction from plant organs)</td>
		</tr>
		<tr>
			<td>Spectrophotometer - Nanodrop 2000/2000c</td>
			<td>ThermoFisher Scientific</td>
			<td>ND2000CLAPTOP</td>
			<td>(for total DNA extraction from plant organs)</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td></td>
			<td></td>
			<td>(=dissecting microscope, from any company, for macromorphological analyses)</td>
		</tr>
		<tr>
			<td>Tetracycline</td>
			<td>Sigma-Aldrich</td>
			<td>T7660</td>
			<td>(for installation of plant fragments)</td>
		</tr>
		<tr>
			<td>Thermoblock</td>
			<td>Merck/Eppendorf</td>
			<td>EP5362000035</td>
			<td>(or from other companies)</td>
		</tr>
		<tr>
			<td>Tissue homogenizer and cell lyzer</td>
			<td>SPEX SamplePrep</td>
			<td></td>
			<td>2010 Geno/Grinder - Automated Tissue Homogenizer and Cell Lyzer (for total DNA extraction from plant organs)</td>
		</tr>
		<tr>
			<td>Toluidine blue O</td>
			<td>Sigma-Aldrich/Harleco</td>
			<td>364-M</td>
			<td>(for hyphae staining)</td>
		</tr>
		<tr>
			<td>Trehalose</td>
			<td>Sigma-Aldrich</td>
			<td>T9531</td>
			<td>(for cryopreservation in vermiculite)</td>
		</tr>
		<tr>
			<td>Tris Base Solution (Tris)</td>
			<td>Sigma-Aldrich</td>
			<td>T1699</td>
			<td>(for total DNA extraction from fungal isolates)</td>
		</tr>
		<tr>
			<td>Unhulled rice grains</td>
			<td></td>
			<td></td>
			<td>(for cryopreservation)</td>
		</tr>
		<tr>
			<td>U-shaped glass rod</td>
			<td></td>
			<td></td>
			<td>(or an adaptation - check step 5.4.1, for fungi slide culture)</td>
		</tr>
		<tr>
			<td>Vermiculite</td>
			<td></td>
			<td></td>
			<td>Fine granulometry (for cryopreservation in vermiculite)</td>
		</tr>
		<tr>
			<td>Vortexer</td>
			<td>Sigma-Aldrich/BenchMixer</td>
			<td>BMSBV1000</td>
			<td>(for total DNA extraction from fungal isolates)</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Sigma-Aldrich</td>
			<td>Y1625</td>
			<td>(for cryopreservation in vermiculite)</td>
		</tr>
	</tbody>
</table>

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.

In the isolation protocol, considering there is contamination from the water used on the last wash and the contamination is also detected in the Petri dishes with inoculated fragments, different actions may be taken, depending on the type of contaminant (Table 1). This procedure must be repeated from the beginning in case of highly sporulating fungal contaminants, which also present accelerated growth, and intense-multiplying bacteria, resistant to the chosen antibiotics....

Watch this Scientific Journal Video about Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants at JoVE.com

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

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 ...

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