Isolation and characterization of wood-decomposing basidiomycetes from the Andean Forest in Boyacá, Colombia

Abstract

This study explores the biotechnological potential of lignocellulolytic fungi collected in an oak forest. Fungal collections were obtained from natural reserves located in Boyacá-Colombia, ranging from 2700 to 3000 m.a.s.l. Twenty-three strains were isolated on malt agar, molecular characterization was performed, and ligninolytic and cellulolytic enzymatic activities were screened. Several white-rot fungi of biotechnological importance were identified as follows: Trametes sp., Trametes versicolor, Trametes villosa, Pycnoporus sanguineus, Bjerkandera adjusta, Lentinula boryana, Panus conchatus, Antrodia neotropica, Brunneoporus malicola, Laetiporus gilbertsonii, Stereum sp., Ganoderma sp., and Dichomitus sp. The strains T. versicolor 0554 and 0583, T. villosa 0562, and B. adusta 0556 showed the highest response in the qualitative enzymatic assays. These strains were used to determine their ability to decolorate the dyes aniline blue and Congo red, and it was found that T. villosa 0562 reached a level of decolorization close to 90% after 48 h of submerged culture. The fungal strains obtained here could offer alternatives to develop a process to accomplish sustainable development objectives.

Introduction

Macromycetes play an essential role in forest ecosystems due to their function in nutrient cycling [1]; for example, they use extracellular enzymatic systems to degrade the main structural compounds of plant cell walls in decaying wood [2]. The most recalcitrant compound, lignin, can also be efficiently degraded by several species of basidiomycetes. Basidiomycetes are also capable of degrading cellulose and hemicellulose, the other components of lignocellulosic material [3]. Lignin-degrading enzymes include phenol oxidases (laccases), lignin peroxidases (LiPs), manganese peroxidases (MnPs), and versatile peroxidase (VPs), while cellulolytic enzymes include endoglucanases, exoglucanases, and β-glucosidases [4, 5]. These fungal enzymes show potential for use in several steps in biofuel production, such as lignocellulosic biomass pretreatment, cellulose saccharification, and fermentation inhibitor removal [6, 7].

Basidiomycetes are also a source of bioactive metabolites with antimicrobial activity [8, 9] and immunomodulatory compounds [10], among others. These capacities provide opportunities to develop several biotechnological applications [11], including the production of edible and medicinal mushrooms by using crop-based agro-industrial wastes [12] and the degradation of xenobiotic compounds [13]. New applications of fungi could lead to the responsible exploration of biodiversity, allowing developing countries harboring this biological diversity to accomplish sustainable development objectives [14–16].

Colombia is recognized as a megadiverse country due to its richness of diverse biological groups [17, 18]. In addition, Boyacá is a Colombian department located in the Eastern Andes Mountains, which is recognized as a hotspot of biodiversity [19], but it is important to mention that Andean ecosystems are among the most threatened in the world [20]. Therefore, it is appropriate to study this biodiversity and promote its conservation, recognizing that establishing culture collections of living fungi is strategic for developing the bioeconomy [21].

This study aimed to identify basidiomycete fungi collected in the Boyacá Forest and explore their biotechnological potential based on their lignocellulolytic enzymatic activities.

Materials and methods

Sample collection and culture of isolates

Twenty-three specimens were collected in the Andean cloud forest dominated by Oak (Quercus humboldtii) in the municipalities of Arcabuco, Combita, and Duitama (Boyacá), located in the east range mountain of Colombia. The sample sites were located between 2300 and 3400 m.a.s.l. with a temperature range of 12 to 14 °C and a mean precipitation of 1564 mm. Collections were made between November 2019 and January 2020. Permission to collect biological samples was granted by Corpoboyacá (Resolution No 2728 de 2019).

Fresh fruiting bodies were removed from decayed wood on the ground. Then, the specimens were transferred to the lab in aluminum foil to avoid contamination and dehydration of the carpophores. Isolates were made from a 0.5–1-cm² piece of the basidiocarp. Before cultivation in MEA (malt extract agar, DIFCO), the piece was cleaned with a solution of NaClO (1%) plus EtOH (70%). Small carpophores were placed on the edge of an MEA plate without touching the agar medium. They were incubated overnight in a moist chamber at room temperature to allow basidiospores to be discharged on the MEA. For each specimen, four explants were cultured from the basidiocarp. If the culture from the same specimen presented differences in morphological features, they were considered different strains. Axenic cultures were maintained at − 80 °C in the Cell Culture Collection of Fungi and Microorganisms of Universidad of Boyacá (Tunja, Colombia).

DNA extraction and PCR amplification

Cryopreserved fungi were activated in Sabouraud dextrose yeast liquid medium and incubated under shaking at 100 rpm and 10 °C for 8 days. They were subsequently collected under vacuum using filter paper No. 1 (Whatman, UK) and frozen at − 80 °C overnight. This fungal biomass was lyophilized for 3 days (Freezone-1-LABCONO, MO). The material obtained was kept in a desiccator until the DNA extraction process began. For DNA extraction, approximately 50 mg of the lyophilized product was taken, and DNA extraction was carried out using a Wizard® Genomic DNA Purification kit according to the procedure for plant tissues recommended by the manufacturer.

Two nuclear loci were amplified, including the nuclear ribosomal internal transcribed spacer region (ITS1-F: 5′ -CTT GGT CAT TTA GAG GAA GTA A-3′ ITS4-R: 5′ -GGA AGT AAA AGT CGT AAC AAG G-3′) and the long subunit of ribosomal RNA (LROR: 5′ -ACCCGCTGAACTTAAGC-3′ LR5: 5′ -ATC CTG AGG GAA ACT TC-3′). Amplification followed the procedure reported by Gardes and Bruns [22] and Vilgalys and Sun [23], with some modifications. The reactions were carried out in 30 µL. The PCR mix contained primers at a final concentration of 0.5 µM, 1 µL of genomic DNA (2 ng/µL), and 12.5 µL of 2 × PCR MasterMix (Applied Biological Materials Inc. (Abm)). The amplification program consisted of one initial cycle of 3 min at 94 °C, followed by 35 cycles comprising denaturation (1 min at 94 °C), annealing (30 s at 56 °C), and extension (1 min at 72 °C), and then a final extension (7 min at 72 °C). The amplified products were analyzed on 1% agarose gels stained with SYBR® Green (Applied Biological Materials Inc. (Abm)).

Sequencing

The PCR products obtained for each marker were purified and sequenced using the Sanger platform. The sequences obtained were edited using Geneious Prime® 2021.0.3 software; subsequently, with the consensus sequences, BLAST was performed in the GenBank database and MycoBank databases to determine the species or genus of each isolate. The identification presented in this article corresponds to the better similarity and overlap percent. The assembled sequences were submitted to the GenBank Database.

Test to identify white-rot fungi

To classify the axenic cultures as a white or brown-rot fungus, aniline agar medium was used according to Alberto and Wright [24]. To prepare this medium, two solutions were employed: solution A (a mixture of 20 mL of aniline, 5 mL of concentrated nitric acid, and 500 mL of deionized water) and solution B (a mixture of 20 g of agar with 500 mL of deionized water). Solutions A and B were sterilized separately and mixed in equal proportions, and then the medium was transferred to Petri dishes. The test was conducted by inoculating a plug of 5 mm of mycelia (7-day-old cultures grown on PDA) on aniline fresh medium. The response was observed after 10 days of incubation. The fungal cultures were classified as a white-rot fungus if a brown halo was observed.

Evaluation of extracellular activities

Extracellular ligninolytic and cellulolytic activities produced by the basidiomycete fungi were assessed in culture. Before each evaluation, the strains were grown in PDA medium for 7 days at 25 °C; an agar pug with mycelia (0.5 cm) was placed on a plate with each evaluation medium. The evaluation was conducted in triplicate, and the cultures were incubated at 25 °C in the dark and examined daily.

Ligninolytic enzyme tests

Three assays were used to identify lignin-modifying enzymes: ABTS agar and tannic acid agar were used to evaluate laccase activity, whereas azure B agar was used for the qualitative determination of lignin peroxidase and manganese-dependent peroxidase activities.

ABTS agar

The composition of the basal medium (BM1) used was described by Pointing [25] and includes slight modifications: sodium–potassium tartrate: 0.77 g/L; NH₄Cl: 0.29 g/L; Fe₂(SO₄)₃*H₂O: 1.05 mg/L; KH₂PO₄: 1 g/L; MgSO₄*7 H₂O: 0.5 g/L; yeast extract: 0.01 g/L; CaCl₂*2H₂O: 10 mg/L; CuSO₄*5H₂O: 1 mg/L; and MnSO₄*H₂O: 1 mg/L. The BM1 medium was supplemented with ABTS (final concentration: 1 g/L) and agar (final concentration: 16 g/L), autoclaved, and then 1 mL (for each 100 mL of the basal medium) of sterilized aqueous glucose solution (200 g/L) was incorporated. Finally, the medium was transferred to Petri dishes and stored at 4 °C until use.

This medium allows the identification of laccases due to the oxidation of ABTS (2,2′-azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid, colorless) to ABTS-azine (green). After 10 days of incubation, the plates were examined, and the magnitude of the response was identified by measuring the green halo diameter. The intensity of the response was defined based on the halo rate (HR: ratio of halo diameter in mm divided # of days culture). Responses were as reported as follows: no presence of halo: no activity ( −); 0.1 < HR = 0.3: ( +); 0.3 < HR = 0.6: (+ +); and HR > 0.6 (+ + +).

Tannic acid agar

The basal medium (BM1), glucose, and agar concentrations used were the same as those used in the ABTS test, but 1 mL of filter-sterilized aqueous tannic acid solution (for each 100 mL of the basal medium; stock concentration: 10 g/L) was mixed with the agar and glucose solution previously autoclaved. Finally, the medium was transferred to Petri dishes and stored at 4 °C until use.

This medium allows the identification of the overall polyphenoloxidase activity by forming a brown halo around the mycelium due to the enzymatic oxidation of tannic acid. After 5–8 days of incubation, the plates were examined, and the magnitude of the response was identified by measuring the brown halo diameter. The intensity of the response was defined based on the HR as follows: no halo presence: no activity ( −); 0.1 < HR = 0.3: ( +); 0.3 < HR = 0.6: (+ +); and HR > 0.6 (+ + +).

Azure B agar

This medium was prepared by supplementation of PDA medium with azure B (final concentration: 15 mg/L), as Montoya [26] recommended. The mixture was autoclaved, transferred to Petri dishes, and stored at 4 °C until use.

After 15 days of incubation, the plates were examined, and the response was identified as the clearance of the medium. The intensity of this response was defined qualitatively based on the magnitude of the decolorization HR as follows: no decolorization: no activity ( −); weak decolorization ( +); medium decolorization (+ +); and total decolorization (+ + +).

Cellulolytic enzyme tests

The endoglucanase and exoglucanase activities were qualitatively determined by using the methods of dye staining of carboxymethylcellulose agar (CMC agar) and cellulose agar clearance (Avicel agar; microcrystalline cellulose), respectively; these methods were made based as recommended by Pointing [25], with some modifications, especially to the last method.

Avicel agar

The composition of the basal medium (BM2) used was described by Pointing [25]: sodium–potassium tartrate: 7.7 g/L; ammonium chloride: 2.9 g/L; KH₂PO₄: 1 g/L; MgSO₄*7 H₂O: 0.5 g/L; yeast extract: 0.1 g/L; CaCl₂*2 H₂O: 1 mg/L. Avicel® PH-101 (final concentration: 5 g/L) and agar (16 g/L) were added to BM2 medium.

Microcrystalline cellulose degradation requires both endoglucanases and exoglucanases; the clearance of Avicel agar is correlated with cellulolytic activities. However, the fungal mycelium is white, as in Avicel agar, and it is difficult to observe a conclusive response. For this reason, we used the procedure presented by Abe et al. [27], based on the capacity of Gram’s iodine to form a colored complex with cellulose.

After 5 days of incubation, the plates were revelated, and then the Petri dishes were flooded with 5 mL of a commercial solution of Gram’s iodine for 5 min. The solution was poured off, and the plates were flooded with 5 mL of deionized water for 5 min. The degradation of microcrystalline cellulose was identified as a yellow-opaque area around the mycelium, while the intact cellulose was identified as a brown area. The intensity of the response was defined based on the growth rate (GR: ratio of growth halo diameter divided by # days of culture) as follows: GR: 0.1 < GR = 0.4: ( +); 0.4 < GR = 0.8: (+ +); and GR > 0.8 (+ + +).

CMC agar

The basal medium (BM2), glucose, and agar concentrations used were the same as those used in the Avicel test, but carboxymethylcellulose (CMC; low viscosity; final concentration 5 g/L) was used instead of Avicel. This medium allows the qualitative determination of endoglucanases, which is possible due to the differences observed between the CMC degraded and intact substrate after dye staining of the medium with Congo red. The procedure applied was described by Kasana et al. [28]. After 5 days of incubation, the plates were revelated, and then the plates were flooded with 5 mL of an aqueous solution of Congo red (10 g/L) for 15 min. Then, the dye was poured off, and the plate was flooded with 5 mL of an aqueous solution of NaCl (1 M) for 15 min. The degraded CMC was identified as a yellow-opaque area around the mycelium, while the intact CMC was identified as a red area. The intensity of the response was defined based on the GR as follows: GR: 0.1 < GR = 0,4: ( +); 0,4 < GR = 0,8: (+ +); and GR > 0,8 (+ + +).

Potential of wild fungal strains to decolor dyes

The strains selected for this assay showed high qualitative laccase and peroxidase activities. Congo red (azo type) and aniline blue (triphenylmethane type) were the dyes used for the decolorization assays. All liquid media were sterilized by autoclaving. Liquid cultures were carried out in an orbital shaker at 25 °C and 100 RPM in the dark.

Growth culture conditions

Five agar plugs (0.5 cm) containing mycelium (7 days old, grown in PDA) were placed into the liquid medium to obtain pellets. The medium used was proposed by Kim et al. [29]: glucose (20 g/L), KH₂PO4 (0.46 g/L), K₂HPO₄ (1 g/L), MgSO₄*7H₂O (0.5 g/L), peptone (2 g/L), and yeast extract (2 g/L). Pellet inoculum was used in this study according to Lueangjaroenkit et al. [30] for the dye degradation assays.

Dye degradation culture conditions

The harvested pellets (7 days old) were washed twice with deionized water under aseptic conditions and later used as inoculum for the dye degradation assays. The degradation medium used was reported by Hernández et al. [31]: KH₂PO₄ (1 g/L), NaH₂PO₄ (0.26 g/L), (NH₄)₂SO₄ (0.317 g/L), MgSO₄*7H₂O (0.5 g/L), CuSO₄ (0.5 mg/L), CaCl₂ (74 mg/L), ZnSO₄ (6 mg/L), FeSO₄ (5 mg/L), MnSO₄ (mg/L), CoCl₂ (1 mg/L), carboxymethyl cellulose (20 g/L), and yeast extract (1 g/L). The pH was adjusted to 6.0.

The initial concentration of each dye was 50 mg/L. The assays were conducted in duplicate. The degradation of the dyes was followed daily by measuring the absorbance (495 nm for Congo red and 590 nm for aniline blue) of the medium. The following equation was used to calculate the % decolorization:

$$\%\mathrm{decolorization}=\frac{AB{S}_0{\mathit{DF}}_0-AB{S}_n{\mathit{DF}}_n}{AB{S}_0{\mathit{DF}}_0}\times 100\%$$

where ABS is the absorbance of the sample, DF is the dilution factor used in the measurement, and the subindices 0 and n refer to the initial time and experimental time, respectively.

Results

Isolation and identification of isolates

Some of the twenty-three specimens collected in the Andean cloud forest are shown in Fig. 1. Thirty-one strains were purified and preserved. Amplification of DNA samples with primers ITS1-ITS4 and LROR and LR5 resulted in approximately 800 bp and 1300 bp, respectively. The sequences were deposited in GenBank, and accession numbers were obtained (Table 1). All PCR products obtained were sequenced and identified to be 96 to 100% similar to the sequences of the ITSI, 5.8S rRNA gene, ITSII, and LSU regions of the respective fungi. The 31 fungal strains that belonged to the Basidiomycota division were identified (Table 1); 19 strains (61.3%) belonged to the Trametes genus, specifically to T. villosa (n = 11), Trametes sp. (n = 1), T. versicolor (n = 5), P. sanguineus (n = 1), and T. hirsuta (n = 1). In addition, two strains each of Lentinula boryana, Antrodia neotropica, and Panus conchatus were identified. Finally, one specimen of each of the following species, Brunneoporus malicola, Bjerkandera adusta, Dichomitus sp., Stereum sp., Ganoderma sp., and Laetiporus gilbertsonii, are reported in this study.

Fig. 1.

Images of the fungi collected in the Boyacá Forest. A Lentinula boryana strain 0548. B Antrodia neotropica strain 0550. C Brunneoporus malicola strain 0549. D Trametes versicolor strain 0554. E Trametes villosa strain 0557. F Trametes sanguinea strain 0559. G Stereum sp. strain 0565. H Panus conchatus strain 0567. I Trametes hirsuta strain 0575. J Laetiporus gilbertsonii strain 0606

Table 1.

Specimen locality, collection number, and type of rot fungus for the strains obtained in this study

Taxon	Locality	Geographic coordinates		Collection # UBCHM-AM	Sequence length (ITS1/ITS4)	GenBank accession number ITS	Sequence length (LR0R/LR5)	GenBank accession number LSU	Aniline classification
		Latitude (N)	Longitude (W)
Lentinula boryana	La Zarza, Duitama	5° 50′ 30.6′′	73° 5′ 23.3′′	541	694	OM400524	863	MZ424266	WRF
Antrodia neotropica	La Zarza, Duitama	5° 51′ 15.5′′	73° 5′ 43.5′′	546	592	OM400525	836	MZ424267	BRF
Lentinula boryana	La Zarza, Duitama	5° 50′ 53.8′′	73° 5′ 18.1′′	548	740	OM400526	834	MZ424268	WRF
Brunneoporus malicola	La Zarza, Duitama	5° 50′ 44.5′′	73° 5′ 13.1′′	549	600	OM400527	820	MZ424269	BRF
Antrodia neotropica	Serranía el Peligro, Arcabuco	5° 48′ 33.9′′	73° 28′ 17.1′′	550	631	OM400528	933	MZ424270	BRF
Trametes versicolor	Serranía el Peligro, Arcabuco	5° 48′ 38.7′′	73° 28′ 26.4′′	552	576	OM400529	833	MZ424271	WRF
Trametes versicolor	Serranía el Peligro, Arcabuco	5° 48′ 38.7′′	73° 28′ 26.4′′	553	625	OM400530	739	MZ424272	WRF
Trametes versicolor	Serranía el Peligro, Arcabuco	5° 48′ 38.8′′	73° 28′ 26.8′′	554	611	OM400531	818	MZ424273	WRF
Trametes sp.	Serranía el Peligro, Arcabuco	5° 48′ 38.8′′	73° 28′ 26.8′′	555	571	OM400532	851	MZ424274	WRF
Bjerkandera adusta	Serranía el Peligro, Arcabuco	5° 48′ 40.1′′	73° 28′ 28.2′′	556	602	OM400533	837	MZ424275	WRF
Trametes villosa	Serranía el Peligro, Arcabuco	5° 49′ 18.0′′	73° 30′ 1.0′′	557	630	OM400534	837	MZ424276	WRF
Dichomitus sp.	Serranía el Peligro, Arcabuco	5° 49′ 12.0′′	73° 39′ 57.0′′	558	586	OM400535	840	MZ424277	WRF
Pycnoporus sanguineus	Serranía el Peligro, Arcabuco	5° 49′ 13.0′′	73′ 30′ 58.0′′	559	531	OM400536	825	MZ424278	WRF
Trametes villosa	Serranía el Peligro, Arcabuco	5° 48′ 3.0′′	73° 28′ 4.0′′	560	630	OM400537	834	MZ424279	WRF
Trametes villosa	Serranía el Peligro, Arcabuco	5° 48′ 3.0′′	73° 28′ 4.0′′	562	548	OM400538	862	MZ424280	WRF
Stereum sp.	Serranía el Peligro, Arcabuco	5° 48′ 21.0′′	73° 28′ 19.0′′	565	588	OM400539	866	MZ424281	WRF
Panus conchatus	Serranía el Peligro, Arcabuco	5° 46′ 26.0′′	73° 21′ 7.0′′	567	654	OM400540	875	MZ424282	WRF
Panus conchatus	Serranía el Peligro, Arcabuco	5° 46′ 26.0′′	73° 21′ 7.0′′	570	556	OM400541	607	MZ424283	WRF
Trametes villosa	El Valle, Combita	5° 50′ 24.0′′	73° 5′ 16.0′′	571	567	OM400542	864	MZ424284	WRF
Trametes hirsuta	El Valle, Combita	5° 50′ 24.0′′	73° 5′ 16.0′′	575	608	OM400543	859	MZ424285	WRF
Trametes villosa	La Zarza, Duitama	5° 50′ 25.0′′	73° 5′ 17.0′′	576	627	OM400544	839	MZ424286	WRF
Trametes villosa	La Zarza, Duitama	5° 50′ 25.0′′	73° 5′ 17.0′′	579	567	OM400545	832	MZ424287	WRF
Trametes versicolor	La Zarza, Duitama	5° 50′ 26.0′′	73° 5′ 18.0′′	583	625	OM400546	818	MZ424288	WRF
Trametes villosa	La Zarza, Duitama	5° 50′ 26.0′′	73° 5′ 18.0′′	584	577	OM400547	828	MZ424289	WRF
Trametes villosa	La Zarza, Duitama	5° 50′ 28.0′′	73° 5′ 21.0′′	587	630	OM400548	804	MZ424290	WRF
Trametes villosa	La Zarza, Duitama	5° 50′ 28.0′′	73° 5′ 21.0′′	592	583	OM400549	379	MZ424291	WRF
Trametes villosa	La Zarza, Duitama	5° 50′ 33.0′′	73° 5′ 19.0′′	599	576	OM400550	820	MZ424292	WRF
Trametes villosa	La Zarza, Duitama	5° 50′ 33.0′′	73° 5′ 19.0′′	602	629	OM400551	826	MZ424293	WRF
Ganoderma sp.	El Valle, Combita	5° 45′ 21.0′′	73° 20′ 9.0′′	603	667	OM400552	857	MZ424294	WRF
Trametes versicolor	El Valle, Combita	5° 45′ 21.0′′	73° 20′ 9.0′′	605	599	OM400553	840	MZ424295	WRF
Laetiporus gilbertsonii	El Valle, Combita	5° 45′ 21.0′′	73° 20′ 9.0′′	606	614	OM400554	639	MZ424296	BRF

WRF white-rot fungi; BRF, brown-rot fungi

Concatenated ITS and LSU gene sequences clustered the 31 strains into ten clades: (1) one strain of P. sanguinea, (2) Dichomitus sp. and Ganoderma sp. with 87.7% similarity, (3) B. malicola with 99.9% similarity to two identical strains of A. neotropica, (4) L. gilbertsonii, (5) B. adusta with 72.9% similarity to two identical strains of P. conchatus, and (6) Stereum sp. with 50.9% similarity to two identical strains of L. boryana. Species of the Trametes genus were grouped into four clades: (7) T. versicolor with 65.3% similarity to Trametes sp., (8) four strains of T. versicolor, (9) two strains of T. villosa, and (10) one strain of T. hirsute and five strains of T. villosa (Fig. 2).

Fig. 2.

Evolutionary relationships were inferred using the neighbor-joining method in Geneious Prime® 2021.0.3 software. The analysis involved 31 nucleotide sequences and an outgroup with nucleotide sequences of D. sulphurellum and L. cinerascens. All positions containing gaps and missing data were eliminated. Numerical values above the internodes are the percentages of 1000 bootstrap replications. Bootstrap values greater than 50% are indicated. The scale bar of 0.08 represents nucleotide substitutions per position

Qualitative evaluation of extracellular activities

All fungal cultures were screened to characterize their ability to produce lignocellulose enzymes. An example of the response and its levels in the qualitative tests are shown in Fig. 3. Two tests were carried out to examine laccase enzyme production, including ABTS and tannic acid oxidation assays, on solid plates. Only three fungal strains exhibited a negative response to ABTS and tannic acid, which correspond to the A. neotropica and B. malicola cultures (0546, 0549, and 0550), as shown in Table 2. L. gilbertsonii, classified as a brown-rot fungus, showed a low response, while the best laccase producers were the strains T. versicolor (0552, 0553, 0554, 0555, 0583, and 0584), T. villosa (0557,0560, 0571, 0576, 0579, and 0602), T. hirsuta (0575), and B. adusta (0556). The azure B test allowed us to determine the best lignin peroxidase and Mn-dependent peroxidase producers. The results showed that the best producers were T. versicolor (0554 and 0583), B. adusta (0556), T. sanguineus (0559), and T. villosa (0562).

Fig. 3.

Response levels in the qualitative enzymatic assays

Table 2.

Qualitative evaluation of lignocellulolytic enzyme production by fungal strains

Strain	Species	Ligninolytic activity			Cellulolytic activity
		ABTS	Tannic acid	Azure B	Avicel	CMC
0541	Lentinula boryana	+  +	+  +	+	+	+
0546	Antrodia neotropica	-	-	+  +	+	+
0548	Lentinula boryana	+  +	+  +	+	+	+
0549	Brunneoporus malicola	−	−	+  +	+	+
0550	Antrodia neotropica	−	−	+  +	+	+
0552	Trametes versicolor	+  +  +	+  +  +	+	+  +	+  +  +
0553	Trametes versicolor	+  +  +	+  +  +	-	+  +  +	+  +  +
0554	Trametes versicolor	+  +  +	+  +  +	+  +  +	+  +	+  +  +
0555	Trametes versicolor	+  +  +	+  +  +	-	+  +  +	+  +  +
0556	Bjerkandera adusta	+  +  +	+  +	+  +  +	+  +  +	+  +  +
0557	Trametes villosa	+  +  +	+  +  +	-	+  +  +	+  +  +
0558	Polyporus sp.	+  +	+	+	+	+
0559	Trametes sanguinea	+  +	+  +	+  +  +	+  +	+  +  +
0560	Trametes villosa	+  +	+  +  +	+  +	+  +  +	+  +  +
0562	Trametes villosa	+  +	+  +	+  +  +	+  +  +	+  +  +
0565	Stereum sp.	+  +	+	+	+	+
0567	Panus conchatus	+  +	+  +	−	+	+
0570	Panus conchatus	+  +	+  +	−	+	+
0571	Trametes villosa	+  +  +	+  +  +	−	+  +  +	+  +  +
0575	Trametes hirsuta	+  +	+  +  +	+	+  +  +	+  +  +
0576	Trametes villosa	+  +  +	+  +  +	+	+  +  +	+  +  +
0579	Trametes villosa	+  +  +	+  +	+  +	+  +	+  +
0583	Trametes versicolor	+  +	+  +  +	+  +  +	+  +  +	+  +
0584	Trametes villosa	+  +	+  +  +	+  +	+  +  +	+  +  +
0587	Trametes villosa	+  +	+  +	+  +	+  +	+  +
0592	Trametes villosa	+  +	+  +	+  +	+  +	+  +
0599	Trametes villosa	+  +	+  +	+  +	+  +	+
0602	Trametes villosa	+  +	+  +  +	+  +	+  +  +	+  +  +
0603	Ganoderma sp.	+  +  +	+	+	+  +	+  +
0605	Trametes versicolor	+  +  +	+  +	+  +	+  +	+  +  +
0606	Laetiporus gilbertsonii	+	+	+	+	+

The number of crosses (“ + ,” “ +  + ,” or “ +  +  + ”) indicates the intensity of the positive response; ( −) indicates no detectable response; NR, not reported

Cellulolytic enzyme production by wild fungal strains was carried out using microcrystalline cellulose (Avicel) or CMC as the only carbon source. CMC is a soluble form of cellulose that can be easily attacked by endoglucanase enzymes, while Avicel has low solubility in water and its assimilation requires exoglucanases and endoglucanases. The use of these substrates allows the identification of fungi that can efficiently utilize the cellulose present in lignocellulosic biomass. All strains studied produce cellulolytic enzymes, as shown in Table 2. No differences were found between the assimilation of Avicel or CMC by each specific strain, and a high rate of growth was always coupled with a high rate of cellulose degradation. Twelve fungal strains presented a high positive response to cellulase production, and the strains of T. versicolor (0553, 0555, 0583), B. adusta (0556), T. villosa (0557, 0560, 0562, 0584, and 0602), and T. hirsuta (0575) were remarkable.

Dye decolorization

T. versicolor (0554 and 0583), B. adusta (0556), and T. villosa (0562) were selected to evaluate their potential to decolor two different types of dyes used in the laboratory and industry (Congo red and aniline blue); see Fig. 4. These strains were chosen because they presented the highest responses in laccase and peroxidase assays, showing their potential to degrade dyes with aromatic chemical structures. Strains 0554 and 0583 (T. versicolor) and 0562 (T. villosa) showed greater than 90% decolorization of aniline blue after 48 h of culture. The highest decolorization level of Congo red was obtained by strain T. villosa (0562), with 89% decolorization after 48 h of culture.

Fig. 4.

Decolorization percentages at 48 h by T. versicolor strains 0554 and 0583, B. adusta strain 0556, and T. villosa strain 0562

Discussion

In this study, taxonomic identification was based on analyzing the ITS1 and LSU nuclear ribosomal DNA sequences. These genetic markers were chosen to be the official barcode for the fungi by a consortium of mycologists, and they allow the species-level identification of fungi [32–34]. This information has previously been used to characterize basidiomycetes, such as Agaricus species [35] and Trametes species [36]. However, the use of nonribosomal markers may be critical to developing a more detailed taxonomic study, for example, with the inclusion of the protein-coding genes RPB1, RPB2, and TEF1 [37].

The collection cultures obtained in this study include several white-rot fungal strains recognized by their biotechnological potentials, such as T. versicolor, T. villosa, T. hirsuta, B. adusta, P. conchatus, and L. boryana. This collection has a significant number of strains of Trametes, a genus of fungi described as cosmopolite due to its presence in almost any wooded ecosystem [37]. According to Paton et al. [38], fungal culture collections are mainly distributed in Europe and North America, and regions recognized as megadiverse are relatively underrepresented. These culture collections could support conservation initiatives, and they are biological resources to continue to study basidiomycete biodiversity. However, to improve the impact of this new Colombian collection, it is necessary to expand the collection to a broader range of taxa.

The Trametes genera could offer interesting opportunities to develop functional foods and other biobased products due to their essential biological activities. For example, T. orientalis and T. robiniophila produce metabolic compounds with antioxidant and antitumoral properties [39, 40] and antimicrobial activity [41, 42]. T. versicolor (also known as turkey tail) is a remarkable ligninolytic fungus that has been used in traditional Asian medicine, and its extracts have been proposed as a food supplement [43]. Moreover, T. versicolor can degrade lignocellulose biomass efficiently due to its capability to secrete several enzymes, including laccase, manganese peroxidase, and lignin peroxidase, enabling it to degrade a great diversity of organic pollutants [44].

In this study, T. versicolor strains 0554 and 0583 showed a good capacity to decolorate aniline and Congo red, which is related to their production of lignin-degrading enzymes. However, the best dye decolorization capacity was obtained by T. villosa strain 0562. Nevertheless, the related enzymatic activity must be quantified to identify which lignin-degrading enzymes are implicated in the biodegradation of these substances, and it could enable the study of culture conditions that promote their production.

Li et al. [45] used the Trametes sp. strain SYBC-14 to decolorize Congo red and aniline blue without the use of laccase mediators, and they found decolorization levels of 57.82% and 92.53%, respectively, after 10 days of culture under solid fermentation conditions. Crude enzymes of T. versicolor (strain ATCC 20,869) have also been used to degrade these compounds, and it was found that these enzymes could decolorize 78.48% of aniline blue and 31.30% of Congo red after 48 h of incubation [46]. In addition, these fungi could offer a promising alternative to process vinasse, a contaminant composed of phenolic compounds that is a byproduct of bioethanol distillation. Following the abovementioned, Tapia-Tussell et al. [47] evaluated laccase in vinasse, reaching a level of discoloration of 69.2% of this contaminant. White-rot fungi efficiently degrade organic matter thanks to their ability to produce extracellular enzymes, including laccase, manganese peroxidase, and lignin peroxidase. As reported by Daassi, a strain of the species Trametes trogii was used to degrade the Acid Orange 51 azo-type dye, also finding high discoloration and low phytotoxicity in the dye products [48]. In studies carried out with other white-rot fungi, low toxicity has also been found in the degradation products of Congo red, for example, with the laccases of Oudemansiella canarii [49], or due to manganese-dependent peroxidase of Pleurotus sajor caju [50]. Likewise, the biodegradation of triphenylmethane dyes such as aniline blue by white-rot fungi has been poorly described. In contrast, for other dyes of the same family, such as methyl green, degradation greater than 80% has been reported by a strain of Ganoderma sp. [51], and for the crystal violet dye, 100% discoloration has been described by a strain of the fungus Dichomitus squalens [52], and low toxicity of the biodegradation products of this group of compounds has been observed in a strain of Bjerkandera adusta [53]. However, the substances obtained during degradation and their toxicity must be characterized since these processes may be incomplete and form more toxic substances for the environment.

On the other hand, these fungi could assimilate CMC and Avicel as the only carbon source, which is related to their capacity to secrete endoglucanase and exoglucanase enzymes. These properties could be used to take advantage of agro-industrial wastes based on lignocellulose biomass, especially to produce fruiting bodies to be used as edible or medicinal mushrooms [54, 55]. Additionally, to produce enzymes required for biofuel and biochemical production, T. hirsuta strains have been studied for their potential [56]. Further assays must be conducted to examine the production of other enzymatic activities, such as xylanases, amylases, and proteases, to confirm the potential use of these fungi in biofuel production [57]. Cellulolytic enzyme production could be strategic to Colombia because this country generates a lot of unused agro-industrial biomass that could be converted to renewable energy such as bioethanol [58].

Other isolates have been reported to have significant biotechnological applications. To illustrate, B. adusta has a versatile peroxidase that has a broad preference for substrates as an example to oxidize halogenated pesticides [59]. Species of the Antrodia genera, such as A. camphorate and A. cinnamomea, have been reported to contain important bioactive compounds with cytotoxic and anti-inflammatory activities in cancer cell lines [60, 61]. Furthermore, Ganoderma spp. have long been used as a medicinal mushroom in Asia, and they have an array of pharmacological properties, including immunomodulatory and antimicrobial activities, among others [62].

Conclusion

In this study, we used qualitative assays to test enzyme production in basidiomycete fungi isolated from the Andean Forest. The strains showed enzymatic activities of biotechnology relevance, including endoglucanase and exoglucanase. In addition, lignin-modifying enzymes, such as laccase, lignin peroxidase, and Mn-dependent peroxidase, were present in several strains. This capacity was used to decolorize two different dyes, Congo red and aniline blue. Finally, ligninolytic fungi were found to be widely distributed in oak forests in Boyacá (Colombia). Despite the effects of anthropogenic intervention, this region remains an essential source of fungi with biotechnological potential.

The department of Boyacá in Colombia has high mountain ecosystems, which have faced deforestation processes due to the expansion of livestock and potato crops. These environments have been little explored, and the biotechnological potential of their saprophytic fungi remains unknown. With the completion of this study, it is evident that protected nature reserves are home to species of basidiomycete fungi with recognized biotechnological potential. It is expected that subsequent studies aimed at using these fungi will promote the valorization of this natural resource. In addition, with the establishment of this biological collection, ex situ conservation processes have been initiated with which it is possible to study fungal biodiversity and use it in reforestation processes that currently involve almost exclusively the restoration of native tree populations without taking into consideration the role that basidiomycete fungi have in the nutrient equilibrium in the forest.

Funding

This research was funded by the Fondo de Ciencia, Tecnología, e Innovación del Sistema General de Regalías (Colombia), the Fondo Nacional de Financiamiento para la Ciencia, la Tecnología y la Innovación Francisco José de Caldas, Minciencias, Programa Colombia BIO, Gobernación de Boyacá, through Contract No. FP80740-545–2019.

Declarations

Conflict of interest

The authors declare no competing interests.