Rhizosphere 7 (2018) 49–56

Contents lists available at ScienceDirect

Rhizosphere
journal homepage: www.elsevier.com/locate/rhisph

Fungal diversity in the roots of four epiphytic orchids endemic to Southwest T

Mexico is related to the breadth of plant distribution
María de los Angeles Beltrán-Namboa, Miguel Martínez-Trujilloa, Juan Carlos Montero-Castrob,
⁎
Rafael Salgado-Garcigliac, Joel Tupac Otero-Ospinad, Yazmín Carreón-Abuda,
a
Genetic and Microbiology Laboratory, Biology Faculty, Universidad Michoacana de San Nicolás de Hidalgo, Francisco J. Múgica s/n, Col. Felicitas del Río, C.P. 58060
Morelia, Michoacán, Mexico
b
Plants´ Molecular Systematics Laboratory, Biology Faculty, Universidad Michoacana de San Nicolás de Hidalgo, Francisco J. Múgica s/n, Col. Felicitas del Río, C.P.
58060, Mexico
c
Vegetal Biotechnology Laboratory, Chemical-Biological Researchs Institute, Universidad Michoacana de San Nicolás de Hidalgo, Francisco J. Múgica s/n, Col. Felicitas del
Río, C.P. 58060, Mexico
d
Biological Sciences Department, Agricultural Sciences Faculty, Universidad Nacional de Colombia, Palmira sede, Colombia

A R T I C LE I N FO A B S T R A C T

Keywords: Fungi associated with plant roots are important for different plant processes, including germination and de-
Nonmycorrhizal fungi velopment. In Mexico, the knowledge about fungal species that interact with orchid roots and their possible roles
Mycorrhizal fungi in plant success and dispersion in different geographical locations is still very limited. For this reason, this work
Shannon-Wiener index aimed to determine the community composition and diversity of fungi associated with orchid roots at seven sites
Beta diversity
in the Transversal Volcanic Belt region in Michoacán, México. The roots of four endemic orchid species were
Fungal diversity
Orchid
analyzed: Laelia autumnalis, L. speciosa, Euchile citrina and P. squalida. In total, 71 isolates were obtained and
Mycorrhiza classified into 20 genera, including one mycorrhizal genus (Tulasnella) and 19 genera classified as basidiomy-
Endemic cetes and ascomycetes, such as Coprinus, Trichoderma and Xylaria. The diversity among different orchid species
and sampling sites was compared using the Chao 1, Shannon-Wiener and Whittaker indexes. In this work, species
such as L. autumnalis, L. speciosa and P. squalida were found in different sites and seemed to be more generalists
with regard to endophytes; they were typically associated with and found to be in symbiosis with species from
the orchid mycorrhizal genus Tulasnella. Other orchids seem to be more specific, such as E. citrina, which showed
the lowest diversity index and poor local distribution. This situation has been reported for species with a high
degree of endemism.

1. Introduction antagonistic relationships with other organisms to provide advantages

A large number of fungi associated with the Orchidaceae family are
described in some reviews (Dearnaley et al., 2012; Liu, et al., 2010;
Rasmussen and Rasmussen, 2007; Xiaoya et al., 2015). However, re-
search about fungal diversity in orchid roots is mainly focused on the
fungi forming orchid mycorrhiza (OM), due to their primordial role in
seed germination and their contribution to plant development and
nutrition (Freudenstein and Chase, 2015; Rasmussen et al., 2015;
Wright et al., 2011). Recently, it was reported that beneficial associa-
tions with other endophytic fungi might also coexist (Herrera et al.,
2017), so interest in the nonmycorrhizal fungi of orchids has increased,
due to their possible physiological functions, the synergistic or
to plants and their potential as a source of bioactive compounds
(Bayman and Otero-Ospina, 2006; Khamchatra et al., 2016;
Nontachaiyapoom et al., 2011).
In other plant families, fungi from both groups (mycorrhizal and
nonmycorrhizal) present in the roots support water and nutrient ab-
sorption, increase plant resistance to various stresses by releasing me-
tabolites, and stimulate germination and seedling development. For this
reason, these organisms have a profound impact on the evolution,
ecology, health, structure and diversity of plant communities (Jiménez
et al., 2011; Mapperson et al., 2014; Otero-Ospina and Bayman, 2009;
Otero et al., 2011; Ye et al., 2014). Therefore, knowledge about the
different fungi associated with orchid roots can provide a better

⁎
Corresponding author.
E-mail addresses: angelesb2008@gmail.com (M.d.l.A. Beltrán-Nambo), codigogenetico@gmail.com (M. Martínez-Trujillo),
cestrum2003@yahoo.com.mx (J.C. Montero-Castro), rafael.salgadogarciglia@gmail.com (R. Salgado-Garciglia), jtoteroo@unal.edu.com (J.T. Otero-Ospina),
ycabud@gmail.com, ycabud@umich.mx (Y. Carreón-Abud).

https://doi.org/10.1016/j.rhisph.2018.07.001
Received 31 May 2018; Received in revised form 22 July 2018; Accepted 23 July 2018
Available online 24 July 2018
2452-2198/ © 2018 Elsevier B.V. All rights reserved.
M.d.l.A. Beltrán-Nambo et al.
perspective on the interactions that occur in orchids’ natural habitats
(Gamboa-Gaitán, 2006) and how these fungi can contribute to orchid
survival and adaptation (Swarts and Dixon, 2009).
Some works have pointed out that the diversity of mycorrhizal and
nonmycorrhizal fungi in roots changes according to the orchid species
and life cycle and that different patterns can be present. Some orchids
maintain an association with a single mycobiont, while others can ex-
pand their associations or can change symbionts during the transition
from the juvenile to adult stage or during some environmental dis-
turbance (Rasmussen et al., 2015). For this reason, it is considered that
the study of endophytes that can stimulate the development of the
embryo and promote plant growth in combination with mycorrhizal
fungi or on their own is a topic that remains little explored, although it
is very promising (Teixeira et al., 2015).
One of the central challenges facing ecology and conservation
biology is the identification of factors influencing the spatial distribu-
tion and abundance of rare or endangered plant species (Waud et al.,
2014). Additionally, little is known about the physiological role and
degree of association or specificity of fungi in the roots of epiphytic
orchids compared with those of terrestrial species. However, most
orchids are epiphytic and tropical or subtropical (Ávila-Díaz et al.,
2013). At a global scale, it is mentioned that mycorrhizal fungi are
found less consistently, but they seem to be strongly associated with
epiphytic orchids compared to their terrestrial counterparts (Bailarote
et al., 2012; McCormick et al., 2018). Furthermore, many of the fungi in
these plants are not mycorrhizal endophytes, and these make up a
largely overlooked component of fungal biodiversity within roots (Yuan
et al., 2009).
In Mexico, the depletion of large areas of pine, oak and mesophilic
mountain forests have caused the loss of entire orchid populations,
whose major diversity is found in this kind of ecosystem (Gual-Díaz and
Rendón-Correa, 2014). A significant proportion of orchid species are
mainly found in the Transversal Neovolcanic Belt mountain chain, and
they are threatened due to the conversion of native forests to orchards
for the cultivation of avocado and other plants (CONABIO and SUMA,
2005), causing approximately 15% of the orchid species to be reported
Fig. 1. Sampling site locations. The locations of the seven collection sites and the limits of the regions (municipalities) in which they are located are shown. (Dark
green = physiographical province; pale green = municipalities; black circles = sampled sites) (Map elaborate using QGIS program V. 9) (QGIS Development Team,
2009).
50
Rhizosphere 7 (2018) 49–56
at risk category at this country (SEMARNAT, Secretaría del Medio
Ambiente y Recursos Naturales, 2010). Because of this, it is important
to develop new strategies for orchid conservation. One of these con-
servation strategies is the use of fungal symbionts that could increase
germination rates and stimulate the orchids’ development and adaptive
capacity, because it was reported that some of the orchid distribution is
influenced by the disturbance degree of sites where they develop, the
species adaptability and their association with widely dispersed fungi,
among other factors (Beltrán-Nambo et al., 2012; Oliveira et al., 2013).
In this country, more than 40% of orchid species are endemic, and
their mycorrhizal and nonmycorrhizal fungi associations have been
poorly studied, although some works have emphasized that the abun-
dance, distribution and heterogeneity of symbiont fungi are important
for orchid adaptation and distribution on a local scale (McCormick
et al., 2018). Therefore, this research aimed to compare the diversity of
fungi associated with the roots of four epiphytic orchid species endemic
to Mexico with those of populations threatened due to their ornamental
and artisanal use: Laelia autumnalis (Lex.) Lindl., L. speciosa (Kunth)
Schltr, Euchile citrina (Lex.) Withner and Prosthechea squalida (La Llave
y Lex.) Soto Arenas and Salazar. Here, we also analyze whether the
presence of a high fungal diversity (generalist association) is related to
a wider orchid distribution or if a more efficient association with fewer
fungi (specific association) is a factor that allows greater adaptive
success for orchids.
2. Materials and methods
2.1. Plants and sample sites
Sampling was performed in seven sites located in different muni-
cipalities of the physiographic province called the Transversal Volcanic
Belt in the state of Michoacán (Fig. 1). Sites were selected based on
distribution records of the four selected species in the state (Instituto de
Ecología AC-INECOL) and field trips to select sites where plants popu-
lations were located. The reference data for the sampling sites and the
orchid species found in each one are shown in Table 1.
M.d.l.A. Beltrán-Nambo et al. Rhizosphere 7 (2018) 49–56

Table 1
Collection sites. Reference data for the different collection sites and orchid species analyzed in each of the sampling points. (Types of vegetation observed and
corroborated according to INEGI, 1985).
Collection sites Reference data (coordinates) Vegetation and altitude Orchid

# Municipality Nearby locality

1 001 Acuitzio Acuitzio N 19°29´36.35´´ Forest of pine- oak, cedar and oyamel. 2145 msnm. L. autumnalis
W 101°20´49.73´´

2 032 Erongaricuaro Oponguio N 19°38´42´´ Forest of oak 2114 msnm. L. autumnalis

W 101°39´43.9´´ L. speciosa

3 053 Morelia Cuanajillo N 19°38´34.18´´ Forest of oak 2200 msnm. L. speciosa

W 101°20´ 45.09´´

4 073 Quiroga Sta. Fe de la Laguna N 19°41´37.37´´ Forest of pine-oak and oak. 2281 msnm L. speciosa
W 101°33´45´´

5 100 Tzintzuntzan Cucuchucho N 19°36´18.4´´ Mixed forest of pine, oak and cedar, some nopal and copal. 2049 msnm L. autumnalis
W 101°37´18.62´´

6 016 Coeneo Carretera N 19°43´02.16´´ Forest of pine-oak. 2239 msnm L. autumnalis

W 101°37´45.53´´ P. squalida

7 111 Ziracuaretiro San Andrés Coru N 19°27´41.5´´ Mixed pine-oak forest, deciduous tropical forest ceiba, cedar, parota, tepeguaje. L. autumnalis
W 101°56´55.2´´ 1700 msnm. E citrina
P. squalida

The orchids Laelia speciosa and Euchile citrina are listed as species at
risk by the Official Mexican Norm 059 (SEMARNAT, 2010) and as
priority species for conservation by the National Commission for
Knowledge and Use of Biodiversity (CONABIO, 2012). In the case of L.
autumnalis and P. squalida, their current conservation status is un-
known. Orchids were located in different phorophytes or substrata,
depending on the species, but mainly on oak (Quercus desertícola Trel.,
Quercus sp. L.). In the case of L. autumnalis, in some areas, it was found
growing on prickly pear (Opuntia ficus-indica Linnaeus Miller), cedar
(Callitropsis lusitanica (Mill.) D.P. Little) and ash (Fraxinus sp. Tourn. Ex
L.), together with P. squalida, and in other sites growing on rocks.
Samples were collected during the flowering season in the years
2014–2016. Their identity was confirmed by INECOL specialists from
photographs and/or flower collection (Fig. A1a–f, Supplementary ma-
terial). The taxonomic names given for the orchid species described in
this work are based on the accepted nomenclature of the Kew Royal
Garden, reported in "The Plant List" (2013), which was consulted
electronically.
A 100 m2 quadrant was established at each collection site and di-
vided into 20 × 33 m areas. A single tree with plants of any of the
chosen species was selected (Fig. A1g, Supplementary material), for a
total of 15 trees sampled per site (when this was possible; not all orchid
species were present in all the sites). Subsequently, three to five roots of
each orchid species were randomly chosen from each tree for a total of
63 roots analyzed from L. autumnalis, 75 from L. speciosa, 30 from P.
squalida and 10 from E. citrina. The roots were properly labeled and
transported to the laboratory under refrigeration.
2.2. Isolation
Roots were cut transversely using a scalpel to obtain 1 cm length
fragments. Then, a 1 mm transversal section from each fragment was
mounted in polyvinyl-lactoglycerol alcohol and observed under an
optical microscope to detect the presence of hyphae or coils. For each
root, three to five colonized fragments were selected for fungal isola-
tion.
The selected fragments were superficially disinfected following the
methodology of Ortega-Larrocea (2008) using commercial sodium hy-
pochlorite diluted at 10%, followed by an antibiotic solution (2% ery-
thromycin and 1% gentamicin), and rinsed three times with sterile
distilled water. To ensure that isolates represented endophytic or
mycorrhizal fungi, fungal coils and hyphae not forming pelotons were
extracted by dissection, and the velamen and exodermis of selected root
fragments were removed under a stereoscopic microscope and laminar
flow hood (Rasmussen and Whigham, 2002). Sterilized distilled water
drops containing coils or hyphae were dispersed in petri dishes con-
taining fungal isolation culture medium (FM) (Clements, 1988;
Mitchell, 1989). Then, samples were incubated at 26 °C under dark
conditions. For morphologic characterization and molecular determi-
nation of the fungal isolates, hyphae segments were cut and transferred
to Petri dishes with potato dextrose agar (PDA) culture medium and
incubated at 26 °C for two or three days until hyphal growth was ob-
served.
2.3. Morphological characterization
The color of the surface colony in culture media was determined
after 15 days using Munsell (2000) soil color charts. Furthermore, other
macroscopic characteristics of fungal culture were analyzed: brightness,
texture, odor and growth mode (Pereira et al., 2005). Additionally,
growth rates were determined using the Currah et al., (1987) and were
represented in average values based on three replicates per strain. After
30 days, the presence of sclerotia, ascospores, basidiospores or mon-
ilioid cells were analyzed, including their dimensions (Shan et al.,
2002). Fixed preparations on slides were stained with trypan blue and/
or acid fuchsin to measure the length and width of monilioid cells and
spores. A Leica microscope with an integrated camera (Z1000) and the
AMScope V. 3.7 software was used for both measurements. Enzymatic
capacity tests were performed by reaction with polyphenyl oxidases in
culture medium with tannic acid as proposed by Davidson et al. (1938)
and Zelmer (1994). Three petri dishes were inoculated with each strain
obtained from the PDA isolation by placing a mycelium fragment of
1 mm3 in the dish and incubating it at 25 °C for 5–15 days. Subse-
quently, the reaction was observed; those that changed the growth
medium coloration were considered positive.
The isolates were grouped using a WARD hierarchical clustering
analysis based on Euclidean distances that were estimated from the
similarity of both qualitative and quantitative characteristics, including
their coefficient of cophenetic correlation. This procedure was per-
formed using R version 3.3 software (R-Development Core Team,
2008).

51
M.d.l.A. Beltrán-Nambo et al. Rhizosphere 7 (2018) 49–56

2.4. Molecular determination

Peroxidase reaction

Representative isolates of each group obtained from clustering

analysis were selected. From the isolates grown in PDA, a 1 mm3
Negative

Negative
Negative
fragment was cut and placed in potato-dextrose broth medium (PDB)
Positive

and incubated for 15–20 days at 25 °C with a 50 rpm agitation speed

and exposure to light. Fungi grown in PDB medium were liquefied in
Morphological characterization. Principal qualitative and quantitative characteristics of fungal strains obtained from epiphytic orchid roots and classified according to WARD clustering analysis.

150 ml of distilled water for 1 min, and aliquots of 1 ml were placed

No. of nuclei

into a dialysis membrane mounted in vacuum pump (manifold). The

2 or more
2 or more

species identification was made based on the internal transcribed

spacers (ITS) of ribosomal DNA. Total DNA was extracted using the
2

DNeasy Plant Mini Kit (Qiagen) from fresh tissue previously grown in
PDB medium and washed with a vacuum pump. For amplification, the
Growth average rates (mm) n = 268 (P≤ 0.05)

universal primers ITS1 and ITS4 (White et al., 1990) were used, fol-
lowing the protocol suggested by Swarts et al. (2010) but using a DNA
polymerase from the Qiagen company. Sequencing in both directions
(5´-3´ and 3´-5´) was performed at Macrogen (Seul, Korea).
All sequences were edited in Sequencher 5.2.4 (GeneCodes, Ann
Arbor, Michigan, USA), and the identification of similar sequences was
performed using the NCBI database (https://www.ncbi.nlm.nih.gov/)
with MegaBLAST option (Morgulis et al., 2008).
0.9 to 2.5 ± 1.4

1.2 to 2.5 ± 0.7

2.5. Diversity analysis

2 to 3 ± 1.2
3 or more

Fungal diversity was compared among orchid species and among

sampling sites. Alpha richness and diversity were determined using the
nonparametric method Chao 1 (Chao, 1984), Shannon-Wiener index
Spores dimensions (µm)

(Baev and Penev, 1995; Shannon and Weaver, 1949) and Pielou (1981)
species evenness index (J´), which refers to how similar in number each
species in an environment is. Additionally, the sampling effort was
determined by cumulative curves using EstimateS V.9.0 software
8.4–13.9

8.3–13.1

(Colwell, 2013). General beta diversity, which indicates the magnitude

8–12.2

10–15

of change in fungal species composition among all studied orchid spe-

cies and among the seven sites was calculated using the Whittaker
Basidiospore

(1972), with PAST V. 3.17 software (Hammer et al., 2001). Further-

Spore type

Ascospore
Ascospore
Monilioid

more, beta diversity was determined for each pair of orchid species and
each pair of sampling sites by using the same index.

3. Results
Waxy flat to plush, irregular growth
Plush to cottony, radial growth

3.1. Isolation and characterization of fungal isolates

A total of 268 fungal samples were isolated from 83 of the 168

processed roots (49.4%) of four orchid species analyzed. Based on
Plush to cottony
Plush to cottony

morphological characterization, the constructed distance tree generated

Colony aspect

four main groups of isolates (A–D) (Fig. A2 Supplementary material).

Group A included isolates with characteristics similar to those of
orchid mycorrhizal fungi, such as 90° branching angles, a slight con-
striction at the branching point, the absence of sporulation, and mon-
ilioid cell formation, among other features (Table 2). Groups B and C
Gray to olive or sepia (3/1 2.5Y, 6/4 10YR)

included isolates with ascospore formation, and group D included those

Colony color (Munsell catalogue, 2000)

isolates with basidiospore and fruiting body formation in some cases as

White to gray (8/1 2.5Y, 3/1 2.5Y)

its main characteristics (Table 2).

Pale yellow (8/2 to 8/3 5Y)

3.2. Molecular determination

Pale yellow (8/2 5Y)

From 268 isolates obtained, 100 were selected for molecular iden-
tification. All isolates from groups A and D (6 and 12, respectively)
were included; 40 isolates from group C were selected according to
colony similarity, and the remaining samples were taken from group B.
Identities were determined for 71 morphotypes (6 from group A, 31
from group B, 30 from group C and 4 from group D) (Table A1,
Supplementary material); 32 morphospecies were classified, with 18
Subgroups

showing identity at the species level in the GenBank database, ten at

Table 2

genus level and four at the order level. The strains were grouped
D
A

C
B

into three genera of Basidiomycota, including one recognized as an

52
M.d.l.A. Beltrán-Nambo et al. Rhizosphere 7 (2018) 49–56

Table 3
Fungal genera by site and orchid species. Distribution of fungal genera identified in axenic cultures from the species of orchids studied by sampling site and number of
fungal strains obtained from each.
Endophyte Host

L. autumnalis (sites) L. speciosa (sites) P. squalida (sites) E. citrine (sites)

Division Order Genera 1 2 5 6 7 2 3 4 6 7 7 Total

Basidiomycota (3 genera) Agaricales Clitopilus giovanellae 2 2

Coprinus sp. 10 10
Tulasnellales Tulasnella calospora 1 1 1 3

Ascomycota (17 genera) Eurotiales Paecilomyces inflatus 1 1

Glomerellales Colletotrichum sp. 1 1
Helotiales Lachnum sp. 1 3 4
Neofabraea actinidiae 1 1
Neofabraea sp. 1 4 5
Ascomycete sp. 4 3 8 1 1 2 19
Hypocreales Trichoderma atroviride 10 10
Trichoderma rossicum 39 39
Trichoderma viride 2 8 13 4 27
Trichoderma viridialbum 2 1 3
N/D 3 6 3 12
Pleosporales Alternaria alternata 6 6
Preussia minima 5 3 6 14
Preussia cymatomera 2 2
Sordariales Chaetomium nigricolor 6 1 7
Chaetomium sp. 3 2 5
Fusarium tricinctum 1 2 3
Fusarium sp. 4 4
Trichocladium opacum 1 1
Trichocladium sp. 2 2
Ascomycete (uncultured) 2 3 1 2 2 2 12
Xylariales Xylaria sp. 4 2 12 7 3 3 31
Ascovirgaria occulta 16 6 22
Muscodor albus 7 1 8
Nemania sp. 3 4 7
Virgaria nigra 3 3
Xylaria enteroleuca 2 2
Not defined Spegazzinia sp. 1 1

Uncultured N/D 1 1
Morphospecies/site (Total) 5 12 14 69 30 8 71 31 10 13 5

Total of strains 130 110 23 5 268

Total genera 15 11 4 1
Total morphospecies 22 18 7 2

orchid mycorrhizal genus (Tulasnella) and 17 genera of Ascomycota
(Table 3).
3.3. Diversity of fungal isolates
The results for alpha diversity showed that orchid species with the
highest richness and diversity of fungi in their roots were those asso-
ciated with the two species of Laelia. However, the evenness index (J´)
indicated the existence of dominant fungal species in some orchid
species (Fig. 2a).
With the analysis of fungal alpha diversity by sampling site, it was
observed that the highest richness and diversity of fungal species was

Fig. 2. Alpha diversity and shared species. A) Graph of richness and fungal diversity among orchid species, b) Graph of richness and fungal diversity among sampling
sites, c) Venn diagram that shows the number of fungal species shared among orchids. (U = Shannon-W Evenness index).

53
M.d.l.A. Beltrán-Nambo et al.
present in site 7 (San Andrés Coru), where three of the orchids analyzed
were found (L. autumnalis, P. squalida and E. citrina) (Fig. 2b). The
lowest species uniformity according to the evenness index occurred in
site 3 (Cuanajillo).
The general beta diversity for the sampled orchids was 2, according
to the calculated Whittaker index (this parameter does not have a
maximum value). When the beta diversity between pairs of orchid
species was analyzed, it was observed that orchids of the same genus or
those that shared a substrate exhibited the lowest index values (Table
A2, Supplementary material) and share fungal species, as L. autumnalis
with L. speciosa, as is demonstrated in Fig. 2c.
When analysis by sampling site was performed, the global beta di-
versity was 3.4. The pair of sites analyzed with the lowest index values
of fungal species turnover was site 2 and sites 1 and 6 (Table A3,
Supplementary material). L. autumnalis was present at all three sites.
The species accumulation curves indicated that in the case of L.
speciosa, a better sampling effort is required since the number of fungal
species would increase if collection sites were expanded (Fig. A3a,
Supplementary material). Finally, the curve for sampling sites indicated
that the sampling effort was satisfactory since the generated curve
is asymptotic, which indicates that the fungal species number
will remain constant even with a greater number of samples (Fig. A3b,
Supplementary material).
4. Discussion
4.1. Fungal isolation and orchid rarity
Because the identification was performed in axenic cultures of fungi
from orchid roots, it is possible that fungal species were underestimated
compared to a metagenomic approach that includes noncultivable
species. The highest number of fungal isolates obtained in this work
were extracted from Laelia autumnalis and L. speciosa. The first orchid
species was present in five of seven sample sites with large populations,
contrasting with the E citrina species that was only located in site 7 (San
Andrés Coru). Although the number of samples of each orchid was an
important factor in the number of fungi obtained (Kassem and
Nanniperi, 1995), other factors have been reported to influence fungal
isolation, such as special requirements preventing their development in
standard culture media (Garden and Whitbeck, 2007) and sensitivity to
environmental changes (Gadd et al., 2007). Plant species that are cri-
tically dependent on these symbionts are more selective and sensitive
than others during symbiosis establishment, so symbiont sensitivity can
be expected to have a major impact on successful colonization and
establishment, and thus ultimately on species rarity (Bonnardeaux
et al., 2007; Otero et al., 2013; Roche et al., 2010; Swarts et al., 2010;
Waud et al., 2017). E. citrina is an at-risk species that has experienced
habitat deterioration, and only two different fungi morphospecies were
isolated from its roots.
4.2. Orchid fungal identity and their reported ecological role
Only one genus of mycorrhizal fungi was obtained, identified as
Tulasnella calospora, which was isolated from three of the orchid species
analyzed. This genus is widely reported, together with Ceratobasidium
and Serendipitia (Jacquemyn et al., 2017), as one of the principal my-
corrhizal fungi of different orchids in the subtribe Laeliinae (which
includes the species in this work), and its role in germinative and de-
velopment processes in these plants is well known (Ding et al., 2014;
Linde et al., 2014; Pereira et al., 2014; Phillips et al., 2014). In Mexico
and Central and South America, Tulasnella is reported to be mainly
associated with some rupicolous and epiphytic orchids such as Epiden-
drum, Coppensia, Vanilla, Acineta and Oncidium spp. (Jacinto-Hernández
and Ortega-Larrocea, 2013; Moreno-Martínez, 2011; Nogueira et al.,
2014; Rendón-Lara et al., 2013; Segundo, 2016). Group A included
Trichocladium Harz, in addition to mycorrhizal fungi. This genus is
54
Rhizosphere 7 (2018) 49–56
comprised of 18 accepted species (Teik-Khiang and Hyde, 1999), most
of them saprotrophs of wood, bark or other plant tissues. Although this
genus is not considered mycorrhizal, many species in this genus un-
dergo asexual reproduction, and it appears to be beneficial in at least
two Trichocladium species (T. lignicola I. Schmidt and T. opacum (Corda)
S. Hughes). It has been observed to have monilioid-like cell formation
(Teik-Khiang and Hyde, 1999) and for this reason, the cladistic analysis
included it in group A. In America, this genus has been found in plants
such as Fraxinus, Quercus, Pseudotsuga and Castanea (Seidl, 2009),
which are like the phorophytes in this work. Due to the limited number
of studies of endophytes associated with orchids, the role that many of
them play is unknown (Rodríguez et al., 2009).
In the present work, most of the fungal isolates were nonmycor-
rhizal. In the Ascomycota phylum, diverse groups were found, in-
cluding the pathogenic species Fusarium, Alternaria, Colletotrichum and
Neofabraea; the beneficial or synergistic endophytes Trichoderma,
Preussia, Chaetomium, and Paecilomyces; and the saprobes Lachnum and
Xylaria. Among the endophyte genera, the most widely reported species
in orchids are Xylaria, Trichoderma and Colletotrichum (Almanza-Álvarez
et al., 2017; Ávila-Díaz et al., 2013; Gamboa-Gaitán and Otero-Ospina,
2016). In this study, the dominant genera in both the number of species
and number of isolated strains were Trichoderma and Xylaria, mostly
isolated from Laelia speciosa and L. autumnalis. These genera are con-
sidered to stimulate plant germination and development by releasing
growth factors or metabolites that stimulate auxin production or act as
antagonists for other organisms that could damage these plant species
(Rivera-Orduña et al., 2011). It has been suggested that replacement of
the Rhizoctonia complex for ectomycorrhizal fungi or other endophytes
could be an orchid strategy because these fungi are more stable and
high-throughput carbon/nutrient resources (Stark et al., 2009).
Group D included 2 genera of the Basidiomycota phylum, Coprinus
and Clitopillus. Some studies have pointed out that some orchids can be
mycorrhized by fungi not included in the Rhizoctonia complex, such as
Coprinus and Russula (Kikuchi et al., 2008; Osgura-Tsujita et al., 2009);
Coprinus have been characterized as a mycorrhizal species of orchids
such as Epipogium roseum (Yamato et al., 2005). Previous reports in-
dicate that endophytic fungi act as a communication bridge between
several plants and fulfil different functions, such as promoting orchid
seed germination (Dearnaley et al., 2012; Marmeisse and Girlanda,
2016; Rasmussen et al. 2015).
4.3. Fungal diversity and orchid distribution
The Shannon-Wiener diversity indexes between orchids and sites
obtained in this study were similar or slightly lower than those reported
in other studies for terrestrial and epiphytic orchids, where values from
1.3 to 3.5 are reported (Stark et al., 2009; Tao et al., 2008). According
to beta diversity results between orchids and sites, it was observed that
the fungal species diversity in sampling sites was related to the orchid
richness and the presence of certain orchid species. Species of Laelia are
evidently capable of maintaining diverse fungi in their roots, and this
ability could be a significant asset as it increases the likelihood of
forming a partnership with whichever fungus is present or obtaining
some benefit at a given site and thus the likelihood of seeds starting to
germinate; it may also enhance dispersion processes (Waud et al.,
2017). L. autumnalis not only showed the highest fungal community
values of richness and diversity but also was the species with the
highest territorial coverage. This confirms what has been established
for some orchid species, which is that their adaptive talent to disperse
and colonize new environments is related to their ability to interact
with a large variety of fungi to gain advantages in the substrate where
they develop and probably with seasonal variation (Ercole et al., 2015;
Waud et al., 2016). Additionally, the plasticity in fungal partners en-
sures their survival during different stages of their life cycle or under
different environmental conditions (Rasmussen et al., 2015). The lower
values showed by E. citrina could be explained by the fact that this
M.d.l.A. Beltrán-Nambo et al.
species was only found in one site, as their natural populations have
been diminished due to habitat deterioration, and that only two dif-
ferent fungi were obtained in culture from its roots. Some researchers
have found that orchids were rarely limited by the availability of ap-
propriate fungi at a geographic scale. The analyzed orchids are asso-
ciated locally with many different fungal partners along their species
distribution range, which allows a local species to remain an ecological
generalist. In such pattern, the association with rare fungi could also
cause an orchid species to be more susceptible to decline, even at a
small scale (Bailarote et al., 2012; McCormick and Jacquemyn, 2014).
Our results also suggest the existence of segregation processes, be-
cause although different orchid species were found to be associated
with the same fungus within a genus, in sites where these plants co-
incided, they were associated with different fungal species. One ex-
ample is the association of Laelia autumnalis and L. speciosa with
Trichoderma viride in different locations. It has been determined that
segregation processes exist between orchids and are mediated by their
interactions with different groups of symbionts. This ensures that nu-
trients are obtained from different fungi, avoiding competition between
orchid species and allowing their coexistence and dispersion (Ercole
et al., 2015; Jacquemyn et al., 2014; Tesitelova et al., 2013; Waud et al.,
2016).
5. Conclusions
The studied orchid species, with local generalist associations and
diverse fungi, managed to develop in different habitats successfully.
Species such as L. autumnalis, L. speciosa and P. squalida seem to be more
generalists with respect to endophytes; they are associated with and
were found in symbiosis with the orchid mycorrhizal genera Tulasnella.
Although they are endemic species of Mexico, they are found and
widely distributed in the central and southern zones of the country,
while other orchids, such as E. citrina, seem to be more specific. This
situation has been reported for species with a high degree of endemism.
Acknowledgments
This work was financed by the CECTI through Project no. 05. The
authors thank Dr. Pilar Ortega Larrocea for transmission of knowledge
and training in techniques for the study of orchid mycorrhiza, which
made the performance of this work possible; and to M.C. Aarón
Giovanni Munguía for helping to improve the translation of this work.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.rhisph.2018.07.001.
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