Pecoraro et al. BMC Microbiology
(2020) 20:236
https://doi.org/10.1186/s12866-020-01906-4
RESEARCH ARTICLE
Open Access
Molecular evidence supports simultaneous
association of the achlorophyllous orchid
Chamaegastrodia inverta with
ectomycorrhizal Ceratobasidiaceae and
Russulaceae
Lorenzo Pecoraro1* , Xiao Wang1, Giuseppe Venturella2, Wenyuan Gao1, Tingchi Wen3, Yusufjon Gafforov4 and
Vijai Kumar Gupta5
Abstract
Background: Achlorophyllous orchids are mycoheterotrophic plants, which lack photosynthetic ability and
associate with fungi to acquire carbon from different environmental sources. In tropical latitudes, achlorophyllous
forest orchids show a preference to establish mycorrhizal relationships with saprotrophic fungi. However, a few of
them have been recently found to associate with ectomycorrhizal fungi and there is still much to be learned about
the identity of fungi associated with tropical orchids. The present study focused on mycorrhizal diversity in the
achlorophyllous orchid C. inverta, an endangered species, which is endemic to southern China. The aim of this work
was to identify the main mycorrhizal partners of C. inverta in different plant life stages, by means of morphological
and molecular methods.
Results: Microscopy showed that the roots of analysed C. inverta samples were extensively colonized by fungal
hyphae forming pelotons in root cortical cells. Fungal ITS regions were amplified by polymerase chain reaction,
from DNA extracted from fungal mycelia isolated from orchid root samples, as well as from total root DNA.
Molecular sequencing and phylogenetic analyses showed that the investigated orchid primarily associated with
ectomycorrhizal fungi belonging to a narrow clade within the family Ceratobasidiaceae, which was previously
detected in a few fully mycoheterotrophic orchids and was also found to show ectomycorrhizal capability on trees
and shrubs. Russulaceae fungal symbionts, showing high similarity with members of the ectomycorrhizal genus
Russula, were also identified from the roots of C. inverta, at young seedling stage. Ascomycetous fungi including
Chaetomium, Diaporthe, Leptodontidium, and Phomopsis genera, and zygomycetes in the genus Mortierella were
obtained from orchid root isolated strains with unclear functional role.
(Continued on next page)
* Correspondence: lorenzo.pecoraro@gmail.com
1
School of Pharmaceutical Science and Technology, Health Sciences
Platform, Tianjin University, 92 Weijin Road, Nankai District, Tianjin 300072,
China
Full list of author information is available at the end of the article
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Pecoraro et al. BMC Microbiology
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(Continued from previous page)
Conclusions: This study represents the first assessment of root fungal diversity in the rare, cryptic and narrowly
distributed Chinese orchid C. inverta. Our results provide new insights on the spectrum of orchid-fungus symbiosis
suggesting an unprecedented mixed association between the studied achlorophyllous forest orchid and
ectomycorrhizal fungi belonging to Ceratobasidiaceae and Russulaceae. Ceratobasidioid fungi as dominant
associates in the roots of C. inverta represent a new record of the rare association between the identified fungal
group and fully mycoheterotrophic orchids in nature.
Keywords: Achlorophyllous orchids, Ceratobasidiaceae, Ectomycorrhizal fungi, Endangered species, Orchid
mycorrhiza, Plant-fungus interactions, Mycoheterotrophy, Russula
Background
Mycoheterotrophic plants are associated with fungi that
support their heterotrophy with varying extents. Indeed,
these peculiar plants acquire carbon through mycorrhizal association with fungal mycelia fetching organic
compounds from different environmental sources [1].
Among mycoheterotrophs, achlorophyllous plants, represented by more than 400 vascular species, are fully
heterotrophic, since they are not able to photosynthesize
and rely solely on carbon from their mycorrhizal fungi
[2]. Some plants that contain chlorophyll are partially
heterotrophic, because they respire more carbon than
they fix and thus obtain part of their organic nutrients heterotrophically via associated fungi and part
autotrophically by photosynthesis [3]. In addition,
many plants are mycoheterotrophic only during their
early stage of development, but become autotrophic
when fully developed [1].
The Orchidaceae family is particularly predisposed to
mycoheterotrophy, encompassing all known levels of nutritional dependence upon fungi. All orchids are mycoheterotrophic during their establishment phase, since
they produce extremely small seeds that lack the nutrient reserves necessary for early growth. For this reason,
orchid seed germination and the following achlorophyllous protocorm stage depend on the presence of a fungal
partner that provides water, mineral nutrients and organic carbon to the juvenile plant [4]. At the adult stage,
most orchid species become fully autotrophic, and although they maintain a relationship with their root associated fungi, this is no longer for carbon nutrition, but
just for water and mineral uptake [5]. In contrast, a significant number of forest orchids that develop a photosynthetic apparatus at adulthood remain dependent on
both organic and inorganic fungal nutrients under low
light availability [6]. This dual nutritional strategy combining mycoheterotrophy and photoassimilation is
known as partial mycoheterotrophy or mixotrophy [7].
Recent studies based on stable isotope analysis, including
2
H, 13C, 15N and 18O, for understanding nutrient exchange between orchids and fungi, have shown that partial mycoheterotrophy plays a far greater role than
previously assumed, not only in forest orchids, but even
in orchids growing in open habitats with full light conditions [8]. Achlorophyllous orchids are at the extreme
point of plant’s nutritional reliance on mycorrhizal fungi,
with about 235 species, i.e. more than 50% of all fully
mycoheterotrophic plants, that completely lack photosynthetic capability even at maturity, thus being obligately mycoheterotrophic throughout their lifetime [9].
Regarding fungal diversity in orchid mycorrhizae, fully
autotrophic orchids generally associate with a wide range
of basidiomycetous fungi of the form-genus Rhizoctonia,
including soil saprotrophs, plant endophytes and pathogens, as well as fungi with poorly known trophic roles
[4, 10]. Mixotrophic and fully mycoheterotrophic orchids
recruit instead ascomycetes and basidiomycetes having
access to large and persistent carbon sources, such as
ectomycorrhizal fungal species that simultaneously
colonize surrounding tree roots [7, 11]. The latter case
constitutes a peculiar tripartite mycorrhizal association
in which a shared fungal mycelium transfers photosynthates from the tree to the orchid, thus the orchid indirectly exploiting the tree as a carbon source [12].
Mycorrhizal specificity, represented by the phylogenetic
diversity of fungi associated with a particular plant, is
low in the majority of green fully photosynthetic orchids,
where an orchid species establishes mycorrhizae with
several phylogenetically distant fungal species in the
Rhizoctonia complex [13]. Sometimes protocorms are
associated with a smaller range of fungal symbionts than
adult plants [5]. Mixotrophic orchids establish either
specific or non-specific mycorrhizal associations, sometimes with the coexistence of rhizoctonias and ectomycorrhizal fungi in the same plant species [14–16]. By
contrast, the level of specificity is normally very high in
achlorophyllous orchids. These orchids often associate
with a single fungal clade constituted by a genus or by a
lower rank taxon, and the fungi involved, with a few exceptions, are not Rhizoctonia species [17, 18]. In particular, achlorophyllous orchids from temperate areas of
northern latidudes have been shown in several works to
associate with non-Rhizoctonia ectomycorrhizal fungi
that allow the orchid to establish an indirect below
Pecoraro et al. BMC Microbiology
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(2020) 20:236
ground connection with nearby autotrophic plants. For
instance, the Eurasian orchid species Epipogium aphyllum has been found associated with basidiomycetes
mainly belonging to the ectomycorrhizal genus Inocybe
[18]. In tropical latitudes instead, achlorophyllous orchids have been recently shown to establish mycorrhizal
relationships with non-Rhizoctonia saprotrophic fungi.
Basidiomycetes belonging to the saprobic Coprinaceae
family were found in association with the Asian achlorophyllous orchid Eulophia zollingeri [19], Gymnopus-related fungal taxa were molecularly identified in the
Australian orchid Erythrorchis cassythoides [20], litterdecaying Mycena in the Caribbean Wullschlaegelia
aphylla [21] and the Japanese Gastrodia confusa [17],
while Marasmius mycobionts were detected in Gastrodia sesamoides from Australia [22]. This peculiar preference for fungal saprotrophs is particularly interesting in
tropical orchid species phylogenetically close to temperate orchids, which are instead associated with ectomycorrhizal fungi. For example, the tropical orchid E.
roseum, showing a very close phylogenetic relationship
with the above-mentioned Eurasian species E. aphyllum,
is associated with saprotrophic Psathyrellaceae [23, 24].
The latter orchid-fungus relationship provides the evidence that the establishment of associations between
achlorophyllous orchids and saprobic versus mycorrhizal
fungi is not phylogenetically constrained. Environmental
factors, such as the warm and humid climate occurring
in tropical areas, may allow saprotrophic fungi to extend
their growing period and improve their decaying activity
on the available organic substrates, thus providing a surplus of nutrients that can be transferred to the fully
mycoheterotrophic orchid partners [25]. However, a few
exceptions are known. For instance, Roy et al. [26] investigated three Asiatic mycoheterotrophic Neottieae species in Thailand and found that all were associated with
ectomycorrhizal fungi, such as Thelephoraceae, Russulaceae and Sebacinales. In a study conducted on fully
mycoheterotrophic orchids from sub-tropical Asia, six
out of seven analysed orchid species established mycorrhizal relationships with either wood- or litter-decaying
saprotrophic fungi, while only one species was associated
with ectomycorrhizal fungi [27]. Another exception is
represented by the Asian non-photosynthetic orchid species
Chamaegastrodia shikokiana, which was found to associate
with ectomycorrhizal fungi belonging to Ceratobasidiaceae.
Mycobionts isolated from C. shikokiana were able to form
ectomycorrhiza in vitro on seedlings of Abies firma, suggesting that the studied orchid may depend on nutrients
supplied from the tree species through the hyphae of the
mycorrhizal fungi in nature [28].
The present study focused on C. inverta (W.W. Smith)
Seidenfaden, one of the five achlorophyllous orchid species in the genus Chameagastrodia, which is endemic to
Yunnan and Sichuan provinces, southern China, usually
growing in damp places in montane forests (1200–2600
m a.s.l.) [29]. The aim of this work was to identify the
main mycorrhizal partners of C. inverta in different
plant life stages, by means of morphological and molecular methods. A better understanding of mycorrhizal
strategies in orchid species belonging to the genus Chamaegastrodia, such as C. inverta, would clarify the biology of achlorophyllous orchids and would be of
relevance to future in situ or ex situ conservation activities of this endangered species.
Results
Mycorrhizal root morphology of C. inverta
Light microscopy on thin sections showed that the roots
of all analysed C. inverta samples (Fig. 1) were extensively colonized by fungal hyphae. Characteristic dense
intracellular hyphal coils (pelotons) were observed in
most orchid root cortical cells (Fig. 2a-b). The majority
of pelotons appeared intact and completely undigested
(Fig. 2a-b). A dominant mycelial morphology characterized the observed pelotons, mainly constituted by dark,
septate, thick-walled (7.5–10.5 μm in diameter) hyphae,
frequently showing branches produced at right angles to
the main hypha, the branch hypha being slightly constricted at the branch origin, and septum often occurring
near the branch origin (Fig. 2c-d).
Fungal isolation
Attempts to isolate in vitro single pelotons extracted
from C. inverta root cells were in most cases unsuccessful. No hyphal growth was observed from the fungal
coils transferred on PDA medium, with a single exception from orchid sample 5. On the contrary, the majority
of surface-sterilized root portions yielded fungal mycelia
that could be assigned to 32 main morphological types
of ascomycetous and zygomycetous fungi by macro- and
microscopic observations. These orchid root endophytes
were subsequently identified using molecular taxonomy,
because the paucity of distinctive morphological characters did not allow a clear taxonomic delimitation, based
on microscopy. However, the morphology of isolated
mycelia was in most cases completely different from the
above-mentioned dominant hyphal morphology observed in the root sections by the microscope.
Identification of C. inverta mycorrhizal symbionts and
other endophytes
Molecular analysis allowed the identification of fungi associated with C. inverta roots. Sequences were produced
from amplicons of both fungal cultures and total root
DNA, using the fungal-specific primer pair ITS1-OF/
ITS4-OF. These primers successfully yielded sequences
from all the 32 fungal isolates and the 8 analysed plants.
Pecoraro et al. BMC Microbiology
(2020) 20:236
Page 4 of 13
Fig. 1 Chamaegastrodia inverta adult and completely developed plants (a, b, c) and young hypogeous individuals (seedlings, d). Green leaves
and stems represented in Fig. 1 a belong to non-orchid surrounding vegetation in C. inverta habitat
Amplicon electrophoretic profiles displayed high intensity bands from 600 to 800 bp. The general primer pair
ITS1F/ITS4 gave, instead, less consistent amplification,
and in some cases, never produced any amplicon, or just
amplified the orchid plant DNA (data not shown).
Total root DNA sequencing revealed that mycorrhizal
tissue was dominated by fungi belonging to the family
Ceratobasidiaceae (Table 1). For 7 out of the total 8 investigated C. inverta plants, amplified sequences were
related to Ceratobasidium sequences in GenBank. The
young orchid plant (seedling) sample 6 yielded sequences 6a (accession no. MT278316) and 6b
(MT278317) with close identity to GenBank accession
sequences of Russulaceae and Ceratobasidiaceae, respectively (Table 1).
Phylogenetic analysis clarified the relationships of C.
inverta root-associated fungi within Russulaceae and
Ceratobasidiaceae. Sequences retrieved from the studied
orchid plants could be aligned with fungal sequences
from various orchid and non-orchid plant hosts, isolated
strains, and sporophores. A neighbour-joining tree revealed that all Ceratobasidium-like sequences obtained
in this study clustered into a single well-supported clade
(Fig. 3) and are closely related to ceratobasidioid fungi
previously found in the rhizome of Chamaegastrodia
shikokiana in Japan and in ectomycorrhizal root tips in
China. These fungi formed a clade with 100% bootstrap
support with another member of Ceratobasidiaceae isolated from the Australian mycoheterotrophic orchid Rhizanthella gardneri (Fig. 3). The phylogenetic tree from
the Russulaceae dataset showed that the sequence amplified from roots of C. inverta plant sample 6 fell in a cluster including Russula cerolens (that was the closest
match) R. pectinata and R. insignis (Fig. 4).
Apart from these basidiomycetes identified from total
root DNA, sequences of ascomycetous and zygomycetous fungi were obtained from the isolated strains
(Table 2). Among them, sequences corresponding to the
genus Mortierella, in the Mortierellaceae family of
Mucoromycota were most commonly recovered from
46.8% of cultured mycelia, including the sole strain obtained from peloton (sample 5, accession no.
MT278348), whereas 25% of isolated fungi yielded sequences with very high ITS similarity to Phomopsis
Pecoraro et al. BMC Microbiology
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Fig. 2 Microscopic characteristics of mycorrhizal roots in Chamaegastrodia inverta. a Hyphal coils (pelotons) extracted from cortical cells. b Fungal
pelotons inside orchid root cells. c, d Details of mycelial morphology characterizing pelotons, with dark, septate, thick-walled hyphae, showing
90° branching, constriction at branch points, and septa near branch origins
species, belonging to the family Diaporthaceae of Ascomycota (Table 2). In the latter family was also found the best
match for sequences obtained from 18.7% of isolates, belonging to the genus Diaporthe. Other ascomycetes from
the genera Leptodontidium (Leptodontidiaceae family)
and Chaetomium (Chaetomiaceae) were also sporadically
found (Table 2).
Discussion
This study represents the first assessment of root fungal
diversity in the rare, cryptic, and narrowly distributed
Chinese orchid species Chamaegastrodia inverta. Microscopic analysis, showing the heavy presence of fungal
pelotons in the root cortical cells of all studied orchid
samples, provided the first evidence of the establishment
of mycorrhizal associations in C. inverta. The observed
extent of fungal colonization in the orchid mycorrhizal
roots was not surprising, given the achlorophyllous condition of the investigated plant species. The studied orchid was expected to be wholly mycoheterotrophic and
therefore highly dependent on soil fungi for its nutrition,
based on results showed from the previously studied
member of the genus Chamaegastrodia, C. shikokiana in
Japan [28], and the general mycorrhizal condition of
non-photosynthetic forest orchids [1, 30].
The use of different experimental approaches, including morphological analysis combined with molecular sequencing, following both culture-dependent methods
and direct total orchid root DNA amplification, allowed
the detection and identification of C. inverta rootassociated fungi, in different plant life stages. Results
showed that the primary mycorrhizal symbionts of C.
inverta are within the family Ceratobasidiaceae. This
family is included in the diverse group of Rhizoctonialike fungi, which comprises a range of rather distantly
related fungal taxa characterised by homogenous asexual
stage hyphal morphology (90° branching of hyphae, a
constriction at the branch point, and a septum near the
point of origin in the branch hyphae), as well as by a
common significant predisposition to establish mycorrhizal symbiosis with orchids [4, 31, 32]. The morphology of mycelia forming pelotons in the root cells of
analysed C. inverta individuals, showing typical Rhizoctonia features, is consistent with molecular identification
of Ceratobasidiaceae fungal symbionts. Ceratobasidioid
fungi have been previously found to associate with several other orchids including both tropical and temperate
species [32–34]. Members of this fungal family have
been recognized as important associates in epiphytic orchids belonging to different genera, such as Oncidium
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Table 1 Fungal diversity molecularly detected in Chamaegastrodia inverta roots, from extracted total root DNA
C. inverta sample
GenBank code
Best BLAST match(es)
Accession code
Overlap length
1b (adult)
MT278309
Vouchered mycorrhizae (Basidiomycota)
AB303058
1160
98%
Uncultured Ceratobasidium
JQ991678
1096
99%
2b (adult)
MT278310
3b (adult)
MT278311
4a (adult)
MT278312
4b (adult)
5a (adult)
5b (adult)
MT278313
MT278314
MT278315
6a (seedling)
MT278316
6b (seedling)
MT278317
7a (seedling)
8a (seedling)
MT278318
MT278319
% match
Vouchered mycorrhizae (Basidiomycota)
AB303057
1162
98%
Uncultured Ceratobasidium
JQ991678
1096
99%
Ceratobasidium sp.
GQ175299
1081
96%
Vouchered mycorrhizae (Basidiomycota)
AB303057
1188
98%
Ceratobasidium sp.
GQ175299
1122
96%
Vouchered mycorrhizae (Basidiomycota)
AB303058
1155
98%
Uncultured Ceratobasidium
JQ991678
1090
99%
Ceratobasidium sp.
GQ175299
1068
95%
Vouchered mycorrhizae (Basidiomycota)
AB303058
2055
98%
Uncultured Ceratobasidium
JQ991678
1994
99%
Ceratobasidium sp.
GQ175299
1920
96%
Vouchered mycorrhizae (Basidiomycota)
AB303058
1088
96%
Uncultured Ceratobasidium
JQ991678
1029
97%
Ceratobasidium sp.
GQ175299
1002
94%
Vouchered mycorrhizae (Basidiomycota)
AB303057
1129
97%
Uncultured Ceratobasidium
JQ991678
1062
98%
Ceratobasidium sp.
GQ175299
1048
95%
Russula cerolens
KX095042
1203
98%
Vouchered mycorrhizae (Basidiomycota)
AB303058
1138
98%
Uncultured Ceratobasidium
JQ991678
1079
98%
Vouchered mycorrhizae (Basidiomycota)
AB303058
1155
98%
Uncultured Ceratobasidium
JQ991678
1090
99%
Ceratobasidium sp.
GQ175299
1068
96%
Phomopsis sp.
KF428571
1161
99%
BLAST search closest matches of fungal internal transcribed spacer DNA sequences amplified from C. inverta. Sample GenBank accession codes, accession codes
for the closest GenBank matches, sequence identity, and overlap of each match are reported
[35], Ionopsis and Tolumnia [36, 37], as well as in terrestrial orchids, of both forest and meadow habitats, including Goodyera [38–40], Anacamptis, Cephalanthera and
Orchis [16, 34, 41]. Although mycorrhizal associations
with Ceratobasidiaceae involve orchids with very different biogeographical and ecological features, the great
majority of orchid species that have been found to establish a trophic relationship with Ceratobasidiaceae fungi
belong to the same physiological category of green
orchids, including species with different degrees of
photosynthetic capability, from fully autotrophic to mixotrophic species [8, 42, 43]. Achlorophyllous nonphotosynthetic orchids, instead, are almost completely
excluded from mycorrhizal partnerships with Ceratobasidiaceae, with very few exceptions [28, 44]. Our finding
of ceratobasidioid fungi as dominant associates in the
roots of the achlorophyllous forest orchid C. inverta represents a new record of the rare association between the
identified fungal group and fully mycoheterotrophic
orchids in nature. Phylogenetic relationships reconstructed from rDNA sequence information suggest that
the C. inverta associated Ceratobasidiaceae have limited
genetic diversity and likely belong to the same species
(Fig. 3). Chamaegastrodia inverta mycobionts are phylogenetically close to a peculiar group of Ceratobasidiaceae
showing ectomycorrhizal capability, such as the fungi
previously found in C. shikokiana in Japan, which were
also able to form ectomycorrhizas on the rootlets of the
woody plant species Abies firma sedlings in pot culture
[28], and the fungi associated with the Australian subterranean orchid (flowering below ground) Rhizanthella
gardneri [44]. Mycorrhizal fungi, isolated from pelotons
extracted from the rhizomes of the latter orchid species,
were tested by Bougoure and collaborators for their ability to form ectomycorrhizal associations with several
plant species belonging to the genus Melaleuca, which
resulted in the undoubted formation of mantle and
Hartig net, typical ectomycorrhizal structures [44].
Pecoraro et al. BMC Microbiology
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Page 7 of 13
Fig. 3 Neighbour-joining phylogenetic tree showing the relationship
between the Ceratobasidiaceae sequences obtained from
Chamaegastrodia inverta (*) and selected database relatives. Kimura
2-parameter distances were used. Bootstrap values are based on
percentages of 1000 replicates. The tree was rooted with Laccaria
bicolor and Tricholoma portentosum as outgroups
Fig. 4 Neighbour-joining phylogenetic tree showing the relationship
between the Russula sequence obtained from Chamaegastrodia inverta
(*) and selected database relatives. Kimura 2-parameter distances were
used. Bootstrap values are based on percentages of 1000 replicates.
The tree was rooted with Gloeocystidiellum aculeatum and Albatrellus
flettii as outgroups
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Table 2 Fungal diversity molecularly detected in Chamaegastrodia inverta roots, from DNA extracted from isolated fungi
C. inverta sample
GenBank code
Best BLAST match(es)
Accession code
Overlap length
% match
1 (1) a
MT278320
Uncultured fungus
AB520571
1184
99%
Mortierella alpina
KJ469836
1168
98%
1 (1) c
1 (1) d
1 (1) e
1 (2) a
1 (2) c
1 (2) e
1 (2) g
1 (3)
1 (3) a
1 (3) b
MT278321
MT278322
MT278323
MT278324
MT278325
MT278326
MT278327
MT278328
MT278329
MT278330
Uncultured fungus
AB520571
1194
99%
Mortierella alpina
KJ469836
1177
99%
Uncultured fungus
AB520571
1203
99%
Uncultured Mortierella
HG936573
1182
98%
Uncultured fungus
AB520571
1994
99%
Uncultured Mortierella
HG936573
1965
98%
Uncultured fungus
AB520571
1190
99%
Uncultured Mortierella
HG936573
1170
98%
Uncultured fungus
AB520571
1229
99%
Uncultured Mortierella
HG936573
1208
98%
Uncultured fungus
AB520571
1214
99%
Uncultured Mortierella
HG936573
1199
99%
Fungal sp. strain
KU838618
1033
99%
Phomopsis sp.
KF428571
1033
99%
Uncultured fungus
AB520571
1190
98%
Uncultured Mortierella
HG936573
1173
98%
Uncultured fungus
KC222700
1037
98%
Mortierella sp.
KX640303
1029
99%
Uncultured fungus
AB520571
1210
99%
Uncultured Mortierella
HG936573
1192
98%
1 (4)
MT278331
Phomopsis sp.
KF428571
1050
99%
1 (6) a
MT278332
Phomopsis sp.
KF428571
1040
99%
1 (6) b
MT278333
Uncultured fungus
AB520571
1218
99%
Uncultured Mortierella
HG936573
1199
98%
1 (6) c
MT278334
Phomopsis sp.
KF428571
1055
99%
3 (3) b
MT278335
Uncultured fungus
KC222831
977
98%
Diaporthales sp.
KF428606
972
99%
Diaporthe sp.
FJ799941
952
96%
3 (3) c
MT278336
3 (3) d
MT278337
3 (3) e
MT278338
3 (4)
MT278339
3 (4) b
MT278340
4 (3) b
MT278341
Uncultured fungus
KC222831
977
98%
Diaporthales sp.
KF428606
972
99%
Diaporthe sp.
FJ799941
946
96%
Diaporthales sp.
KF428606
972
99%
Diaporthe sp.
FJ799941
944
96%
Diaporthales sp.
KF428606
972
99%
Diaporthe sp.
FJ799941
950
96%
Uncultured fungus
KF296769
1127
98%
Uncultured Leptodontidium
JF519497
1127
98%
Uncultured fungus
KF296769
1133
98%
Uncultured Protoventuria
JQ346991
1133
98%
Uncultured Leptodontidium
JF519497
1133
98%
Diaporthales sp.
KF428606
1002
100%
Diaporthe sp.
FJ799941
979
97%
Pecoraro et al. BMC Microbiology
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(2020) 20:236
Table 2 Fungal diversity molecularly detected in Chamaegastrodia inverta roots, from DNA extracted from isolated fungi (Continued)
C. inverta sample
GenBank code
Best BLAST match(es)
Accession code
Overlap length
% match
4 (3) c
MT278342
Uncultured fungus
AB520571
1195
99%
Uncultured Mortierella
HG936573
1179
98%
4 (3) d
MT278343
Diaporthales sp.
KF428606
1002
100%
Diaporthe sp.
FJ799941
987
97%
5 (1) a
MT278344
Phomopsis sp.
KF428571
1048
99%
5 (1) b
MT278345
Phomopsis sp.
KF428571
1042
99%
5 (3)
MT278346
Uncultured fungus
AB520571
1177
98%
Mortierella alpina
KJ469836
1164
98%
5 (4) a
MT278347
Chaetomium nigricolor
JF439467
1057
99%
5 (Pel)
MT278348
Uncultured fungus
AB520571
1190
98%
Uncultured Mortierella
HG936573
1173
98%
7 (2) a
MT278349
Fungal endophyte isolate
KR015900
1026
98%
Phomopsis sp.
JQ341094
1020
99%
7 (2) b
MT278350
Fungal endophyte isolate
KR015900
1022
98%
Phomopsis sp.
JQ341094
1020
98%
8 (1)
MT278351
Uncultured fungus
AB520586
479
84%
Uncultured Mortierella
FJ197919
473
83%
BLAST search closest matches of fungal internal transcribed spacer DNA sequences amplified from C. inverta. In sample codes, first numbers (from 1 to 8) indicate
the plant samples, numbers in brackets the root portions, letters the isolated fungal strain. Sample GenBank accession codes, accession codes for the closest
GenBank matches, sequence identity, and overlap of each match are reported
Ectomycorrhizal taxa constitute a rare exception among
Ceratobasidiaceae, the great majority of members within
this fungal family, as well as Rhizoctonia-forming Agaricomycotina in general, being regarded as saprotrophs
and plant pathogens [28, 31, 43–45]. The previously
reported cases of tripartite relatioships C. shikokianaCeratobasidium-A. firma [28] and R. gardneri-Ceratobasidium-Melaleuca [44] involved ceratobasidioid fungi
which were able to establish orchid mycorrhizas with
fully mycoheterotrophic orchid species and ectomycorrhizas with autotrophic tree or shrub hosts simultaneously, the photosynthetic plant partner being the
provider of carbon for the system. An additional example of ectomycorrhizal Ceratobasidiaceae was provided by the fungi isolated from the roots of the
mixotrophic orchid Platanthera minor, which were also
found to show ectomycorrhiza-forming ability on Pinus
densiflora, the sole ectomycorrhizal tree species in the
sampling sites in Japan [45]. The P. minor associated
Ceratobasidium fungi formed a highly supported clade
with mycobionts from C. shikokiana and R. gardneri in
the phylogenetic analyses performed by Yagame et al.
[45]. The Ceratobasidiaceae associated with C. inverta,
in this study, are also closely related and cluster with the
above mentioned ectomycorrhizal ceratobasidioid fungi
from C. shikokiana and R. gardneri. It is therefore possible that C. inverta establishes a tripartite relationship
with some of the surrounding autotrophic plants in the
forest habitats where it grows, using the ceratobasidioid
associated fungi to exploit the ectomycorrhizal plant as a
carbon source. Further studies are necessary to test this
hypothesis, by determining C. inverta natural abundance
in 13C and 15N compared with those of surrounding
photosynthetic plants, in order to confirm and quantify
the contribution of fungal source to the orchid carbon
nutrition [46]. Besides, experiments of ectomycorrhiza
formation on roots of potential tree hosts available in C.
inverta habitats, using Ceratobasidiaceae isolated from
the studied orchid as inoculum, would clarify the ability
of the orchid fungal associates to establish ectomycorrhizal symbiosis with surrounding autotrophic partners.
Our attempt of fungal isolation from pelotons extracted
from the cortical cells of C. inverta roots was unsuccessful. The sole strain observed to apparently grow from a
peloton isolated from orchid sample 5 was molecularly
similar to an uncultured fungus detected in agricultural
soil from Japan [47] and to a Mortierella sequence amplified from Zea mays field soil samples in Germany [48]
(Table 2). This isolated mycelium may actually reflect a
fungal contaminant from spore or a non-mycorrhizal
root endophyte from hyphal fragment accidentally
trapped in the cultured peloton. The difficulty in isolating in axenic culture the real C. inverta fungal symbionts
from pelotons was confirmed by the result of isolation
attempts from root fragments, which provided a number
of ascomycetous and zygomycetous strains, but did not
yield any ceratobasidioid basidiomycete. This result is in
agreement with previous studies on C. shikokiana and R.
Pecoraro et al. BMC Microbiology
(2020) 20:236
gardneri, involving similar Ceratobasidiaceae mycorrhizal fungi. In the former study on C. shikokiana the
authors reported that no fungal growth was observed on
Czapek-Dox medium, which they normally used for
saprobic fungi isolation, while active mycelial growth
was obtained from pelotons cultured on a Modified
Melin-Norkrans medium, specific for ectomycorrhizal
fungi [28]. Similarly, in the study on R. gardneri, Bougoure et al. [44] found that isolation and growth of fungal pelotons from the roots of the studied orchid were
mostly unsuccessful, with extracted hyphal coils failing
to grow or colonies suddenly dying after initial growth.
The absence of hyphal growth from C. inverta mycobiont extracted pelotons on PDA medium suggests that
the analysed fungi belong to the ectomycorrhizal trophic
group and may require very specific media to be
isolated. However, the ecology and lifestyle of Ceratobasidiaceae associated with achlorophyllous mycoheterotrophic orchids is difficult to predict and generalize, and
requires in-depth analyses to be understood, in every
single association with different plant species. For instance, Bougoure et al. [49] showed that Ceratobasidiaceae associated with R. gardneri obtained carbon by
both saprothrophic and mycorrhizal means, simultaneously. In the latter work, isotopically labelled tracers,
13
CO2 and double-labelled [13C-15N]glycine were used
to assess the direction of carbon and nitrogen transfers
between the plants involved in the investigated tripartite
association via the fungal connections, showing that R.
gardneri obtained nutrients from the associated mycorrhizal ceratobasidioid fungi, which were able to derive
carbon not only from surrounding autotrophic shrubs
via ectomycorrhizas, but also from soil organic matter
via saprotrophic activity [49].
One out of the three analysed C. inverta young seedlings, sample 6, showed an interesting association with
another basidiomycete (Fig. 4) highly similar to Russula
cerolens in GenBank from an unpublished study by Ma
et al. on Russulaceae diversity in Heilongjiang Province,
in China, and to sequences from specimens of R. insignis
and R. pectinata collected in Europe [50]. The amplified
Russula mycorrhizal fungus was co-occurring with Ceratobasiaceae in the roots of the same C. inverta individual
(Table 1). Species in the large mushroom genus Russula
are well documented as ecologically important ectomycorrhizal symbionts with forest tree species [50, 51]. Russulaceae fungi have been found to be mycorrhizal
partners of a variety of orchids belonging to different
physiological types, such as the fully mycoheterotrophic
species Corallorhiza maculata [11] and Dipodium
hamiltonianum [52], the partially mycoheterotrophic orchids Epipactis microphylla [7] and Limodorum abortivum [53], as well as green photosynthetic Cypripedium
species [54]. In a comprehensive study on mycorrhizal
Page 10 of 13
diversity in the fully mycoheterotrophic orchid genus
Hexalectris, Kennedy et al. [55] found that H. brevicaulis
collected in Mexico and H. grandiflora from USA displayed mixed associations with Russulaceae and Sebacinaceae symbionts. To our knowledge the simultaneous
presence of ectomycorrhizal Russulaceae and Ceratobasidiaceae in the same achlorophyllous orchid species
represents a new finding from the present work, where
the two mycorrhizal fungi were even detected in the
same C. inverta individual. This result supports the hypothesis of C. inverta preference for ectomycorrhizal
fungi to establish trophic relationships that may also involve other plants interconnected by underground fungal
network. This specificity toward ectomycorrhizal fungal
partners needs to be confirmed with additional analyses
on C. inverta samples from different localities. It is interesting that the presence of Russula in C. inverta roots
was only detected at young seedling stage, while all analysed adult plants only yielded Ceratobasidiaceae sequences. It is possible that the analysed orchid species
associates with different fungi during different stages of
its development, as it was previously shown in a variety
of orchids characterized by changing level of mycorrhizal
specificity through life stages, from germinating seeds
and protocorms to adult plants [5, 30]. Experiments
using orchid seed baits buried in sites characterized by
the presence of C. inverta populations may be crucial to
identify fungal taxa able to stimulate seed germination
and to sustain protocorm development in nature [56], as
well as to clarify whether or not the identity of mycorrhizal fungi associating with C. inverta can be affected
by the plant life stage.
Besides typical orchid mycorrhizal taxa, some other
fungi with unclear functional roles were also detected in
the studied orchid roots. They were mainly uncovered
using the culture-dependent approach, thus demonstrating the importance of culture-based method for the accurate analysis of fungal community associated with
orchid roots (Table 2). Among them, the most abundant
were in the Mucoromycota, which represent an exceptional report as orchid associates, and were previously
considered root pathogens or saprotrophic fungi in orchid dead cells [57]. Ascomycota from different families
were more sporadically detected, the best matches for
sequences amplified from C. inverta being fungi from
various sources, including Phomopsis sp. from roots of
Populus trees (Bonito et al. unpublished), Diaporthe sp.
from leaves of Ipomoea philomega [58], uncultured Leptodontidium from roots of Fagus sylvatica (Schnecker
et al. unpublished), and Chaetomium nigricolor from forest soil in Zijin Mountain, China [59] (Table 2). These
ascomycetes may represent root fungal endophytes [43,
60]. However, some of them, such as Leptodontidium
fungi, are very frequently selected during in vitro
Pecoraro et al. BMC Microbiology
Page 11 of 13
(2020) 20:236
isolation or PCR amplification from orchid tissues using
fungus-specific primers [34, 61, 62]. Further physiological characterization of these fungi is needed to
understand whether they play any trophic role in their
association with C. inverta.
Conclusions
Our results provide new insights on the spectrum of
orchid-fungus symbiosis suggesting an unprecedented
mixed association between the achlorophyllous forest orchid C. inverta and ectomycorrhizal fungi belonging to
Ceratobasidiaceae and Russulaceae. This urges for more
in-depth investigations of mycorrhizal diversity and
physiology in the five orchid species belonging to the
genus Chamaegastrodia, which may represent useful
models to understand the evolution and specificity of
mycoheterotrophic interactions, even if their rarity constitutes a challenge for performing detailed, large scale
experiments.
Methods
Orchid collection
Chameagastrodia inverta root samples were collected in
September 2015, from a forest with coniferous species of
Pinus, mixed with various broad-leaved trees dominated
by Eucalyptus sp. and Quercus sp., located in the western part of Kunming Botanical Garden, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming,
Yunnan Province, China. No permissions were necessary
to collect plant samples, using a protocol that allows
plant survival. Plants were identified by the authors.
After harvesting the root material necessary for the
study, plants were replaced in the sampling site, in the
exact location where they were found. In total, eight orchid individuals were sampled, including 5 adult and
completely developed plants, at the end of flowering
stage, and 3 hypogeous young plants (seedlings) at an
initial stage of development (Fig. 1a-d). Although the
sampling size for the studied species was limited due to
its rarity, small population size and the cryptic nature of
its inflorescence, which is the only above ground organ,
the investigated site provided a significant number of
samples representing different life stages of the analysed
orchid. All collected roots were washed under running
water and carefully brushed in order to remove soil debris. Mycorrhizal morphology of fresh root samples was
observed on thin cross-sections under a light microscope
at 100 to 1000-fold magnification. Root fragments
exhibiting high fungal colonization were immediately
processed for fungal isolation. Part of root material was
frozen in liquid nitrogen and stored at − 80 °C for
molecular analysis.
Fungal isolation
Isolation of fungi from fresh orchid root fragments was
performed immediately after sampling. Single pelotons
(hyphal coils within orchid root cells) were dissected
from the outer cortex of C. inverta roots following Rasmussen [4], rinsed twice in double distilled water, transferred in 1 μl water, and cultured on potato dextrose
agar (PDA, Solarbio, Beijing, China) for both morphological and molecular identification.
Fungi were also isolated from root segments [41]
surface-sterilized with consecutive washes of 1:5 sodium
hypochlorite (30 s), rinsing in three changes of sterile
water, cut in 3–5 mm long pieces, and cultured on PDA.
Petri dishes were incubated at room temperature (20–
25 °C) in the dark for up to 2 months to allow the development of slow-growing mycelia.
Molecular identification of root fungal associates
Both DNA from isolated fungi and total DNA from orchid root samples were extracted following the cetyltrimethyl ammonium bromide (CTAB) method [63].
Fungal ITS regions were amplified by polymerase
chain reaction (PCR), using the primer pairs ITS1F/ITS4
[64] and ITS1-OF/ITS4-OF [65] in 50 μL reaction volume, containing 38 μL steril distilled water, 5 μL 10 ×
buffer (100 mM Tris-HCl pH 8.3, 500 mM KCl, 11 mM
MgCl2, 0.1% gelatin), 1 μL of dNTP mixture of 10 mM
concentration, 0,25 μM of each primer, 1.5 U of RED
TaqTM DNA polymerase (Sigma) and approximately
10 μg of extracted genomic DNA. Amplifications were
performed in a PerkinElmer/Cetus DNA thermal cycler,
under the following thermal conditions: 1 cycle of 95 °C
for 5 min initial denaturation before thermocycling, 30
cycles of 94 °C for 40 s denaturation, 45 s annealing at
various temperatures following Taylor & McCormick
[65], 72 °C for 40 s elongation, followed by 1 cycle of
72 °C for 7 min extension. The resulting PCR products
were electrophoresed in 1% agarose gel with ethidium
bromide and purified with the QIAEX II Gel Extraction Kit (QIAGEN) following the manufacturer’s instructions. Controls with no DNA were included in
every amplification experiment in order to test for
the presence of laboratory contamination from reagents and reaction buffers.
DNA sequencing was performed at the GENEWIZ
Company, Tianjin, China.
Sequences were edited, assembled using the program Sequencher 4.1 for MacOS 9, and analysed with
BLAST searches against the National Center for Biotechnology Information (NCBI) sequence database
(GenBank) [66]. Fungal DNA sequences amplified
from C. inverta were submitted to GenBank under
accessions MT278309 - MT278351.
Pecoraro et al. BMC Microbiology
Page 12 of 13
(2020) 20:236
Phylogenetic analysis was conducted with Mega v. 5.0
[67]. DNA sequences were aligned with Clustal X v. 2.0
[68] and neighbour-joining trees against selected database sequences were constructed using Kimura 2parameter distances, with bootstrapping of 1000 replicates [69]. Distinct phylogenetic analyses were performed for the phylogenetically distant fungi identified
from the roots of investigated orchids. The ceratobasidioid fungi tree was rooted with Laccaria bicolor and
Tricholoma portentosum, while Gloeocystidiellum aculeatum and Albatrellus flettii were used as outgroups to
root the Russula fungi tree.
Abbreviations
CTAB: Cetyltrimethyl ammonium bromide; PCR: Polymerase chain reaction
Acknowledgements
We are grateful to Prof. Zhu Liang Yang for precious help in sampling and
preliminary analysis. We thank Prof. Robert P. Borris, the Handling Editor Prof.
Bala Rathinasabapathi, and the anonymous reviewers for critical and useful
comments to the original version of this manuscript.
Authors’ contributions
Conception and design of the research LP, acquisition of data LP, analysis
and interpretation of data LP, XW, drafting the article LP, revising the article
critically for important intellectual content XW, GV, WYG, TCW, YG, and VKG.
All authors approved the manuscript version to be published.
Funding
Not applicable.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Availability of data and materials
The Fungal DNA sequences amplified during this study are available in
GenBank under accessions MT278309 - MT278351. No permissions were
necessary to collect plant samples, using a protocol that allows plant
survival. Plants were identified by the authors. After harvesting the root
material necessary for the study, plants were replaced in the sampling site, in
the exact location where they were found.
16.
Ethics approval and consent to participate
Not applicable.
17.
Consent for publication
Not applicable.
18.
Competing interests
The authors declare that they have no competing interests. Two of the
authors, LP and VKG, are members of editorial board of this journal.
19.
Author details
1
School of Pharmaceutical Science and Technology, Health Sciences
Platform, Tianjin University, 92 Weijin Road, Nankai District, Tianjin 300072,
China. 2Department of Agricultural, Food and Forest Sciences, University of
Palermo, Palermo, Italy. 3The Engineering Research Center of Southwest
Bio-Pharmaceutical Resources, Ministry of Education, Guizhou University,
Guiyang, China. 4Laboratory of Mycology, Institute of Botany, Academy of
Sciences of Uzbekistan, Tashkent, Uzbekistan. 5AgroBioSciences and Chemical
& Biochemical Sciences Department, University Mohammed VI Polytechnic,
Hay Moulay Rachid, Ben Guerir, Morocco.
15.
20.
21.
22.
23.
Received: 15 April 2020 Accepted: 14 July 2020
24.
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