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 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.
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 (>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.
Figure 1: Schematic summarization of the presented methods. Plant collection and fungal isolation, preservation, and molecular identification by culture-dependent and -independent methodologies. Please click here to view a larger version of this figure.
1. Plant sample collection
2. Isolation of endophytic fungi associated with plant organs14
NOTE: Every material, solution, and reagent used in this section must be sterile. Ones that cannot be purchased already sterilized should be autoclaved at 121 °C for 20 min.
3. Preservation of purified fungal isolates
NOTE: Every material, solution, and reagent used in this section must be sterile. Ones that cannot be purchased already sterilized should be autoclaved at 121 °C for 20 min.
4. Macromorphological characterization of filamentous fungi (colony morphology)
5. Micromorphological characterization of filamentous fungi (hyphal morphology)
NOTE: The micromorphological techniques are compared in the discussion section, considering their possible uses and disadvantages.
Figure 2: Procedures for slide culture of filamentous fungi. (A) Schematic configuration of a slide culture kit, where the numbers indicate the order of arranging the elements. (B) Detaching the square of the culture medium after hyphal growth is observed in the glass slide and the coverslip. Please click here to view a larger version of this figure.
6. Total DNA extraction from fungal isolates (homemade protocol26 with modifications 27)
NOTE: Every material, solution, and reagent used in this section must be sterile. Ones that cannot be purchased already sterilized should be autoclaved at 121 °C for 20 min. Wear gloves during the whole protocol and perform some stages inside a fume hood.
7. Total DNA extraction from plant organs for metabarcoding methodology (commercial kit)
NOTE: For the following methodology, it is necessary to purchase the commercial kit indicated in the Table of Materials as a soil DNA extraction kit. Every material, solution, and reagent used in this section must be sterile. Ones that cannot be purchased already sterilized should be autoclaved at 121 °C for 20 min. It is highly recommended to wear gloves during the whole protocol, and the steps can be conducted inside a laminar flow hood. The described protocol is modified from De Souza et al.12, from the protocol detailed by the manufacturer.
8. DNA quantification in a spectrophotometer (check the Table of Materials)
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. Instead, if the fungal contaminant presents slow growth or does not sporulate, it is possible to simply avoid isolating it in the subsequent steps of the protocol. Finally, contaminations with bacterial strains that multiply slowly may also be easily avoided in the posterior steps, especially in purification, when recuperating hyphae.
In all the cases previously presented, the ideal scenario is redoing the disinfestation procedure using other samples and installing the plant fragments in a culture medium that combines other antibiotics, with a broader spectrum of action, in cases of resistant bacteria contaminating the first dishes. However, considering the difficulties involved in collecting new samples and obtaining organs from mycoheterotrophic plants, some cases may be successfully solved in the posterior stages of the protocol, as presented in Table 1.
Contaminant | Contaminant characteristic | Possible consequence of contamination | Required Action | |||
Bacteria | Intense multiplication | Total or partial inhibition of the growth of the endophytes of interest | Restart isolation from the beginning | |||
Slow multiplication | Isolation of the bacteria along with the endophyte of interest | Avoid bacteria colonies during purification | ||||
Fungi | Vigorous growth and/or highly sporulating | Makes it impossible to isolate and/or purify the endophytes of interest | Restart isolation from the beginning | |||
Slow growth and/or non-sporulating | Isolation of the contaminant by mistake | Avoid isolation of the contaminant |
Table 1: Description of the possible contaminants in the process of endophytic fungi isolation, contamination consequences, and mitigation procedures.
Following 5 days from the installation of organ fragments in the PDA medium, it is possible to visualize the growth of small groups of filamentous fungi mycelia, emerging from the interior of the fragments (Figure 3). Each mycelial morphotype should result in a fungal isolate by the end of the isolation protocol, considering that each one of them must be striated in a new Petri dish with AA medium for purification. It is advised that the original dishes from the fragment installation are not discarded before completing the whole isolation protocol. They may be kept refrigerated at 4 °C until the purified colonies are obtained. Also, regarding this part of the protocol, the IF analysis can be greatly helpful in evaluating the percentage of tissue samples that result in isolated fungi among the total installed samples.
Figure 3: Root fragments for fungal isolation in potato dextrose agar. Culturable endophytic fungi growing from the root fragments of a mycoheterotrophic orchid after ca. 5 days of incubation. Each Petri dish (A, B, and C) contains five root fragments. Please click here to view a larger version of this figure.
After 3 days following incubation of the AA dishes for isolate purification, minute hyphal colonies, almost imperceptible, should have grown from the striae in the AA medium. At this stage, in case there was any contamination from slow-growing bacteria in the isolation dishes, it is possible that these bacteria were also transferred to the AA dishes with the selected fungal endophyte hyphae. If so, bacteria will be restricted to stria regions, with little growth when compared to the fungus, allowing the recuperation of solely the fungus of interest to new PDA dishes.
During 7-14 days of inoculation of the purified fungal isolates in PDA dishes, the pure colony must grow centrifugally (from the center of the dish to its periphery), forming a sole circular mycelium (Figure 4). Possible contaminations are easily identified in this stage, as they considerably compromise the colony homogeneity concerning its growth, form, aspect, color, pigment production in the medium, etc. (Figure 5). Assuming the final colonies obtained are not pure, the fungi initially grown from the organ fragments must be submitted again to purification procedures, by striation and subculturing (step 2.4). It is possible to recuperate the isolates from either the initially installed organs or the final isolation dishes containing heterogenous cultures.
Figure 4: Representation of purified fungal isolates, grown for 7-14 days in a PDA culture medium. In the first and third columns (A, C, E, G, I, and K), the registered colonies are seen from the upper side; the second and fourth columns (B, D, F, H, J, and L) present the same colonies, respectively seen from the underside. Please click here to view a larger version of this figure.
Figure 5: Representation of unpure fungal isolates, grown for 7-14 days in PDA culture medium. In the first column (A, D, and G), a general view of the colonies from the upper side is seen; the second column (B, E, and F) shows the colonies in detail; the third column (C, F, and I) shows the colonies from the underside. The numbers represent different fungal morphotypes present in each dish, and the lines represent a subtle delimitation between the different fungal isolates. Please click here to view a larger version of this figure.
The preservation of isolates should not be made using one sole storage method, as each fungus may present more or less sensibility to each method described. It is highly advisable to choose at least two types of storage for each fungal isolate, guaranteeing more chances of success in preserving it. We highlight that the unviability of fungi preserved in Castellani or mineral oil is expected after 6 months or less. Researchers should consider if these are the only preservation methods chosen, to avoid possible unexpected losses of isolates. To extend this limited period, it is possible to reactivate the fungal isolates by growing them in the PDA medium (39 g/L) for 2-3 weeks and then storing them again. After a period of storage, it is expected that the isolates present a slower growth, compared to the observed growth rate before its preservation, and colonies with less vigorous aspects (i.e., less dense, different in color). Subculturing a few times in a nutrient-rich culture medium is enough to reestablish such characteristics.
When evaluating the macromorphology of the colonies obtained in the isolation procedure, qualitative data should be collected, considering as many characteristics as possible: (a) color of the colony (top and underside), which can be analyzed using printed color guides (e.g., Rayner28, Kornerup & Wanscher29, Ridgway30); (b) colony opacity: transparent, opaque, translucent; (c) diffusible pigments in the medium31 (presence/absence and color); (d) exudates31 (presence/absence, color, general appearance); (e) macroscopic structures31 (presence/absence, type, and appearance; e.g., sclerotia, pycnidia); (f) aerial and submerged mycelia19,32,21 (presence/absence, appearance-scant or abundant); (g) margin appearance19 (color, form, uniformity – submerged or aerial); and (h) colony topography (heaped, wrinkled, crateriform, flat, etc.), the general appearance and texture – cottony, velvety, powdery, woolly, sebaceous (waxy), glabrous, chalky, slimy, leathery, prickly, etc.19,21.
Figure 6: Macro- and micromorphology of an unidentified fungus isolated from the mycoheterotrophic orchid Wullschlaegelia aphylla. (A) Upper and (B) lower aspects of the colony, (C) amplified view of the aerial hyphae (as seen in a stereomicroscope). Micromorphology of the hyphae (D) without a stain and stained with (E) LPCB and (F) TBO. Abbreviations: c = conidiophore, p = phialide, s = septum. Scale bars: A,B = 2 cm; C = 2 mm; D–F = 20 µm. Please click here to view a larger version of this figure.
In Figure 6, a colony isolated from the fusiform roots of the mycoheterotrophic orchid Wullschlaegelia aphylla, applying the described methodology, is shown. The colony is whitish to grey on the upper side (Figure 6A) and brownish on the lower face (Figure 6B). It is opaque, with neither diffusible pigments in the medium nor exudates. The mycelia are aerial and abundant, the margins are irregular and aerial, and the colony has a velvety texture and a wrinkled topography (Figure 6A). Macroscopic structures are absent.
The slide culture method is traditional and advantageous, and although time-consuming, may be applied to produce permanent slides that can be examined afterward. The different dyes presented here may be used to evince important structures, and they require tests in the samples, to determine the most adequate incubation time. Congo red and TBO are general-purpose stains. Congo red is adequate to demonstrate some delicate structures (further reading: Malloch22). TBO stains the cell content more intensely than the fungal cell wall (Figure 6F). Although being a usual dye for plant structures and not so common in fungal microscopy, TBO has an important metachromatic property33, which favors other applications, such as fungal analysis in plant tissues10. LPCB is a vastly applied dye in fungal analysis, although demanding caution because of phenol. Cotton blue (synonym: methyl blue) is the stain, lactic acid is a clearing agent, and phenol is a killing agent21. LPCB has an affinity for chitin and evinces spore walls and ornamentations23,24. The fungal septa are stained neither by LPCB (Figure 6E) nor TBO (Figure 6F), facilitating the identification of such structures. The application of stains is advantageous to evince structures that are not easily seen when hyphae are not stained (Figure 6D).
For DNA extraction from fungi and root samples, the amount of ground sample in liquid nitrogen to be added to the extraction buffer must be respected, not exceeding the indicated amount in the protocol. We should highlight that a large amount of processed sample does not simply represent the obtention of a high concentration of DNA without major drawbacks, as it can considerably compromise the final DNA quality. This occurs when saturating the following stages of DNA purification. Besides, concentrating putative compounds produced by plants or fungi (e.g., pigments, secondary metabolites), present in the final solution with DNA, may act to reduce DNA quality and/or inhibit polymerase chain reaction (PCR). Another issue that can significantly reduce DNA quality is scrapping the culture medium altogether with fungal mycelium and grinding it, which must be strongly avoided. Some reagents from the extraction procedure (e.g., phenol, ethyl alcohol, SDS) and other substances (e.g., fungal pigments) may be inhibitors interfering with PCR.
In the DNA cleansing stages, when using phenol and chloroform, the supernatant must always represent the phase with fewer impurities, normally comprehending the clearest phase. During such a stage, the inferior phase must not be recuperated when aspiring the supernatant that will be transferred to new tubes. By the end of the protocol, the pellet (constituted by DNA) should present a translucid (highly desirable) to whitish color. In the drying stage, proceeding with drying the DNA in a thermoblock is fundamental to completely evaporating ethanol. After 12 h at 4 °C, the pellet should have eluted completely in the aqueous solution added to the tubes, not being visible after that. In case the elution is not complete, and the DNA concentration is far under the ideal concentration, it is possible to keep the solution refrigerated for 12 h more, without significant losses in its quality.
The DNA analyses in the software generate a table, as represented in Table 2, where data related to DNA quantity and quality are indicated. The DNA quantification is given by the concentration of nucleic acids in nanograms per microliter of a sample. Considering the molecular identification of isolated fungi, DNA samples should have approximately 200 ng/µL in concentration, ideal for PCR stages. In cases of samples with higher concentrations of nucleic acids, an aliquot from the sample should be diluted, approximating the concentration to the value previously mentioned. Samples with excessively high concentrations, such as samples 1 and 2 in Table 2, may suggest a false detection due to contaminants in the solution. Meanwhile, samples with lower nucleic acid concentrations, such as number 7 (Table 2), may still be used for PCR, being important to apply a higher volume of sample in the reactions. Regarding the quality parameters, it is satisfactory that the quotients 260/280 and 260/230 are between 1.8 and 2.2, as indicated in samples 3, 5, and 8 (Table 2). Samples that present such values (being much below or above) should be submitted to new DNA extractions, as indicated in samples 9 and 10 from Table 2. In cases of samples with an adequate quantity of DNA and minimally exceeding the ideal quality range, as suggested by the 260/280 and 260/230 values in numbers 4 and 6 (Table 2), diluting is the most beneficial procedure to make the nucleic acid concentration adequate for PCR and dilute possible inhibitors, such as vestigial reagents from the extraction procedure, in the samples.
Sample | Nucleic Acid Conc. | Unit | A260 | A280 | 260/280 | 260/230 | Sample Type |
1 | 14491.7 | ng/µL | 289.833 | 141.175 | 2.05 | 1.8 | DNA |
2 | 13359.3 | ng/µL | 267.187 | 124.607 | 2.14 | 2.15 | DNA |
3 | 1137.6 | ng/µL | 22.751 | 10.574 | 2.15 | 2.13 | DNA |
4 | 1472.6 | ng/µL | 29.452 | 13.287 | 2.22 | 2.16 | DNA |
5 | 3464.8 | ng/µL | 69.295 | 33.329 | 2.08 | 1.88 | DNA |
6 | 1884.2 | ng/µL | 37.684 | 17.912 | 2.1 | 1.78 | DNA |
7 | 187.6 | ng/µL | 3.751 | 1.834 | 2.05 | 2.06 | DNA |
8 | 1580.3 | ng/µL | 31.607 | 15.281 | 2.07 | 1.98 | DNA |
9 | 923.3 | ng/µL | 18.466 | 9.196 | 2.01 | 1.37 | DNA |
10 | 2414.4 | ng/µL | 48.287 | 21.008 | 2.3 | 3.45 | DNA |
Table 2: Generated results in the software from fungal DNA samples analyzed in a spectrophotometer.
Different from DNA samples from isolated endophytic fungi, the DNA concentration in samples for metabarcoding analyses should present massive amounts of extracted DNA molecules, with high quality and molecular weight. The samples should be evaluated by electrophoresis in agarose gel, obtaining the maximum integrity possible, with little or no smearing in the gel.
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 organ fragments is essential. Better results are achieved when combining different antibiotic types, resulting in a broader spectrum of action. Another important consideration is the intrinsic bias in isolating the fungi, as using PDA and AA will inevitably select certain species and disfavor others. The combination of other culture media may minimize the bias, although not eliminate such limitations9.
Generally, fungi that produce spores while growing in Petri dishes present higher survival rates in preservation protocols, either in lower temperatures or not, and non-sporulating isolates may not tolerate Castellani's or mineral oil preservation34,35,36, so a cryopreservation method may be crucial in such cases. Additionally, some isolates may be sensitive to freezing, and the addition of cryoprotectants aids the preservation, as in the presented method using vermiculite18. The colony and hyphal characteristics may be useful to group the isolates with similar traits, facilitating the selection for uses in symbiotic germination treatments (as detailed by Pena-Passos et al.10). These characteristics also contribute to choosing the preservation methods to be applied, especially considering sporulation or its absence.
Morphologically characterizing the isolates also improves and complements morphological descriptions available in specialized literature, especially when associated with molecular characterization. Despite being a challenge with numerous isolates, morphologically characterizing fungi is time-consuming and depends on published data. Even if only the molecular characterization is planned to be conducted, it is advisable to maintain a photographic register of the colony appearance (macromorphology) and publish it whenever possible, to contribute to future identification procedures. Maintaining photos of the dishes where fragments are installed is also important, to allow comparison between the final obtained isolates and the morphotypes observed growing in installation dishes. In such a case, the compared fungi must be cultured in the same type of medium. Additional methodologies are found in Currah et al.16 to evaluate common orchid mycorrhizal fungi, such as culture on tannic acid medium (TAM) to differentiate Epulorhiza (teleomorph: Tulasnella) and Ceratorhiza (teleomorph: Ceratobasidium) genera, and culture on CMA to stimulate monilioid cells for the characterization of fungi from the Rhizoctonia complex.
Considering the detailed methods to observe fungal hyphae under a light microscope, tease mount and adhesive tape mount are faster than slide culture, although the tease mount method is not adequate for analyzing spores and measuring branch angles. Adhesive tape mount preserves the mycelium organization better than the tease mount technique, although using adhesive tape is not as reliable as slide culture for measuring branch angles (authors' observation). Slide culture, despite being time-consuming, is the most adequate technique to produce semi-permanent and permanent slides. Macro- and micromorphology are analyzed considering the literature. Webster and Weber32 is an extensive general source of information and additional references. Currah et al.16 and Zettler and Corey37 provide helpful information on orchid mycorrhizal fungi. Although describing fungi of medical importance, Walsh et al.21 and McGinnis31 are also helpful on general characteristics of filamentous fungi colonies, visual/descriptive information, and additional references for fungal micromorphology.
According to Yu et al.38, the DNA quantification analyses in a spectrophotometer present great precision and accuracy, being consistent with quantitative real-time PCR analyses. An important observation is that some isolates may not provide DNA samples adequately pure for the subsequent molecular identification stages, as mycoheterotrophs' fungal endophytes are extremely diverse, especially those associated with tropical plants39, as are the produced metabolites. Spectrophotometer analyses may be used to evaluate such cases and different protocols, or commercial kits applied for DNA purification of the samples. The suggested subsequent stages in the molecular identification of isolates are the amplification of the internal transcribed spacer (ITS) region and sequencing. The ITS region is a ribosomal RNA spacer DNA extensively employed in the specialized literature and formally accepted as the main barcode for fungal identification9,40. It may be amplified by PCR with the primers ITS1 and ITS441, then sequenced in the Sanger platform42.
Metabarcoding analyses, besides permitting investigation of the richness, abundance, and taxonomic composition of microorganisms in environmental and plant samples, may provide results of positive or negative interactions between these microorganisms12. The maceration stage using liquid nitrogen added to the DNeasy PowerSoil standard protocol aims to break as many fungal cells as possible in relation to plant tissues, enabling better sampling of the fungal community in the macerated tissue11. Primer choices for fungal sequences (barcodes) in molecular identification must consider the interference with plant DNA. We advise the use of primers that amplify variable regions of the fungal 18S gene, for instance, ITS1-5,8S-ITS243. After obtaining the DNA samples, the subsequent steps are preparing the metagenomic libraries, which depend on the chosen sequencing platform, posterior sequencing of the libraries, and analyzing the obtained data using bioinformatic tools. We recommend using the platform Illumina MiSeq, which represents a less expensive option with high output, generating 250 bp sequences in short time periods11,44.
The authors have nothing to disclose.
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's degree scholarship, process 88887.600591/2021-00) and CNPq.
Adhesive tape | (from any company, for adhesive tape mount in micromorphological analyses) | ||
Ampicillin | Sigma-Aldrich | A5354 | (for installation of plant fragments; other antibiotics may be used – check step 2.2.1) |
Autoclave | (from any company, for materials sterilization in many steps) | ||
Bacteriological agar | Sigma-Aldrich | A1296 | (for many steps) |
C1, C2, C3, C4, C5, and C6 solutions | Qiagen | 12888-50 | (purchased with DNeasy PowerSoil kit) |
Centrifuge | Merck/Eppendorf | 5810 G | (for total DNA extraction from fungal isolates) |
Centrifuge tubes | Merck | CLS430828 | (for samples collection) |
Chloroform | Sigma-Aldrich | C2432 | (for total DNA extraction from fungal isolates) |
Congo red | Supelco | 75768 | (for hyphae staining) |
Cryotubes | Merck | BR114831 | (for many steps) |
Ethanol | Supelco | 100983 | It will be necessary to carry out the appropriate dilutions (for many steps) |
Ethylenediaminetetraacetic acid (EDTA) | Sigma-Aldrich | 3609 | (for total DNA extraction from fungal isolates) |
Filter paper | Merck | WHA10010155 | (for many steps) |
Glass test tubes | Merck | CLS7082516 | (for cryopreservation in unhulled rice grains) |
Glass wool | Supelco | 20411 | (for cryopreservation in unhulled rice grains) |
Glucose | Sigma-Aldrich | G8270 | Or dextrose (for cryopreservation in vermiculite) |
Glycerol | Sigma-Aldrich | G5516 | Or glycerin (for cryopreservation in vermiculite, for preparing LPCB) |
Isopropanol | Sigma-Aldrich | 563935 | (for total DNA extraction from fungal isolates) |
Lactic acid | Sigma-Aldrich | 252476 | (for preparing LPCB – hyphae staining) |
Lactophenol blue solution (LPCB) | Sigma-Aldrich | 61335 | (for hyphae staining) |
Laminar flow hood | (class I, from any company, for many steps) | ||
Light microscope | (from any company, for hyphae observation) | ||
MB Spin Columns | Qiagen | 12888-50 | (purchased with DNeasy PowerSoil kit) |
Methyl blue (cotton blue) | Sigma-Aldrich | M5528 | (for preparing LPCB – hyphae staining) |
Microcentrifuge tube (1.5 mL) | Merck | HS4323 | (for total DNA extraction from fungal isolates) |
Microcentrifuge tube (2 mL) | Merck | BR780546 | (for many steps) |
Mineral oil | (for preservation of fungal isolates) | ||
Paper bags | Average size 150 mm x 200 mm (for samples collection) | ||
Petri dish (Glass, 120 mm x 20 mm) | Merck/Pyrex | SLW1480/10D | (autoclavable, for fungi slide culture, prefer higher ones) |
Petri dish (Glass, 50 mm x 17 mm) | Merck/Aldrich | Z740618 | (for purification of fungal isolates); alternatively: polystyrene petri dishes (sterile, γ-irradiated, non-autoclavable) |
Petri dish (Glass, 80 mm x 15 mm) | Merck/Brand | BR455732 | (for installation of plant fragments); alternatively: polystyrene petri dishes (sterile, γ-irradiated, non-autoclavable) |
Phenol | Sigma-Aldrich | P1037 | (for total DNA extraction from fungal isolates, for preparing LPCB) |
Porcelain mortar | Sigma-Aldrich | Z247464 | (for total DNA extraction from fungal isolates) |
Porcelain pestle | Sigma-Aldrich | Z247502 | (for total DNA extraction from fungal isolates) |
Potato dextrose agar (PDA) | Millipore | P2182 | (for many steps) |
PowerBead tubes | Qiagen | 12888-50 | (purchased with DNeasy PowerSoil kit) |
Rapid mounting medium (Entellan) | Sigma-Aldrich | 1.0796 | (for fungi slide culture) |
Silica gel | Supelco | 717185 | (for cryopreservation in unhulled rice grains) |
Sodium chloride (NaCl) | Sigma-Aldrich | S9888 | (for total DNA extraction from fungal isolates) |
Sodium dodecyl sulfate (SDS) | Sigma-Aldrich | L3771 | Lauryl sulfate sodium salt (for total DNA extraction from fungal isolates) |
Sodium hypochlorite (w/ 2% active chlorine) | (commercial product, for superficial desinfestation) | ||
Soil DNA extraction kit (DNeasy PowerSoil kit) | Qiagen | 12888-50 | (for total DNA extraction from plant organs) |
Spectrophotometer – Nanodrop 2000/2000c | ThermoFisher Scientific | ND2000CLAPTOP | (for total DNA extraction from plant organs) |
Stereomicroscope | (=dissecting microscope, from any company, for macromorphological analyses) | ||
Tetracycline | Sigma-Aldrich | T7660 | (for installation of plant fragments) |
Thermoblock | Merck/Eppendorf | EP5362000035 | (or from other companies) |
Tissue homogenizer and cell lyzer | SPEX SamplePrep | 2010 Geno/Grinder – Automated Tissue Homogenizer and Cell Lyzer (for total DNA extraction from plant organs) | |
Toluidine blue O | Sigma-Aldrich/Harleco | 364-M | (for hyphae staining) |
Trehalose | Sigma-Aldrich | T9531 | (for cryopreservation in vermiculite) |
Tris Base Solution (Tris) | Sigma-Aldrich | T1699 | (for total DNA extraction from fungal isolates) |
Unhulled rice grains | (for cryopreservation) | ||
U-shaped glass rod | (or an adaptation – check step 5.4.1, for fungi slide culture) | ||
Vermiculite | Fine granulometry (for cryopreservation in vermiculite) | ||
Vortexer | Sigma-Aldrich/BenchMixer | BMSBV1000 | (for total DNA extraction from fungal isolates) |
Yeast extract | Sigma-Aldrich | Y1625 | (for cryopreservation in vermiculite) |