diversity
Article
The Diverse Assemblage of Fungal Endophytes from Orchids in
Madagascar Linked to Abiotic Factors and Seasonality
Kazutomo Yokoya 1 , Lawrence W. Zettler 2 , Jake Bell 1 , Jonathan P. Kendon 1 , Alison S. Jacob 1 , Emily Schofield 1 ,
Landy Rajaovelona 1 and Viswambharan Sarasan 1, *
1
2
*
Citation: Yokoya, K.; Zettler, L.W.;
Bell, J.; Kendon, J.P.; Jacob, A.S.;
Schofield, E.; Rajaovelona, L.; Sarasan,
V. The Diverse Assemblage of Fungal
Endophytes from Orchids in
Madagascar Linked to Abiotic Factors
and Seasonality. Diversity 2021, 13, 96.
https://doi.org/10.3390/d13020096
Academic Editor: Michael Wink
Received: 3 January 2021
Accepted: 6 February 2021
Published: 23 February 2021
Royal Botanic Gardens Kew, Richmond, London TW9 3AE, UK; kazyokoya@gmail.com (K.Y.);
jakebell93@gmail.com (J.B.); j.kendon@kew.org (J.P.K.); ali.jacob@virginmedia.com (A.S.J.);
emily_schofield2@outlook.com (E.S.); L.Rajaovelona@kew.org (L.R.)
Department of Biology, Illinois College, Jacksonville, IL 62650-2299, USA; lwzettle@ic.edu
Correspondence: v.sarasan@kew.org
Abstract: The inselbergs of the Central Highlands of Madagascar are one of many ‘micro-hotspots’
of biodiversity on the island, particularly for Orchidaceae. In this region are several genera that have
a large number of endemic species that are in serious decline or edging towards extinction. Studies
relating to diversity of orchids and their fungal partners (both mycorrhizal and non-mycorrhizal root
associates) deserve more attention, as climate change and human induced decline in resilience of
species in the wild is at an all-time high. Identification of mycorrhizal fungi (MF) via conventional
seed baited-protocorms has limitations for large scale studies and its application for time-bound
conservation projects. The paper describes the value of understanding fungal diversity in the roots of
orchids at different stages of maturity. The first part of the study was a preliminary investigation
mainly to identify culturable Rhizoctonia endophytes, and the second part looked at all life forms of
available taxa together with associated soil characteristics. We isolated and identified 19 putative
MF from 18 of the 50 taxa spread over an area of 250 sq. km, covering three life forms, growth
phases of the orchid taxa, and habitat types. In the rest of the taxa, we were unable to detect
any putative MF, but had varying numbers of non-mycorrhizal endophytes. We also found that
diversity of putative MF was higher in plants from soils with the lowest P levels recorded. Putative
mycorrhizal OTUs were predominantly from the Tulasnella lineage, followed by Ceratobasidium and
Serendipita. Within a small subset of samples, a difference in colonised endophytes depending on the
collection season was observed. In vitro germination studies using 10 OTUs of mycorrhizal fungi
in 14 orchid species showed mostly generalist associations. When orchid seed and fungal sources
were studied irrespective of habitat, life form, and distance from each other (orchid seed and fungal
source), compatibility for symbiotic seed germination was observed in most cases. Issues with the
identification of compatible MF and symbiotic system of seed germination are discussed.
Keywords: conservation; biodiversity hotspot; in vitro; orchid; mycorrhiza
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1. Introduction
Madagascar harbours more than 1000 species of orchids, nearly 90% of which are
endemic, adding to the island’s international reputation as a unique biologically diverse
resource. Many of these species are clustered within ‘micro-hotspots’ throughout the island,
including the rocky domes (inselbergs) of the Central Highlands. Not only are the orchids
of the Central Highlands largely inaccessible due to the formidable terrain, Madagascar
itself is remote, further hindering study by specialists. The Itremo Massif within the Central
Highlands is one such ‘micro-hotspot’, home to more than 86 orchid taxa of which the
majority are endemic, and we have studied fungal diversity in 41 species in the past [1].
More than 80% of Madagascar’s natural vegetation has been cleared or permanently
altered [2] due to illegal mining and other anthropogenic activities [3]. Despite that region’s
rugged terrain, many orchids in the Central Highlands are exposed to several threats.
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Illegal collecting, pollinator decline, and fragmentation due to inbreeding depression are
causing population decline in some orchids. These include several well-known species
(e.g., Angraecum longicalcar) that cling to survival within the rocky landscape year after year
in small, isolated populations. Many of these populations appear to be ‘senile’, i.e., lack
biotic and/or abiotic factors necessary for generating spontaneous seedlings [4], and only
a select few have received sufficient study.
Orchids are particularly vulnerable to decline in population resilience given their
dependency on mycorrhizal fungi (MF) for seed germination [5] and further establishment [5–7]. To effectively conserve the orchids of the Central Highlands with respect to
seed germination, more information is needed about the diversity and distribution of the
fungal associates intimately tied to the orchid life cycle, and the abiotic factors needed by
these fungi in each habitat (e.g., substrate pH, nutrient levels). A logical first step is to
identify culturable mycorrhizal fungi for establishing associations with orchids spanning
different growth stages (protocorms, seedlings, mature plants).
The fungi that associate with photosynthetic orchids typically belong to the Rhizoctonia
complex [8], which usually includes members of the Tulasnellaceae, Ceratobasidiaceae,
and Sebacinales. These fungi are assumed to exist as free-living saprotrophs subsisting
on organic matter that they decompose when moisture is available. As endophytes in
living orchid tissues, they form dense intracellular masses of fungal hyphae (pelotons) that,
once digested, release carbohydrates and nutrients to the orchid ‘host’. Yet not all pelotonforming fungi are assignable to the Rhizoctonia complex. For example, species of Fusarium
and Trichoderma have been acquired from pelotons of orchids in Ecuador [9], China [10],
as well as Madagascar [11], but their physiological role(s) on orchid development has
yet to be determined. Yagame et al. [12], however, did determine that at least one nonRhizoctonia basidiomycete (Coprinellus sp.) was capable of serving as an orchid mycorrhizal
associate. Thus, the possibility exists that these lesser-known fungi may augment the
orchid’s mycotrophic needs. Given that MF are increasingly recognized as one of the most
important ecological components influencing orchid distributions [13,14], studying the full
breadth of fungal endophytes has merit for conservation.
This paper presents an overview of a wide range of fungal endophytes identified from
50 orchid species from the Central Highlands in Madagascar following a study spanning
five years. These fungi include members of the Rhizoctonia complex as well as lesser-known
groups. To verify that selected fungal endophytes were indeed mycorrhizal associates,
symbiotic germination experiments were also carried out and results described. In addition
to the sampling of fungi, soil from the habitats were assessed for soil pH, nutrients (N, P,
K) and moisture content during both the rainy and dry season. In doing so, we attempted
to correlate the fungal diversity observed to these abiotic components and how these
factors have the potential to influence orchid distribution and growth stages. The major
impediment for the study throughout the five-year period was the limited number of plant
roots and seed capsules that could be collected due to the small population number of the
taxa studied, and the restrictions imposed by government permits.
2. Materials and Methods
2.1. Soil Analysis
Soil/substrate samples were collected at several locations and consisted of soil and
humus in the case of terrestrial habitats, and organic/inorganic debris on the surface of bark
and in the cracks of rocks where epiphytic/lithophytic orchids were found. Single samples
of soil were taken at each collection site. On return to Kew Madagascar Conservation Centre
(KMCC), the collected samples were analysed using a LaMotte STH Series Combination
Soil Testing Outfit (LaMotte, Chestertown, MD, USA). This provided quantitative data
on the condition of the soil/substrate in the vicinity of each orchid sampled. Parameters
tested were pH, nitrate, phosphorous, and potassium. Organic content (humus) level was
also tested, giving rise to a category value of 1–5, where 1 is no organic content and 5 is
very high organic content.
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2.2. Fungal Isolation, Initial Identification, and Deposition
In addition to the seven sites visited in 2013 [1] (dry season), seven further sites in the
Central Highlands were visited in 2015 (rainy season) to collect orchid roots and seeds,
some of the taxa collected are included in Figure 1. Single plants were sampled according to
the collecting permit issued by the Madagascan government, whereby a maximum of five
juvenile and five mature plants could be collected for each species. Material was imported
to the UK and USA with all relevant permissions and authorizations, including CITES
and phytosanitary certification. All root samples were placed in refrigeration (4–6 ◦ C)
immediately upon arrival at Royal Botanic Gardens, Kew (RBG Kew), and Illinois, USA,
from Madagascar after collecting. Samples were promptly processed for isolating fungi
present in the juvenile and mature phase plant roots. This process continued for 10 days
while viable pelotons were available from healthy roots. Colonization of mycorrhiza as
pelotons in the cortical region of root sections was scored as percentage of colonized
root cortical cells, observed in cross-section under a dissection stereomicroscope, for all
three life-forms: epiphtytic, lithophytic, and terrestrial. From root sections with visible
colonization where pelotons were observed, either single pelotons or clumps of cells were
teased out using a sterile scalpel assisted by dissection microscope and plated directly onto
FIM [15] supplemented with 100 mg/L streptomycin sulphate [16] to isolate and identify
the peloton-forming fungi. Culturing of the pelotons was achieved in vitro according to
Zettler et al. [17] and Yokoya et al. [1].
After 1–4 days, hyphal tips that were observed emerging from cortical cells and/or
pelotons under a dissection microscope were subcultured to FIM (RBG Kew) or Potato
Dextrose Agar (PDA, Difco™, Becton, Dickinson and Co., Sparks, MD, USA) (Illinois)
using a sterile scalpel. To safeguard the mycorrhizal OTUs for the purposes of future work
(e.g., symbiotic seed germination) and long-term conservation, they were cryopreserved in
vapour phase of liquid nitrogen, as described by Batty et al. [18].
2.3. Molecular Identification of Fungi by ITS Sequencing
Pure cultures of fungi were positively identified using DNA sequencing as described
in Yokoya et al. [1]. Briefly, DNA from fresh mycelia was extracted in 96-well plates using
the Extract-N-Amp™ Plant Tissue PCR Kit (Sigma Aldrich, UK). PCR amplification of
the ITS region using primer ITS1F with ITS4 and ITS1 with ITS4-tul [19–21] was followed
by Sanger sequencing using the same forward and reverse primers. The forward and
reverse sequences were checked for accuracy and consensus and compared with database
sequences using BLAST (National Center for Biotechnology Information, Bethesda, MD,
USA). Sequences were aligned and grouped into operational taxonomic units (OTUs) based
on a conservative similarity threshold of 95% (Supplementary Materials). Representative
sequences of each OTU were used to re-query the GenBank database using BLAST.
2.4. Seed Germination
Seeds from 14 orchid taxa covering three life forms were cultured in vitro with putative
MFs from different life forms of orchids. On average, 5000–8000 seeds of each orchid taxon
were used with 10 different OTUs of putative mycorrhizal fungi. Seeds were sown in vitro
on standard oatmeal agar (OMA; 7 g agar, 2.5 g rolled oats, 1 L water) as a control medium
tested against OMA inoculated with different fungal isolates. Seed germination data were
collected up to 12 weeks to ensure uniformity in data collecting, although different life
forms of orchids were studied. Seed germination and protocorm development stages
were assigned as defined by Yamazaki and Miyoshi [22]. Stages 3–4 (protocorm) and leaf
forming Stage 5 were the main responses analysed.
2.5. Data Analysis
The number of germinated seeds and number of seeds that developed to Stage 5
were analyzed using the Kaplan–Meier statistical test using the Survival package (R statistical software, Version R 3.0.1, The R Foundation for Statistical Computing) as used by
Diversity 2021, 13, 96
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McNair et al. [23,24]. At 24 weeks, the germination analysis was carried out on each stage
using Chi2 (Microsoft Excel, Microsoft, Reading, UK). The program PVCLUST was used in
R to produce a hierarchical cluster analysis using the Ward method.
Figure 1. Images of some of the orchids studied. (A)—Polystachya sp.; (B)—Angraecum sororium; (C)—Aerangis fastuosa
(growth phase of plants collected); (D)—Habenaria sp.; (E)—Aerangis punctata; (F)—Habenaria simplex; (G)—Polystachya
concreta; (H)—Liparis ochracea; (I)—Jumellea pachyceras; (J)—Eulophia plantaginea; (K)—Disa incarnata.
3. Results
Majority of putative MFs collected during dry season were from juvenile plants while
all MFs collected during rainy reason were from mature plants. Out of six mature plants
yielding putative MFs, five of them were terrestrial orchid taxa sampled in both dry and
rainy seasons (Tables 1 and 2). The remaining one was an epiphyte during rainy season
(Table 2). However, six out of seven taxa yielding putative MFs collected during the dry
season were juvenile plants (Table 1). A full list of Rhizoctonia MFs is included in the
Supplementary Materials.
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Table 1. Life form and growth phase of orchids collected during dry season and OTUs identified.
Taxa (Epiphytes)
Aerangis sp. 1
Aerangis sp. 2
Growth
Phase
OTU
Taxa (Lithophytes)
Growth
Phase
OTU
Aerangis ellisii
Juvenile
cer2
Benthamia sp.
Mature
-
Mature
-
B. cinnabarina
Mature
tul2
Juvenile
-
B. glaberrima
Mature
-
Mature
-
B. rostrata
Juvenile
-
Juvenile
cer1
Juvenile
cer3
Angraecum coutrixii
Taxa (Terrestrial)
Growth
Phase
OTU
Mature
-
Aerangis ellisii
Mature
-
Aerangis punctata
Juvenile
cer1, tul7
Angraecum longicalcar
Mature
-
Calanthe sp.
Mature
-
Angraecum
magdalenae
Juvenile
tul1
Cynorkis gibbosa
Mature
-
Juvenile
tul3
cer4
ser1
Aeranthes sp.
Mature
-
Angraecum sp. 1
Juvenile
-
Angraecum sp. 2
Juvenile
-
Angraecum protensum
Mature
-
Juvenile
-
Angraecum coutrixii
Mature
-
Angraecum
rutenbergianum
Angraecum
protensum
Juvenile
-
Angraecum sororium
Juvenile
-
Angraecum
rutenbergianum
Mature
-
Bulbophyllum sp. 1
Mature
-
Disa incarnata
Mature
-
Bulbophyllum sp. 2
Juvenile
-
Oeceoclades calcarata
Mature
-
Eulophia macra
Mature
-
Bulbophyllum sp. 3
Mature
-
Graphorkis concolor
Mature
-
Bulbophyllum sp. 4
Mature
-
Habenaria sp.
Mature
-
Bulbophyllum sp. 5
Mature
-
Satyrium trinerve
Mature
-
Cynorkis purpurea
Juvenile
tul3
tul4
Jumellea densefoliata
Juvenile
-
Tylostigma sp.
Mature
tul5
Polystachya concreta
Juvenile
ser2, ser3
T. nigrescens
Mature
tul6
P. cultriformis
Juvenile
-
T. tenellum
Juvenile
tul4
Figure 2. Number of putative mycorrhizal and non-mycorrhizal fungi from epiphytic, lithophytic, and terrestrial taxa
studied during rainy season.
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Table 2. Soil data collected during the rainy season from 12 orchids representing three life forms (Figure 2).
Host
Growth
Phase
Life Form
OTU
pH
Nitrogen
(ppm)
Phosphorus
(ppm)
Potassium
(ppm)
Soil Comments
Aerangis fastuosa
Seedling
Epiphyte
-
6.8
20
37
50
Very little soil. Live moss
and dead leaves
Angreacum punctata
Seedling
Epiphyte
-
6.8
20
37
50
Very little soil. Live moss
and dead leaves
Angraecum sororium
Seedling
Lithophyte
-
5.2
20
12
150
Dark, moist, live grass
Disa incarnata
Mature
Terrestrial
-
5
30
5
70
Dry, brown, sandy
Eulophia plantaginea
Mature
Terrestrial
tul9, tul10
5.2
30
5
70
Dry, brown, sandy
Habenaria sp.
Juvenile
Terrestrial
-
5.2
20
5
50
Dark, moist, grass
and moss
Habenaria simplex
Mature
Terrestrial
tul5
5
30
5
70
Dry, brown, sandy
Jumellea pachyceras
Mature
Terrestrial
-
5
30
37
50
Lots of grass roots, wood
and mulch. Dark, moist
Liparis ochracea
Mature
Terrestrial
tul12, cop1,
cop2
6.8
20
37
50
Very little soil. Live moss
and dead leaves
Orchid sp.
Mature
Terrestrial
tul5
6.8
30
12
65
Heavy clay. Dark
Polystachya concreta
Mature
Epiphyte
tul5, tul 13
5
50
12
175
Dark, slightly moist,
some grass roots
Polystachya sp.
Seedling
Epiphyte
-
5
50
12
175
Dark, slightly moist.
some gr ass roots
3.1. Orchid MF Collected during Dry Season
Orchid MF of all three genera of the Rhizoctonia complex were identified from root
collections. This consisted of seven Tulasnella, four Ceratobasidium, and one Serendipita, as
summarised in Table 1. Putative MFs were obtained from only juvenile plants in epiphytes
and lithophytes except Polystachya concreta, however, fungal colonization was observed
from the pelotons extracted from the collected roots of mature epiphytes and lithophytes.
3.2. Orchid MF Collected during Rainy Season
Tulasnella and Serendipita OTUs were identified from root collections during the rainy
season from lithophytes and terrestrials (Table 2). Two putative MFs, Tulasnella, were
obtained from juvenile samples of Polystachya concreta, while one MF obtained from mature
plants in the dry season. However, in all other cases, pelotons grown in culture failed to
identify any putative MFs.
The majority of the orchid species that yielded MF during rainy season were terrestrial
taxa (Table 2, Figure 2). MFs were not obtained from the two terrestrial taxa (Disa incarnata
and Habenaria sp.) out of six taxa studied. Both epiphytes studied (Aerangis fastuosa and
A. punctata) and one lithophyte out of three taxa studied, Polystachya sp, yielded no MFs
(Figure 2).
3.3. Soil Characteristics and Fungal Diversity in Orchids Collected during Rainy Season
More non-mycorrhizal fungi were found in plants grown at lower nitrogen content
(Figure 3A). Except in Liparis ochracea, MF were found in plants grown at phosphorus content of 12 ppm or below (Figure 3B). In orchids from soil with 37 ppm of phosphorus, either
no MFs or greater numbers of non-MF were identified. In Liparis ochracea, the diversity of
fungi was significantly different compared to all other orchids studied (Supplementary
Materials). Tulasnella (tul12) and Coprinellus sp. and C. disseminatus (putative MF) were
found in addition to several dark septate endophytes and other endophytes in this terrestrial species. The fungal diversity in Aerangis punctata is presented as a comparison
(Supplementary Materials).
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Figure 3. Fungal diversity, both putative mycorrhizal and non-mycorrhizal, in 12 orchids collected during the rainy season
relative to nitrogen (A) and phosphorus (B) content in the soil.
Immediately after rains, when L. ochracea roots were collected and fungi were identified, the fungal diversity data shows an interesting picture. The plants were found in shady,
moist forest with moss and litter as the substrate with very little soil (pH 6.2). Many of the
orchids collected during this period were from soil with comparatively low phosphorus
content (5–12 ppm), while substrate associated with L. ochracea had comparatively higher
phosphorus content (37 ppm).
Aerangis punctata was collected in both dry and rainy seasons and showed seasonal
variability in the recruitment of fungi. The species yielded MFs Ceratobasdium and Tulasnella
(OTUcer1 and OTUtul7) during the dry season (Table 1), while no orchid MF were isolated
from the rainy season collection (Supplementary Materials). Dothideomycetes, Lophiostoma,
Pleosporales, Capronia, Cladiophialophora, and Virgaria nigra were the fungi isolated during
the dry season, while 15 completely different non-MF endophytes were identified in the
rainy season. Roots with even higher colonisation sometimes failed to yield MF. Even
where pelotons were present, they were not be viable, because they are seasonally active.
Tulasnella was the only MF lineage from both Angraecum magdalenae and A. protensum.
Angraecum sororium sp. collected during the rainy season also yielded tul5, Tulasnella MF
(Table 2). Although this is a small selection of Angraecum taxa, in the majority of our
samples, we observed pelotons in <60% of the cortical cells, always on the side of the root
that was in close contact with the substrate. In the several species of Bulbophyllum that were
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collected, none of the roots yielded any MF. Eulophia plantaginea and Polystachya concreta
yielded two Tulasnella OTUs, both from soil with low levels of phosphorus (5 and 12 ppm,
respectively) (Table 2, Figure 3B). Our findings show that most of the MF OTUs were found
at the tip of the roots.
3.4. Symbiotic Seed Germination
Diverse orchids were sown with the available range of MFs to see how compatible
they are to produce symbiotic seedlings. However, MFs from all 14 seed sources were
not available for this study (Table 3). Nine out of 14 orchid taxa were able to germinate
and develop to Stage 5 (seedling stage, Figure 4) with at least one of the 10 selected
isolates. Aerangis ellisii and Cynorkis purpurea were germinated by their own MF (in bold
in Table 3). In a previous study as part of this project, Yokoya et al. [1] showed MFmediated symbiotic seed germination in Tylostigma nigrescens. In the lithophytic Angraecum
rutenbergianum, the Tulasnaella OTUs tul1 (from A. magdalenae, a lithophyte) and tul2
(from the terrestrial Benthamia cinnabarina) were able to facilitate germination to Stage 5,
suggesting that germination can be promoted by isolates of distant species of orchid
spanning different life forms. The epiphytic Bulbophyllum peyrotii seeds reached seedling
stage (Stage 5) with cer1 from Cynorkis purpurea (terrestrial). Both species of Habenaria, from
montane grassland, produced seedlings with orchid MF from Aerangis sp. (cer3) collected
from gallery forest.
Figure 4. Symbiotic seed germination in vitro of Bulbophyllum peyrotii (A,B), Angarecum rutenbergianum (C), and Tylostigma
nigrescens (D).
Development to the protocorm stage alone was not regarded as complete germination
induced by the presence of fungi, given that water imbibition and other factors (e.g.,
nutrient requirements for seed germination fulfilled by OMA components) may also be
involved. Embryos in many the orchids studied here developed to the protocorm stage on
oatmeal agar controls without fungal inoculation.
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Table 3. Epiphytic (E), lithophytic (L), and terrestrial (T) seed sources (14 taxa), fungal source (nine taxa covering all three life forms), and germination stages reached by the seeds after 12
weeks. Number 5 represents seedling development (bold number 5 represents germination with own mycorrhizal fungus).
cer1
cer2
cer3
ser2
tul1
tul2
tul3
ser3
tul5
Cynorkis
purpurea (T)
Polystachya
concreta (E)
Angraecum
magdalenae
(L)
Benthamia
cinnabarina
(T)
Cynorkis
purpurea (T)
Polystachya
concreta (E)
Tylostigma sp.
(T)
5
-
-
-
-
-
-
5
5
5
5
4
-
5
5
5
4 (74)
-
3
3
-
3
-
-
-
-
2 (2.1)
5
4
-
3
4
4
-
-
Data not available
5
4
-
-
4
-
4
-
4 (18.2)
4
4
-
-
-
5
4
-
4 (14.8)
Aerangis
punctata
(E)
Aerangis
ellisii (E)
Aerangis
sp. (E)
Aerangis ellisii (E) *
-
5
Cynorkis purpurea (T) **
5
Angraecum coutrixii (L)
-
Habenaria quartziticola (T)
4
4
Habenaria ambositrana (T)
4
4
Benthamia cinnabarina (T)
4
-
OTU and Fungal Source
ser1
Oatmeal Control- Most
Advanced Stage (Total
Germination Percentage
of Stages 2–4)
Seed source
0
Disa incarnata (T)
4
-
4
-
4
4
4
4
-
-
4 (10.8)
Satyrium trinerve (T)
-
-
4
-
3
-
3
-
-
-
3 (19.2)
Tylostigma nigrescens (T)
4
-
4
4
4
-
5
4
-
-
3 (21.4)
Aerangis citrata (E)
-
-
4
-
-
3
-
-
-
-
1 (0)
Bulbophyllum peyrotii (E)
-
-
-
5
4
-
4
4
-
-
4 (98)
Bulbophyllum sp. (E)
-
-
-
-
-
4
-
-
-
-
1 (0)
Angraecum
rutenbergianum (E/L)
-
-
-
3
-
5
5
3
-
-
2 (5.1)
Tylostigma sp. (T)
4
-
4
4
4
5
5
4
4
-
4 (37.8)
* Aerangis ellisii seeds were germinated by Cer2 and Cer3 (from Aerangis sp.), as reported by Kendon et al. [25]. ** Cynorkis purpurea seeds were germinated better by non-Cynorkis orchid mycorrhizal fungi than
own mycorrhizal fungus (ser1), as reported by Rafter et al. [24].
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4. Discussion
This study, encompassing three long-distance trips to the Central Highlands of Madagascar during a five-year period, documents, for the first time, fungal diversity associated
with roots of 50 different taxa found on rocks (lithophytes), on host trees (epiphytes) and
in soil (terrestrials). In addition, we noted some correlations between fungal diversity
and substrate nutrients (N, P, K), pH, or dry vs. rainy season, suggesting patterns in the
environmental conditions that these orchids and their fungi may require for their survival
in situ. While our sampling was limited, we did reveal several preliminary findings that we
think warrant more study. For example, during the dry season, five of the epiphytes and
lithophytes sampled (Aerangis, Angraecum sp.) harboured mycorrhizal OTUs assignable to
Ceratobasidium and Tulasnella that were present in juvenile plants, but none were detected
in roots of mature plants (Table 1). It is conceivable that these juvenile-plant-derived fungi
were root-inhabiting ‘relics’ that played a role in seed germination and early seedling
development, and simply persisted in the root system at the time they were collected.
As to why roots from the mature stages were devoid of fungi, it is not known but may
be attributed to a number of factors [5]. For example, Dycus and Knudson [26] concluded
that the velamen and exodermis in aerial roots of epiphytic orchids serve as a barrier to the
uptake of water and certain solutes including available N and P. However, passage into
the cortex was noted through root surfaces in contact with the substratum (bark, clay pots)
where cells in the velamen layer were notably thinner. Bayman et al. [27] noted that fungal
pelotons in the cortical region of epiphytic orchid roots were positioned closest to the
bark substrate, suggesting that this is the entry point for fungal colonization into the root.
During our sampling in Madagascar, the majority of roots from epiphytes and lithophytes
that were acquired were in close contact with the substratum. Thus, we hypothesize that
the lack of fungi in samples from mature epiphytes and lithophytes may be attributed to a
reduced dependency on MF of mature plants, which would already have developed root
systems that are well associated with their substrate. Conversely, juvenile lithophytes and
epiphytes might be more dependent on water and nutrient uptake via their mycorrhizal
associates, particularly during the dry season. Although the sample sizes in this study are
small, our work provides some evidence that juvenile epiphytes are more reliant on their
root symbionts more during the dry season (as shown in Aerangis spp. and Angraecum
magdalenae).
In contrast, roots of terrestrials have a reduced velamen layer and remain underground
where access to moisture is more readily available, and it is not surprising that even mature
terrestrials (Benthamia, Tylostigma) harboured mycorrhizal OTUs (Tulasnella) during the dry
season (Table 1). However, deeper into the dry season, most terrestrials undergo dormancy
as drought-resistant tubers, and would be less dependent on MF during this period. There
were several terrestrial species collected in the dry season that had good root colonisation
but failed to yield MF in culture, which could be explained by the imminent dormant
period. The emerging picture is that terrestrial orchids harbour MFs throughout their life
cycle but are apparently not associated with them during dormancy, while lithophytes and
epiphytes are more reliant on MF during the juvenile stages of development but did not
have detectable MF in mature specimens. From the standpoint of conservation, it would
be suggested that reintroduction of symbiotically grown seedlings of any life form should
be performed during the rainy season as the best option to ensure that there is adequate
moisture for both organisms (seedlings and fungi) leading up to establishment. Correlating
seasonality with orchid growth stages and the fungi that are involved has the potential to
render conservation methods more effective.
Availability of compatible fungi within the ecosystem underpins the distribution of
orchids, irrespective of their life forms. Conversely, resilience of the fungal populations
in the ecosystem may depend on the abundance of host plants in a given population. It
has been shown that these fungi persist within the orchid even after the germination event.
Shrinking orchid populations will directly contribute to declining fungal diversity and
density, and vice versa. Our limited sampling of a large number of orchid species is not
Diversity 2021, 13, 96
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fully sufficient to prove this point. Analysing the influence of ecological constraints and
the relationship between orchids and orchid MF is highly relevant. Plants harbouring
MF displayed more advanced growth attributes, as reported in the temperate orchid
Cephalanthera rubra [28], although the isolated mycorrhizal OTUs were not successful in
germinating the C. rubra seeds. This shows orchids recruit and harbour some of the MF,
but not all of them contribute to successful seed germination.
Two species studied in detail in in vitro germination studies, Aerangis ellisii (epiphyte/lithophyte) and Cynorkis purpurea (terrestrial), displayed contrasting results under
in vitro symbiotic germination conditions. C. purpurea turned out to be a generalist [24]
while A. ellisii was a specialist [25] in terms of compatible mycorrhizal associates. Seeds of
Cynorkis purpurea germinated successfully with its own MF, but more advanced seedling
development occurred with MF sourced from other orchids. Specificity varies among
species, in terrestrial orchids [29–32]; in neotropical, epiphytic orchids [33–38]; and in lithophytes [25]. It is argued that generalist orchids might be more prevalent in nutrient-poor or
drought-stressed niches [31]. Without reintroduction trials, in vitro seed germination supported by MF (Table 3) is not a fully reliable test, as compatible fungi may differ in the wild
environment. Future studies using different growth phases of orchids offer opportunities to
identify compatible MF for seed germination that can potentially be used for conservation.
Screening of orchids to culture and identify lineages of a wide spectrum of fungi will help
support in vitro germination research and therefore improve the conservation prospects of
these species.
Considering that 80% of Madagascar’s natural vegetation has been cleared or permanently altered [2] due to illegal mining and other anthropogenic activities [3], documenting
the nutrients (N, P, K) present in orchid substrata was another focus of our study. This
is especially true in the Central Highlands, where the controlled burning of tapia forests
and grasslands can have a profound negative impact on vegetation [39] and presumably
soil nutrients. Not surprisingly, more is currently known about the impact of nutrients on
terrestrial orchids. Compared to other plants, orchids seem to favor organic forms of N
over inorganic forms [40], and higher levels of the latter may actually inhibit growth or lead
to toxicity [41,42]. Similarly, Jacquemyn et al. [31] suggested that generalist orchids might
be more prevalent in nutrient-poor or drought-stressed niches. Recently, Figura et al. [43]
determined that orchids inhabiting oligotrophic habitats were more sensitive to nitrate
levels compared to those found in eutrophic habitats. They also reported that nitrate levels
had a negative impact on orchid distribution by inhibiting seed germination. In our study,
N levels (ppm) were relatively consistent among the substrata for epiphytes, lithophytes,
and terrestrials alike (Table 2), possibly because our sampling took place in more remote
areas with little intensive agriculture. Given the findings reported by other researchers, we
advocate that future conservationists focus their efforts on regions of the Itremo Massif that
remain free of human activity as much as possible. Analysis of soil/supporting substrate
for all life forms of orchids to enable successful repatriation back to the wild must also be
included in the list of future research priorities.
While we still know fairly little about how nutrients in the landscape affect orchid
distribution, even less is known about the role of nutrient sources (mineral or organic) on
orchid MF. Studies by Nurfadilah et al. [44], however, explored the impact of C, N, and P
on the growth of all three major genera of orchid mycorrhizal associates—Ceratobasidium,
Sebacina, and Tulasnella—and determined that these fungi exploited a ‘wide and variable
menu’ of C, N, and P sources. Additionally, all three genera utilised ammonium as a source
of inorganic N. In tropical regions that are subjected to rainy and dry seasons, nutrient
levels could potentially fluctuate and have the potential to influence the distribution and
survival of orchid mycorrhizal associates and the seedlings they support. In the present
study, we attempted to correlate nutrient levels with the presence of MF and non-MF
and seasonality. Although highly preliminary, our findings seem to suggest that lower P
content (12 ppm or below) may promote greater colonisation of orchid roots by Rhizoctonia
fungi compared to the non-mycorrhizal endophytes (Figure 3B). To our knowledge, this
Diversity 2021, 13, 96
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potentially important aspect of orchid ecology has not been addressed in the literature and
warrants study.
5. Conclusions
Diverse orchids of Central Highlands harbour Tulasnella lineage of MF as the dominant
group. The majority of non-juvenile samples of epiphytic and lithophytic orchids failed
to yield any culturable putative MF during the dry season. This means that seed baiting,
a time-consuming and labour-intensive method, cannot be completely ruled out as an
alternative route to identify the compatible mycorrhizal associates as part of a longer
duration conservation action planning for orchids that are at the brink of extinction. On
the other hand, terrestrial species yielded MF during both dry and rainy seasons from all
growth stages. We also found that putative MFs isolated from closely related or distant
taxa can initiate germination of seeds in vitro. The implications of using putative MF from
distantly related orchids is a point to be explored further, as it brings up the question of
whether it is critically necessary to pursue the original mycorrhizal associate, particularly
when isolation and culture of an endangered orchid’s own MF becomes difficult due to
them having been depleted to small numbers in the wild. In addition to the compatibility
assessment for mycorrhizal symbionts, reintroduction/reinforcement in the wild requires a
detailed study on soil characteristics. Both the fungus and orchid collected from a specific
habitat must be studied for their resilience in supporting each other during and after
reintroduction to achieve high survival rates of the orchid–fungus complex and attain
sustainable success in the conservation of rare orchids.
Supplementary Materials: The following are available online at https://www.mdpi.com/1424-2
818/13/2/96/s1, Table S1: Closest match in Genbank of mycorrhizal and non-mycorrhizal fungi
identified from Liparis ochracea; Closest match in Genbank of mycorrhizal and non-mycorrhizal
fungi identified from Aerangis punctata; Orchid taxa studied, accession of putative mycorrhizal fungi
isolated, name of OTUs and sequence.
Author Contributions: Conceptualization, V.S. and L.W.Z.; methodology, V.S., K.Y., L.W.Z.; validation, K.Y., V.S.; formal analysis, K.Y., J.B., J.P.K., A.S.J.; investigation, K.Y., L.W.Z., L.R., J.P.K., J.B.,
A.S.J., V.S., E.S.; resources, V.S., L.W.Z.; data curation, J.P.K., V.S.; writing—original draft preparation,
V.S.; writing—review and editing, K.Y., L.W.Z., L.R., J.P.K., J.B., A.S.J., V.S.; supervision, V.S., L.W.Z.;
project administration, V.S., L.W.Z.; funding acquisition, V.S., L.W.Z. All authors have read and
agreed to the published version of the manuscript.
Funding: We acknowledge the assistance received from Jacky Andriantiana (Parc Botanique et
Zoologique de Tsimbazaza), Edward Jones (Royal Botanic Gardens, Kew), and Amanda Wood
(Illinois College) for field support during the collection. The technical support from Margaret Ramsay,
Ebrailon Masetto, and James Pickering (Royal Botanic Gardens, Kew) is also much appreciated.
Institutional Review Board Statement: Not Applicable.
Informed Consent Statement: Not Applicable.
Data Availability Statement: Publicly available datasets were analyzed in this study.
Acknowledgments: The authors acknowledge funding from the Sainsbury Orchid Project, BenthamMoxon Trust, and Illinois College.
Conflicts of Interest: The authors declare no conflict of interest.
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