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Article

Root-Associated Endophytic and Mycorrhizal Fungi from the Epiphytic Orchid Maxillaria acuminata in a Tropical Montane Forest in Southern Ecuador

by
Stefania Cevallos
1,*,
Paulo Herrera
1,
Johanna Vélez
2 and
Juan Pablo Suárez
1
1
Microbial Systems Ecology and Evolution (MS2E) Research Group, Departamento de Ciencias Biológicas y Agropecuarias, Universidad Técnica Particular de Loja, San Cayetano Alto s/n., C.P., Loja 1101608, Ecuador
2
Carrera de Bioquímica y Farmacia, Universidad Técnica Particular de Loja, Loja 1101608, Ecuador
*
Author to whom correspondence should be addressed.
Diversity 2022, 14(6), 478; https://doi.org/10.3390/d14060478
Submission received: 2 April 2022 / Revised: 25 May 2022 / Accepted: 26 May 2022 / Published: 13 June 2022
(This article belongs to the Topic Fungal Diversity)
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Abstract

:
In natural environments, it has been shown that orchids interact with multiple microorganisms including various species of fungi that colonize their tissues. The diversity of these fungi associated with orchid roots is still being described along with the ecological role they play when interacting with the orchids. In this study, we evaluated the richness and diversity of the endophytic and mycorrhizal fungi associated with the roots of Maxillaria acuminata, a common epiphytic orchid species from a tropical montane forest in southern Ecuador. We characterized the fungal communities by sequencing the ITS2 region of the nrDNA with Illumina MiSeq technology. In total, 843 fungal OTUs were uncovered using a 97% sequence similarity. The highest percentage of OTUs belonged to the Agaricomycetes class, Basidiomycota. Interestingly, the most frequent trophic guild from the analyzed OTUs was assigned as saprophytic. Also, some groups of orchid mycorrhizal-forming fungi were detected, including members within the families Ceratobasidicaceae, Serendipitaceae, Tulasnellaceae, and in the order Atractiellales. We discuss the potential influence of this diverse group of root-associated endophytic fungi on the development and survival of M. acuminata in the tropical forests of southern Ecuador.

1. Introduction

Plants in the family Orchidaceae harbor a vast array of endophytic fungi in their roots that colonize the velamen and the root cortical tissue [1]. Fungi inhabiting the root velamen are considered opportunists without a clear relationship with the fungi present in the cortical tissue [2,3], and because of this, the velamen is removed during sample preparation in some studies [3,4]. In contrast, the fungi from the cortical tissue are the target of many studies of fungi associated with orchid roots; some of them have a symbiotic relationship with orchids (orchid mycorrhizal fungi) and others can live in the roots without causing either damage or benefits to the host (non-mycorrhizal endophytic fungi) [1,5]. On the one hand, orchid mycorrhizal fungi could increase the water and mineral nutrient absorption in the adult chlorophyllous plants, and in exchange, fungi use the photosynthesis products of the plants [6] or have an increased C supply [7]. On the other hand, the ecological roles of non-mycorrhizal fungi are still unclear (e.g., protective or nutritive effects) [8], moreover, many of these fungal species are still unknown [9].
Orchidaceae is considered one of the largest families of vascular plants and is found in almost every ecosystem worldwide [10]. Although a great number of orchid species are found in Ecuador, their survival is endangered due to climate change but also because of anthropic disturbances [11] such as habitat loss due to deforestation, soil erosion, and illegal commerce [12]. Even though they produce several thousand seeds in one pod, they are small and lack a nutrient supply for germination [6]. Maxillaria acuminata Lindl. is a miniature-sized orchid (~2.85 cm flower size), native to tropical South America that is distributed between 400 and 2500 m a.s.l. and mostly found growing as epiphytes but they can be terrestrial or lithophytic [13]. In Ecuador, this species has been reported in all continental regions [14].
Although the diversity and community assembly of root-associated endophytic fungi from epiphytic orchids are poorly understood, several studies have reported that members of Ascomycota and Basidiomycota are the dominant groups (e.g., [15,16]). Therefore, it is necessary to identify and classify the fungal diversity associated with epiphytic orchids [17] as a baseline for future investigations into the potential roles these fungi could be playing in the plant’s lifecycle [8].
With current advances in molecular technologies such as next-generation sequencing (NGS), a large number of orchid endophytic and mycorrhizal fungi have been identified, offering more precise inferences in regard to fungal biogeography, ecology, and community structure [18]. With NGS, we can achieve more precise detection of a large number of fungal operational taxonomic units (OTUs) based on the amplification of the nuclear ribosomal internal transcribed spacer DNA region (nrDNA, ITS) [19,20]. These methods can help to identify the structural patterns of fungal communities that normally are undetected with first-generation sequencing. In contrast to molecular technologies, studies based on fungal isolation on media are limited by the ability of fungi to grow in cultures, therefore, reducing the estimation of the existing diversity. However, this technique is necessary for some in vitro tests and other experiments [21].
To contribute to the understanding of the composition and the structural patterns of mycorrhizal and endophytic fungal communities, the colonization of the roots of M. acuminata was assessed. We used metabarcoding via the Illumina MiSeq technology to amplify the fungal ITS2 region in order to (1) identify mycorrhizal and root-associated endophytic fungi and (2) determine the potential ecological role of root-associated endophytic fungi from M. acuminata.

2. Materials and Methods

2.1. Study Site and Sample Collection

The study was carried out in the site known as “Curva Misteriosa” (3°59′ S, 79°08′ W), located 22 km from the Loja-Zamora road, Zamora-Chinchipe province, in the Podocarpus National Park (PNP), with an elevation range from 2000 to 2050 m a.s.l. The site is classified as a tropical montane forest, characterized by a steep slope (51%), with tree heights ranging between 5 to 8 m, a mean annual temperature of 20.8 °C, and mean annual precipitation of 2193 mm [22,23].
In October 2012, seven flowering individuals of Maxillaria acuminata were sampled in an area of approximately 1 ha (no other individuals of M. acuminata were identified in the study area). Three roots in direct contact with the phorophyte bark were collected per individual.

2.2. Root Analysis

Transversal sections from the roots of each plant were cut using a scalpel. Sections were stained with methyl blue 0.05% solution in lactic acid for 3 min and examined under an Axiostar plus microscope (Carl Zeiss, Göttingen, Germany) to detect the presence of fungal pelotons.
The root samples with evidence of fungal endophytic hyphae and pelotons were sterilized by immersion in a solution of 70% ethanol for 30 s, then in a solution of 3% sodium hypochloritefor for 5 min. Finally, root samples were rinsed with sterile distilled water for 3 min [17]. The velamen was then removed, leaving only the cortical tissue. For each sample, two to three pieces (approx. 2 cm each) of root sections were washed with sterile distilled water and stored in 1.5 mL microtubes for immediate DNA extraction.

2.3. Molecular Analysis

DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions. The ITS2 region of nrDNA was amplified using the primer pair ITS86F [24] and ITS4 [25]. These primers are helpful in recovering a vast array of mycorrhizal and root-associated endophytic fungi in orchids [19,26,27,28,29]. The PCR amplifications were carried out in a total reaction volume of 25 µL, which contained 21.5 µL of Platinum PCR Supermix (Invitrogen, Carlsbad, CA, USA), 0.5 µL of each primer, 0.5 µL of 1% BSA, and 2 µL of total DNA. The PCR was run under the initial denaturalization at 95 °C for 5 min, followed by 35 cycles, each consisting of 30 s at 94 °C, 30 s at 55 °C, 2 min at 72 °C, and a final extension at 72 °C for 10 min. All amplifications were tested on 1% agarose gels stained with GelRed (Biotium, Hayward, CA, USA). These amplicons were purified using the Kit Wizard SVClean (PROMEGA, Madison, WI, USA), and concentrations were determined using a NanoDrop 2000c spectrophotometer (Thermo Scientific, Wilmington, NC, USA). Amplicons were sequenced using Illumina MiSeq in the IMGM laboratory (Munich, Germany).

2.4. OTU Delimitation

Using the raw data from the sequencing, we delimited the OTUs using the software UPARSE version 8.0.1623 [30], as suggested in previous studies [19]. First, we used the command “fastq_mergepairs” to merge the obtained sequences with each primer in one consensus sequence. Quality filtering was done using the command “fastq_filter” with a margin of error of a maximum of 0.5. Singleton sequences were discarded using “derep_fulllength”, and sequences with 97% homology were grouped as OTUs using the command “cluster_otus”.
Once the OTU sequences were obtained, those with less than 240 pairs of bases as well as the chimerical OTUs were discarded. Each OTU was classified taxonomically using the database BLAST [31]. Only OTUs corresponding to fungi were kept and deposited in GenBank (Bioproject PRJNA794265). The MYCOBANK database [32] was used to enrich the OTUs’ taxonomic group assignment. These OTUs were used to describe the community of mycorrhizal and endophytic fungi associated with M. acuminata roots.

2.5. Data Analysis

The richness of root-associated mycorrhizal and endophytic fungi associated with M. acuminata was evaluated using the software EstimateS 9.1.1 [33]. We calculated the accumulation curve with confidence intervals at 95%, adjusting them to Clench’s equation using Statistica (StatSoft, Tulsa, OK, USA). The asymptote of the curve was calculated as a/b, where a and b are both Clench´s curve parameters [34]. Additionally, the sampling effort was measured from the estimated proportion of OTUs [34].
Finally, to assign the potential trophic strategies, the taxonomically defined OTUs were compared to those of the FunGuild v1.0. database [35], which suggests the ecological group to which each OTU belongs.

2.6. Phylogenetic Analysis and OTU Delimitations of Orchid Mycorrhizal Fungi

Sequences of potential mycorrhizal fungal OTUs were compared with the GenBank database using BLAST [31] to identify the closest matching sequences to the ITS2 region. Four datasets were created, comprising sequences closest to members of Tulasnellaceae, Ceratobasidiaceae, Serendipitaceae (previously known as Sebacinales clade B), and Atractiellales that, according to previous studies, were documented as mycorrhizal fungi of epiphytic orchids [17,36,37].
Sequences of each dataset were aligned using MAFFT Ver. 6.602b [38] under the Auto option. Resulting alignments were evaluated visually in SEAL Ver. 2.0 and BLAST [31] to eliminate remaining chimeric sequences from further analyses. For each alignment, we performed a Bayesian approach based on Markov chain Monte Carlo (B/MCMC) analysis in MrBayes Ver. 3.1 [39] under the GTR+I+G model, including two runs each, involving four incrementally heated Markov chains over 4,000,000 generations using random starting trees [40,41,42]. Trees were sampled every 100 generations resulting in 40,000 trees from which the last 24,000 were used to construct a 50% majority-rule consensus tree, enabling the use of Bayesian Posterior Probabilities (BPP) as node support. We considered well-supported clades to be those with posterior probabilities equal to or greater than 0.90 [43]. The final consensus trees were visualized using FigTree Ver. 1.4.3. [44].
Finally, we recalculated OTUs based on the sequence similarity of the ITS2 region. A table of p-distances was calculated using PAUP 4.0b10 and sequences were grouped with OPTSIL [45] at 96% (Tulasnellaceae) and 97% (Ceratobasidiaceae, Atractiellales y Serendipitaceae) threshold sequence similarity.

3. Results

3.1. Diversity of Mycorrhizal and Root-Associated Endophytic Fungi

Raw data libraries corresponding to the seven individuals sampled are available in the Sequence Read Archive (SRA) database under the codes SRX14024162, SRX14024143, SRX14024142, SRX14024141, SRX14024140, SRX14024139, and SRX14024138. From these, the sequencing of the root samples yielded a total of 208,128 good quality-filtered sequences, with a length of ≥240 bp. These sequences were grouped into a total of 843 OTUs, using a 96% or 97% sequence similarity threshold. The calculated accumulation curve did not achieve the asymptote (Supplementary Material Figure S1), which is estimated at 2720 OTUs, meaning at least 299 samples of M. acuminata need to be collected in order to achieve 95% of the estimated fungal OTUs.
The mycorrhizal and root-associated endophytic fungi identified in M. acuminata included 392 OTUs (46.50%) corresponding to Ascomycota, 218 OTUs (25.86%) in Basidiomycota, 23 OTUs (2.72%) in Glomeromycota, 5 OTUs (0.59%) in Chytridiomycota, 3 OTUs (0.36%) in Mucoromycota, and 2 OTUs (0.24%) in Mortierellomycota. The remaining 200 OTUs (23.72%) were fungi unassigned to a phylum (Figure 1).
The most OTU-rich class of Ascomycota was Dothideomycetes with 101 OTUs (25.8%), followed by Sordariomycetes with 92 OTUs (23.5%), Leotiomycetes with 66 OTUs (16.8%), and Eurotiomycetes with 52 OTUs (13.3%) (Figure S2). Other classes of Ascomycota were detected but in low numbers (Figure S2).
Agaricomycetes were the most OTU-rich class from Basidiomycota, with 117 OTUs (53.7%), followed by Tremellomycetes with 38 OTUs (17.4%), Microbotryomycetes with 15 OTUs (6.9%), and others in low numbers (Figure S3). The mycorrhizal OTUs corresponded to 2.72% of the total identified OTUs and were represented by the orders Atractiellales with five OTUs and Cantharellales with 10 OTUs, and the family Serendipitaceae had 18 OTUs.
We identified 92 OTUs that corresponded to 77 fungal families, where Nectriaceae (Ascomycota) was the most abundant with 5 OTUs, followed by Aspergillaceae, Bionectriaceae, Glomerellaceae, Icmadophilaceae, Polyporaceae, and Septobasidiaceae with 3 OTUs each.

3.2. Ecological Roles (Trophic Guilds)

It was possible to assign a trophic guild using the FunGuild v1.0. database to 157 mycorrhizal and endophytic fungi identified at the species level. The analyzed OTUs were assigned to the following trophic guilds: saprophytes with 66 OTUs (42%), pathotrophs with 25 OTUs (16%), pathotroph-saprophytes-symbiotes with 20 OTUs (13%), pathotroph-saprophytes with 15 OTUs (10%), symbiont with 8 OTUs (5%), pathotroph-symbiont with 3 OTUs (2%), saprophytes-symbiont with 1 OTU (1%), and 19 OTUs (12%) with an unknown trophic guild (Figure 2). For the remaining 686 root-associated endophytic fungal OTUs, there is no taxonomic information at the species level; their ecological role remains unclear.

3.3. Phylogenetic Analysis of the Orchid Mycorrhizal Fungi

The phylogenetic analysis of the datasets corresponding to Tulasnellaceae, Ceratobasidiaceae, Serendipitaceae, and Atractiellales resulted in 5, 5, 18, and 5 OTUs, respectively (Figures S4–S7). Within the Tulasnellaceae dataset, OTUs T1, T2, and T3 were grouped with sequences from Tulasnellaceae obtained from other orchid species around the world (Figure S4). Sequences of Ceratobasidiaceae formed 5 OTUs (OTU C1–C5), which were grouped in distinct well-supported clades. OTU C1 belonged to the genus Thanatephorus (Figure S5). OTUs C2, C3, and C4 were similar to species of Rhizoctonia, whereas OTU C5 was similar to sequences of Thanatephorus (Figure S5). The Serendipitaceae phylogenetic analysis resulted in 18 OTUs (OTU S1–S18), all of them grouped in well-supported clades into the family Serendipitaceae (previously called Sebacinales clade B) (Figure S6). All OTUs were similar to sequences previously detected in orchid mycorrhizae (Figure S6). Finally, sequences of Atractiellales were grouped into 5 OTUs (OTU A1–A5; Figure S7). OTUs A1 to A4 were similar to the sequences of Atractiellales obtained from other orchid species from southern Ecuador (Figure S7). OTU A1 was closest to the genus Helicogloea, whereas OTU A5 was similar to the genus Bourdotigloea (Figure S7).

4. Discussion

M. acuminata mycorrhizal and root-associated endophytic fungi have been evaluated for the first time in this study. The whole mycorrhizal and root-associated endophytic fungi included 810 OTUs (95.8%) that have no clear role in orchid ecology, and the remaining 33 OTUs (4.2%) corresponded to fungi previously reported as orchid mycorrhizal fungi, with Basidiomycota members of Ceratobasidicaceae, Serendipitaceae, Tulasnellaceae, and Atractiellales. Ascomycota was the most OTU-rich group, most of them assigned as saprophytes.

4.1. Richness of Mycorrhizal and Endophytic Fungi

A great diversity of mycorrhizal and endophytic fungi was detected in association with the roots of M. acuminata. Despite the few samples, 843 OTUs were recovered, with a threshold of 96% or 97% sequence similarity, which is the commonly used threshold for the clustering of OTUs of fungi [15,27]. Using next-generation sequencing was advantageous for the amplification of fungi associated with the root tissue because far more sequences per sample were obtained compared to traditional methods (e.g., Sanger sequencing) [17,36], leading to a more accurate characterization of fungal communities [46].
As mentioned, our results did not reach the asymptote of the accumulation curve. This was due to the low number of orchid samples caused by the difficulty of identifying M. acuminata when plants are not in bloom and due to the rarity of this species in the study area, suggesting a vast array of fungi are still undetected. Nevertheless, it is necessary to explore additional populations because the mycorrhizal and endophytic fungal diversity associated with M. acuminata roots can be affected by multiple biotic and abiotic factors, including the site where the orchids grow [19] and nutrient availability [47]. The capability of plants to associate with multiple fungi at the same time gives them the advantage of increased resistance to environmental conditions and allows them to obtain supplementary nutrients [48,49]. More studies about the factors that could affect the composition of orchid-associated fungal communities are needed for deeper ecological insights.
Most of the detected endophytic fungi in this study (~50% of the fungi) belonged to the phylum Ascomycota, mainly in the class Dothideomycetes. These results are congruent with previous studies [15,50], likely due to the advantages of next-generation sequencing technology. In contrast, previous studies using Sanger sequencing reported Sordariomycetes as the most abundant class of endophytic fungi associated with other epiphytic and terrestrial orchid species from tropical and temperate areas (e.g., [8,51]).
The functional knowledge of endophytic fungi throughout the life cycle of orchids is limited [52]. Further investigations are crucial to clarify if endophytes undergo ecological and physiological activity in orchids or break down dead cells without affecting orchid health. It has been shown that some endophytes produce active compounds that could be beneficial for orchids that face environmental stress [51].

4.2. Richness of Orchid Mycorrhizal Fungi

In addition to endophytic fungi, we also detected fungi cataloged as orchid mycorrhizal fungi. The richest group was Serendipitaceae (Basidiomycota), and in a lower proportion were members of Tulasnellaceae, Ceratobasidiaceae, and Atractiellales. These results are likely limited by the area where the samples of M. acuminata were collected because our findings are similar to those of previous studies carried out in the nearby tropical montane forest reporting the same orders of fungi associated with various species of tropical orchids [15,17,19,36,37,43,53,54,55,56].
Our results are congruent with previous studies and show that Serendipitaceae members were the most abundant mycorrhizal fungi associated with epiphytic orchids in tropical forests in southern Ecuador [19,56]. Eighteen OTUs were related to sequences from Serendipitaceae, which are widely recognized as mycobionts inhabiting tropical orchids [36]. Moreover, our results supported the hypothesis of the wide distribution of this fungal family since many of the observed Serendipitaceae OTUs were shared between the studied geographic regions of southern Ecuador [15] and the rest of the world [57,58].
In the case of Tulasnellaceae, the phylogenetic analysis allowed us to consider OTUs T1 to T3 as symbionts of epiphytic orchids [17,56,59]. Conversely, OTUs T4 and T5 were not grouped into clades with reference sequences assigned to orchid mycorrhizal fungi, and therefore, we are not confident in their function as mycorrhizae.
The well-supported clades from the family Ceratobasidiaceae, OTUs C1 and C3 to C5, included sequences previously reported as fungi associated with epiphytic and terrestrial orchids from tropical and temperate areas of Ecuador [21,56], the United States [60], and Chile [61], supporting their mycorrhizal potential.
The phylogenetic analysis of Atractiellales showed that OTU A1 to OTU A4 were exclusively reference sequences from tropical orchids in Ecuador [37,54]. The reports of Atractiellales in association with orchids are becoming more frequent because of the use of more accurate primers and other molecular tools [19,37,54,55,62].

4.3. Ecological Roles (Trophic Guilds)

The largest trophic guild found in the root fungal community of M. acuminata was saprophytes, which concurs with the results of previous investigations in tropical forests [15]. Similar results were also reported for orchids in premontane wet forests [62] and neotropical forests [63]. In this study, 686 OTUs remain without classification at the species level, and it is possible that fungi with different ecological roles are present. Additionally, other fungal groups, such as hyperparasitic fungi [64], are not considered in the FunGuild database, thus, the trophic guilds assigned to our OTUs are preliminary results and need to be investigated further.
The taxonomic assignment or characterization of mycorrhizal and endophytic fungi still presents inconsistencies due to a lack of available data, incomplete data, or unidentified OTUs [51,65]; therefore, it is of great importance to improve public DNA databases and add sequences of fungi identified up to the species level [66], in addition to identifying and describing new fungal species.

5. Conclusions

In conclusion, of the fungi cataloged as mycorrhizal, Serendipitaceae was the group with the highest richness. Meanwhile, we found a wide richness of mycorrhizal and endophytic fungi associated with M. acuminata in southern Ecuador, including a high richness of Agaricomycetes (Basydiomycota) OTUs. A high number of these OTUs were identified as saprophytes, however, most of them did not have a clear role in the orchid life cycle. The results support that the great diversity of fungi is a potential source of associated mycobionts used for the conservation of threatened species.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/d14060478/s1. Figure S1: Species accumulation curve showing the number of OTUs of mycorrhizal and root-associated endophytic fungi as a function of the sampled individuals of Maxillaria acuminata (continuous black line) with confidence intervals at 95% (continuous grey line). Figure S2: Operational taxonomic units (OTUs) frequency distribution from Ascomycota phylum. Figure S3: Operational taxonomic units (OTUs) frequency distribution from phylum Basidiomycota. Figure S4: Phylogenetic tree of the ITS2 nrDNA of sequences derived from the mycorrhizal group Tulasnellaceae from Maxillaria acuminata and closest matching sequences available at GenBank. The phylogenetic tree was inferred with an MCMC analysis. Codes from OTU T1–OTU T5 (marked in grey color) indicate the Tulasnellaceae OTUs obtained from Maxillaria acuminata and determined by 96% of sequence similarity of ITS2 region. Values close to the nodes correspond to BPP analysis, only values larger than 0.5 are shown. The tree was rooted with Multiclavula mucida. Figure S5: Phylogenetic tree of the ITS2 nrDNA of sequences derived from the mycorrhizal group Ceratobasidiaceae from Maxillaria acuminata and closest matching sequences available at GenBank. The phylogenetic tree was inferred with an MCMC analysis. Codes from OTU C1–OTU C5 (marked in grey color) indicate the Ceratobasidiaceae OTUs obtained from Maxillaria acuminata and determined by 97% of sequence similarity of ITS2 region. Values close to the nodes correspond to BPP analysis, only values larger than 0.5 are shown. The tree was rooted with Serendipita sp. Figure S6: Phylogenetic tree of the ITS2 nrDNA of sequences derived from the mycorrhizal group Sebacinales from Maxillaria acuminata and closest matching sequences available at GenBank. The phylogenetic tree was inferred with an MCMC analysis. Codes from OTU S1–OTU S18 (marked in grey color) indicate the Serendipitaceae OTUs obtained from Maxillaria acuminata and determined by 97% of sequence similarity of ITS2 region. Values close to the nodes correspond to BPP analysis, only values larger than 0.5 are shown. The tree was rooted with Geastrum saccatum. Figure S7: Phylogenetic tree of the ITS2 nrDNA of sequences derived from the mycorrhizal group Atractiellales from Maxillaria acuminata and closest matching sequences available at GenBank. The phylogenetic tree was inferred with an MCMC analysis. Codes from OTU A1–OTU A5 (marked in grey color) indicate the Atractiellales OTUs obtained from Maxillaria acuminata and determined by 97% of sequence similarity of the ITS2 region. Values close to the nodes correspond to BPP analysis, only values larger than 0.5 are shown. The tree was rooted with Infundibura adhaerens.

Author Contributions

Conceptualization, J.P.S. and S.C.; methodology, J.P.S. and S.C.; software, P.H. and S.C.; validation, P.H. and S.C.; formal analysis, P.H., J.V. and S.C.; data curation, P.H. and S.C.; writing—original draft preparation, J.V. and S.C.; writing—review and editing, J.P.S., P.H. and S.C.; supervision, J.P.S.; project administration, J.P.S.; funding acquisition, J.P.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by ACADÉMIE DE RECHERCHE ET D’ENSEIGNEMENT SUPÉRIEUR WALLONIE-BRUXELLES (ARES), within the frame of a PRD project.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

All data are available in this article.

Acknowledgments

We would like to thank Alberto Mendoza for his contribution to the fieldwork. Additionally, we thank Pablo Ramírez Castillo and Kelsey Huisman for their contribution to the English review.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Figure 1. OTU frequencies of the mycorrhizal and root-associated endophytic fungi associated with Maxillaria acuminata.
Figure 1. OTU frequencies of the mycorrhizal and root-associated endophytic fungi associated with Maxillaria acuminata.
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Figure 2. Relative frequency of the mycorrhizal and root-associated endophytic fungal OTUs corresponding to the different trophic guilds as assigned by FunGuild.
Figure 2. Relative frequency of the mycorrhizal and root-associated endophytic fungal OTUs corresponding to the different trophic guilds as assigned by FunGuild.
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Cevallos, S.; Herrera, P.; Vélez, J.; Suárez, J.P. Root-Associated Endophytic and Mycorrhizal Fungi from the Epiphytic Orchid Maxillaria acuminata in a Tropical Montane Forest in Southern Ecuador. Diversity 2022, 14, 478. https://doi.org/10.3390/d14060478

AMA Style

Cevallos S, Herrera P, Vélez J, Suárez JP. Root-Associated Endophytic and Mycorrhizal Fungi from the Epiphytic Orchid Maxillaria acuminata in a Tropical Montane Forest in Southern Ecuador. Diversity. 2022; 14(6):478. https://doi.org/10.3390/d14060478

Chicago/Turabian Style

Cevallos, Stefania, Paulo Herrera, Johanna Vélez, and Juan Pablo Suárez. 2022. "Root-Associated Endophytic and Mycorrhizal Fungi from the Epiphytic Orchid Maxillaria acuminata in a Tropical Montane Forest in Southern Ecuador" Diversity 14, no. 6: 478. https://doi.org/10.3390/d14060478

APA Style

Cevallos, S., Herrera, P., Vélez, J., & Suárez, J. P. (2022). Root-Associated Endophytic and Mycorrhizal Fungi from the Epiphytic Orchid Maxillaria acuminata in a Tropical Montane Forest in Southern Ecuador. Diversity, 14(6), 478. https://doi.org/10.3390/d14060478

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