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Journal ArticleDOI

A fine-scale spatial analysis of fungal communities on tropical tree bark unveils the epiphytic rhizosphere in orchids.

TL;DR: In this article, the authors conducted environmental metabarcoding of the ITS-2 region to understand the spatial structure of fungal communities of the bark of tropical trees, with a focus on epiphytic orchid mycorrhizal fungi.
Abstract: Approximately 10% of vascular plants are epiphytes and, even though this has long been ignored in past research, are able to interact with a variety of fungi, including mycorrhizal taxa. However, the structure of fungal communities on bark, as well as their relationship with epiphytic plants, is largely unknown. To fill this gap, we conducted environmental metabarcoding of the ITS-2 region to understand the spatial structure of fungal communities of the bark of tropical trees, with a focus on epiphytic orchid mycorrhizal fungi, and tested the influence of root proximity. For all guilds, including orchid mycorrhizal fungi, fungal communities were more similar when spatially close on bark (i.e. they displayed positive spatial autocorrelation). They also showed distance decay of similarity with respect to epiphytic roots, meaning that their composition on bark increasingly differed, compared to roots, with distance from roots. We first showed that all of the investigated fungal guilds exhibited spatial structure at very small scales. This spatial structure was influenced by the roots of epiphytic plants, suggesting the existence of an epiphytic rhizosphere. Finally, we showed that orchid mycorrhizal fungi were aggregated around them, possibly as a result of reciprocal influence between the mycorrhizal partners.

Summary (3 min read)

1. Introduction

  • The authors hypothesized that (i) as described in soils, these communities have no random distribution on the bark .
  • Due to the ability of many fungi to colonize plant roots, (ii) their distribution should be modulated by the distance to roots of vascular epiphytes.
  • Particularly, (iii) communities of OMF should be aggregated around their orchid hosts.

2.1 Study area

  • The elevation provides frequent fogs throughout the year, and the humidity is around 80%, even in the dry season.
  • The climate of the region is humid subtropical mesothermic, with temperatures ranging from 17 to 23°C and annual rainfall averaging 1300 mm (Rolim & Ribeiro, 2001) .
  • This forest is characterized by medium to large trees, and a high diversity of orchid species, the majority of which are epiphytic (Lana et al., 2018) .

2.2 Bark and root sampling

  • Two trees belonging to Siparuna sp. (Siparunaceae; tree 1) and Himathanthus sucuuba (Apocynaceae; tree 2) were selected in February 2015 and February 2016 (95 m away from each other) respectively because they had epiphytic orchids growing on their lower trunk, namely Isochilus linearis and Epidendrum armeniacum.
  • Bark was also collected under each root sample.
  • All samples were frozen at -20°C within few hours in the nearby field laboratory of the Serra do Brigadeiro State Park headquarters for downstream molecular analyses.
  • Two additional thin sections of orchid roots surrounding each sampled piece were collected to check for mycorrhizal fungal colonization on the following day under the microscope and all, without exception, displayed hyphal coils in at least one of each inspection section.

2.3 High-throughput sequencing of fungal communities

  • Tagging system negative controls were performed at this step (Hornung et al., 2019; Zinger et al., 2019) , i.e., pairs of barcoded primers were intentionally omitted in the final sequencing to control for cross-contamination.
  • Plate designs were randomized in order to avoid possible cross-contamination leading to misinterpretation in subsequent spatial analysis.
  • After visualization on gel, the positive amplicons were purified with NucleoMag® NGS Clean-up and Size Select (Macherey-Nagel, GmbH & Co KG.), quantified by fluorescence with Qubit TM dsDNA High-Sensitivity (Invitrogen TM ), and pooled in equimolar ratios prior to library preparation and 2x250 bp paired-end sequencing on an Illumina MiSeq platform at Fasteris (Geneva, Switzerland).
  • Three positive controls (mock community) and three negative controls (ultrapure water) were used per PCR trial , resulting in a total of 36 positive and 36 negative controls in total.

2.5 Fungal functional guilds

  • OTUs found in at least one orchid root sample were considered as endophytes.
  • Among them, those Basidiomycota belonging to Tulasnellaceae, Ceratobasidiaceae (Veldre et al., 2013) , Serendipitaceae (Weiß et al., 2016) , and Atractiellales (Kottke et al., 2010) were recognized as orchid mycorrhizal fungi (OMF) (Dearnaley et al., 2012) .
  • Besides, trophic guilds were assigned to all OTUs using FunGuild (Zanne et al., 2019) : the authors chose to keep those which were either exclusively saprotrophs, symbiotrophs, plant pathogens, or lichenized fungi.
  • For the remaining OTUs, guilds provided by FunGuild were validated based on the author's expertise.
  • The OMF were kept in a separate category despite their saprotrophic and symbiotrophic ability (Dearnaley et al., 2012; Selosse & Martos, 2014) .

2.6 Statistical analyses

  • As the similarities between samples are not independent of one another, coefficients of the binomial GLM were obtained using a leave-one-out Jackknife procedure as described in (Millar et al., 2011) .
  • The significance of the distance decay of similarity was tested using a permutational Mantel test (Spearman method, 9999 permutations; Anderson et al., 2013) , while the significance of the distance decay of richness was assessed by ANOVA (F-test).

3.1 Roots and bark harbored distinct, but partially overlapping fungal communities

  • Among the 31 OMF OTUs that were found, encompassing the four OMF families (see 2.5, Fig. 3 ), only five OTUs were shared between the two trees after rarefaction (Table S4 ) and only one OTU (Tulasnellaceae, TUL-1) when considering the roots only (Table S4 , S5).
  • The sharing of OMF between grids was not statistically different to that of other fungi, meaning that the trees harbored different fungal communities overall.
  • On grid 2, where two orchid species co-exist, OMF OTUs belonging to Ceratobasidiaceae (CER-1) and Serendipitaceae (SER-1) were shared between the two species when they were spatially close (Table S5 , Fig. S1 ).

3.2 All fungal communities were spatially structured

  • Spatial autocorrelation of single OTUs showed that only OMF on grid 2 tend to be more frequently spatially clustered than other fungi (Table S6 ).
  • OMF families showed vertical stratification on grid 2 that covered a greater height on the tree (1.7 m), whereas this pattern was not obvious on grid 1 (covering 0.7 m only; Fig. S9 ).

3.3 Epiphytic roots influenced all fungal communities

  • The Jaccard similarity between roots and bark fungal compositions significantly decreased with increasing distance from the roots for the whole fungal community on both grids (Fig. 5 ).
  • This was also observed for endophytes on both grids, for non-OMF symbiotrophs on grid 1 only, and for OMF, plant pathogens and saprotrophs on grid 2 only (Table 1, Fig. 5 ; see also Fig. S10 and Table S8 for details).
  • The distance decay of bark fungal richness showed contrasting results with either non-significant or opposite results between grids (Fig. S12 -14, Table S9 ).
  • By comparing the density distribution of OMF versus endophytes (distance from roots beyond which 80% of the occurrences of a given OTU are limited), the OMF were not statistically closer to roots than other endophytes (Wilcox tests, W = 370, p = 0.423 and W = 1142, p = 0.397 for grid 1 and 2, respectively).

4.1 Features of bark fungal communities compared to the soil's

  • This thinness allowed us to exhaustively sample fungal communities at a given position.
  • Whether these communities are spatially structured or are either homogeneously or randomly distributed remained an open question, which the authors investigated in this study.

4.5 Fungal communities could modulate epiphytic plant population dynamics

  • Here, the OMF were more spatially clustered than any other fungi (Table S6 ), reflected in the vertical stratification on grid 2 (Fig. S9 ), which suggests that they could strongly constrain orchid seed germination.
  • In soil, it has also been proposed that the patchiness of orchid individuals (Jacquemyn et al., 2007) could be due to that of their mycorrhizal partners (Jacquemyn et al., 2012) .

4.6 Conclusion and perspectives

  • The authors observed a vertical niche differentiation for OMF communities, but not for other fungal guilds, probably because their sampling design was not appropriate to investigate such vertical gradients.
  • Yet, a possible trend for lower vertical than horizontal structure was observed.

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A ne-scale spatial analysis of fungal communities on
tropical tree bark unveils the epiphytic rhizosphere in
orchids
Remi Petrolli, Conrado Augusto Vieira, Marcin Jakalski, Melissa F Bocayuva,
Clément Vallé, Everaldo da Silva Cruz, Marc-andré Selosse, Florent Martos,
Maria Catarina M. Kasuya
To cite this version:
Remi Petrolli, Conrado Augusto Vieira, Marcin Jakalski, Melissa F Bocayuva, Clément Vallé, et al.. A
ne-scale spatial analysis of fungal communities on tropical tree bark unveils the epiphytic rhizosphere
in orchids. New Phytologist, Wiley, In press, �10.1111/nph.17459�. �hal-03279090�

1
A fine-scale spatial analysis of fungal communities on tropical tree bark unveils the
1
epiphytic rhizosphere in orchids
2
3
REMI PETROLLI
1*
, CONRADO AUGUSTO VIEIRA
1,2*
, MARCIN JAKALSKI
3
, MELISSA
4
F. BOCAYUVA
2
, CLEMENT VALLE
1
, EVERALDO DA SILVA CRUZ
2
, MARC-ANDRÉ
5
SELOSSE
1,2,3
§
, FLORENT MARTOS
1
§
, MARIA CATARINA M. KASUYA
2
§
6
7
1
Institut de Systématique, Évolution, Biodiversité (ISYEB), Muséum national d’Histoire
8
naturelle, CNRS, Sorbonne Université, EPHE, CP 39, 57 rue Cuvier, F-75005 Paris, France
9
2
Department of Microbiology, Viçosa Federal University (UFV), P. H. Rolfs street CEP:
10
36570-900, Viçosa, Minas Gerais, Brazil
11
3
University of Gdańsk, Faculty of Biology, ul. Wita Stwosza 59, 80-308 Gdańsk, Poland
12
*
These authors contributed equally to this work.
13
§
These authors supervised equally this work.
14
15
Rémi Petrolli (Corresponding author)
16
Muséum National d’Histoire Naturelle
17
UMR 7205, Institut de Systématique, Évolution et Biodiversité (ISYEB),
18
12 rue Buffon, CP 39, 75005 Paris, France
19
Email : remi.petrolli@mnhn.fr
20
21
6162 words: 842 words (Introduction), 1916 words (M&M), 1146 words (Results) and 2258
22
words (Discussion). 5 colored figures, 1 table, and 23 supplementary figures and tables.
23
24
We declare no conflict of interest regarding this work.
25

2
Abstract
26
Approximately 10% of vascular plants are epiphytes and, even though this has long
27
been ignored in past research, can interact with a variety of fungi, including mycorrhizal
28
ones. However, the structure of fungal communities on bark, as well as their relationship
29
with epiphytic plants, is largely unknown.
30
To fill this gap, we conducted environmental metabarcoding of ITS-2 region to
31
understand the spatial structure of fungal communities of the bark of tropical trees, with
32
a focus on epiphytic orchid mycorrhizal fungi, and tested the influence of root
33
proximity.
34
For all guilds, including orchid mycorrhizal fungi, fungal communities were more
35
similar when spatially closed on bark, i.e., displayed positive spatial autocorrelation.
36
They also showed distance decay of similarity from epiphytic roots, meaning that their
37
composition on bark increasingly differed, compared to roots, with distance from roots.
38
We first showed that all the investigated fungal guilds presented a spatial structure at
39
very small scales. This spatial structure was influenced by the roots of epiphytic plants,
40
suggesting the existence of an epiphytic rhizosphere. Finally, we showed that orchid
41
mycorrhizal fungi were aggregated around them, possibly resulting from a reciprocal
42
influence between the mycorrhizal partners.
43
44
45
Key words
46
epiphytism; fungal guilds; metabarcoding; fungal spatial distribution; orchid mycorrhizal fungi;
47
Tulasnellaceae
48
49
50

3
1. Introduction
51
52
Although globally distributed, microorganisms present a highly variable local richness and a
53
spatial structure at every scale (from centimeters to thousands of kilometers), especially in soils
54
(Green et al., 2004; Green & Bohannan, 2006). Much of the soil microbial biodiversity appears
55
to be intrinsically linked with plants in the rhizosphere and controls their community structure
56
by monitoring soil-root interactions (Bever et al., 2010). Reciprocally, soil microorganisms that
57
develop nutritional and protective symbioses with roots are especially structured by host
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presence and diversity (Peay et al., 2013) such as the mycorrhizal fungi that associate with
59
approximately 90% of the vascular land flora (Van Der Heijden et al., 2015; Brundrett &
60
Tedersoo, 2018). Fungal metabarcoding studies in soils have shown that the mycorrhizal taxa
61
are not randomly distributed, but exhibit spatial structure at rather fine scales, in temperate as
62
in tropical systems (Anderson et al., 2014; Bahram et al., 2016; Coince et al., 2013; Pickles et
63
al., 2010; Tedersoo et al., 2010; Zhang et al., 2017), i.e., a patchiness due to host distribution,
64
but also other factors such as spore dispersal and community interactions (Hanson et al., 2012).
65
However, the characterization of the underground distribution of soil fungi (mycorrhizal fungi,
66
saprotrophs or pathogens) is complicated by the three-dimensional nature of soils, since
67
differences may exist between soil horizons (Anderson et al., 2014; Bahram et al., 2015).
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69
Unlike soils, tree barks can be easily investigated as their multiple layers can be sampled and
70
sequenced at once, especially on young trees where the bark is usually thin. Thus, young barks
71
can be seen as virtually two-dimensional and are ideal systems for surveying the spatial
72
distribution of fungal communities and mycorrhizal taxa around their epiphytic plant hosts.
73
Indeed, ca. 10% of vascular plant species root on barks in the tropical wet forests around the
74
globe (Zotz, 2016). These plants have long been considered as essentially non-mycorrhizal in
75

4
such aerial substrates (Lehnert et al., 2017; Brundrett & Tedersoo, 2018; but see Rowe &
76
Pringle, 2005) and their fungal partners have thus so far largely been ignored. However, there
77
is now growing interest in the field of epiphytic fungal endophytes which could strongly
78
influence the dynamics of epiphyte plant populations (Leroy et al., 2019). One symbiosis that
79
regularly occurs in the epiphytic habitats is the orchid mycorrhiza (Martos et al., 2012; Herrera
80
et al., 2018; Novotná et al., 2018). Epiphytic orchids, representing no less than 80% of this
81
hyper-diverse plant family (with over 25 000 species (Givnish et al., 2015)), harbor typical
82
hyphal coils within their root cortical cells, which are formed by the same families but different
83
species of saprotrophic basidiomycetes (Dearnaley et al., 2012; Martos et al., 2012; Xing et al.,
84
2019) compared to soil. The fungi are also required for germination of the minute, nutrient-
85
poor orchid seeds (Smith & Read, 2008). It was therefore hypothesized that the distribution of
86
orchids must be constrained by that of their mycorrhizal fungi (McCormick & Jacquemyn,
87
2014; McCormick et al., 2018)
88
89
The distribution of orchid mycorrhizal fungi (OMF) has been investigated in soils (Jacquemyn
90
et al., 2014, 2017; McCormick & Jacquemyn, 2014; McCormick et al., 2016, 2018; Voyron et
91
al., 2017), but only marginally on barks (Kartzinel et al., 2013), perhaps because most studies
92
focus on temperate and Mediterranean ecosystems where orchids are strictly terrestrial. For
93
example, two recent studies (Waud et al., 2016b,a) showed a decline in abundance and
94
similarity composition of OMF with distance from adult orchids, which likely explains the
95
patchy distribution of grassland orchids (Jacquemyn et al., 2007, 2014). Still in grassland
96
habitats, Voyron et al., (2017) found that communities of OMF are more similar in nearby soil,
97
i.e., display spatial autocorrelation (Hanson et al., 2012). As for the epiphytic environment,
98
very little is known on the spatial distribution of mycorrhizal fungi on bark [but see (Izuddin et
99
al., 2019) for a first approach]. Similarly, the evolution of their community structure by distance
100

Citations
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Journal ArticleDOI
TL;DR: The root mycobiome plays a fundamental role in plant nutrition and protection against biotic and abiotic stresses as discussed by the authors , and it is known that the numerous fungi involved in root symbioses are often shared between neighboring plants, thus forming complex plant-fungus interaction networks of weak specialization.
Abstract: The root mycobiome plays a fundamental role in plant nutrition and protection against biotic and abiotic stresses. In temperate forests or meadows dominated by angiosperms, the numerous fungi involved in root symbioses are often shared between neighboring plants, thus forming complex plant-fungus interaction networks of weak specialization. Whether this weak specialization also holds in rich tropical communities with more phylogenetically diverse sets of plant lineages remains unknown. We collected roots of 30 plant species in semi-natural tropical communities including angiosperms, ferns, and lycophytes, in three different habitat types on La Réunion island: a recent lava flow, a wet thicket, and an ericoid shrubland. We identified root-inhabiting fungi by sequencing both the 18S rRNA and the ITS2 variable regions. We assessed the diversity of mycorrhizal fungal taxa according to plant species and lineages, as well as the structure and specialization of the resulting plant-fungus networks.The 18S and ITS2 datasets are highly complementary at revealing the root mycobiota. According to 18S, Glomeromycotina colonize all plant groups in all habitats forming the least specialized interactions, resulting in nested network structures, while Mucoromycotina (Endogonales) are more abundant in the wetland and show higher specialization and modularity compared to the former. According to ITS2, mycorrhizal fungi of Ericaceae and Orchidaceae, namely Helotiales, Sebacinales, and Cantharellales, also colonize the roots of most plant lineages, confirming that they are frequent endophytes. While Helotiales and Sebacinales present intermediate levels of specialization, Cantharellales are more specialized and more sporadic in their interactions with plants, resulting in highly modular networks.This study of the root mycobiome in tropical environments reinforces the idea that mycorrhizal fungal taxa are locally shared between co-occurring plants, including phylogenetically distant plants (e.g. lycophytes and angiosperms), where they may form functional mycorrhizae or establish endophytic colonization. Yet, we demonstrate that, irrespectively of the environmental variations, the level of specialization significantly varies according to the fungal lineages, probably reflecting the different evolutionary origins of these plant-fungus symbioses. Frequent fungal sharing between plants questions the roles of the different fungi in community functioning and highlights the importance of considering networks of interactions rather than isolated hosts.

7 citations

Journal ArticleDOI
TL;DR: Zhang et al. as discussed by the authors compared the fungal colonization process among two compatible and two incompatible fungi during seed germination of Dendrobium officinale and found that compatible fungi could effectively promote seed growth up to seedlings, while incompatible fungi may stimulate germination but do not support subsequent seedling development.
Abstract: Orchids highly rely on mycorrhizal fungi for seed germination, and compatible fungi could effectively promote germination up to seedlings, while incompatible fungi may stimulate germination but do not support subsequent seedling development. In this study, we compared the fungal colonization process among two compatible and two incompatible fungi during seed germination of Dendrobium officinale. The two compatible fungi, i.e., Tulasnella SSCDO-5 and Sebacinales LQ, originally from different habitats, could persistently colonize seeds and form a large number of pelotons continuously in the basal cells, and both fungi promoted seed germination up to seedling with relative effectiveness. In contrast, the two incompatible fungi, i.e., Tulasnella FDd1 and Tulasnella AgP-1, could not persistently colonize seeds. No pelotons in the FDd1 treatment and only a few pelotons in the AgP-1 treatment were observed; moreover, no seedlings were developed at 120 days after incubation in either incompatible fungal treatment. The pattern of fungal hyphae colonizing seeds was well-matched with the morphological differentiation of seed germination and seedling development. In the fungal cocultural experiments, for both orchids of D. officinale and Dendrobium devonianum, cocultures had slightly negative effects on seed germination, protocorm formation, and seedling formation compared with the monocultures with compatible fungus. These results provide us with a better understanding of orchid mycorrhizal interactions; therefore, for orchid conservation based on symbiotic seed germination, it is recommended that a single, compatible, and ecological/habitat-specific fungus can be utilized for seed germination.

6 citations

Posted ContentDOI
11 May 2022-bioRxiv
TL;DR: In this article , the root mycobiome plays a fundamental role in plant nutrition and protection against biotic and abiotic stresses, and the diversity of mycorrhizal fungal taxa according to plant species and lineages, as well as the structure and specialization of the resulting plant-fungus networks.
Abstract: Background The root mycobiome plays a fundamental role in plant nutrition and protection against biotic and abiotic stresses. In temperate forests or meadows dominated by angiosperms, the numerous fungi involved in root symbioses are often shared between neighboring plants, thus forming complex plant-fungus interaction networks of weak specialization. Whether this weak specialization also holds in rich tropical communities with more phylogenetically diverse sets of plant lineages remains unknown. We collected roots of 30 plant species in semi-natural tropical communities including angiosperms, ferns, and lycophytes, in three different habitat types on La Réunion island: a recent lava flow, a wet thicket, and an ericoid shrubland. We identified root-inhabiting fungi by sequencing both the 18S rRNA and the ITS2 variable regions. We assessed the diversity of mycorrhizal fungal taxa according to plant species and lineages, as well as the structure and specialization of the resulting plant-fungus networks. Results The 18S and ITS2 datasets are highly complementary at revealing the root mycobiota. According to 18S, Glomeromycotina colonize all plant groups in all habitats forming the least specialized interactions, resulting in nested network structures, while Mucoromycotina ( Endogonales ) are more abundant in the wetland and show higher specialization and modularity compared to the former. According to ITS2, mycorrhizal fungi of Ericaceae and Orchidaceae , namely Helotiales , Sebacinales , and Cantharellales , also colonize the roots of most plant lineages, confirming that they are frequent endophytes. While Helotiales and Sebacinales present intermediate levels of specialization, Cantharellales are more specialized and more sporadic in their interactions with plants, resulting in highly modular networks. Conclusions This study of the root mycobiome in tropical environments reinforces the idea that mycorrhizal fungal taxa are locally shared between co-occurring plants, including phylogenetically distant plants (e.g. lycophytes and angiosperms), where they may form functional mycorrhizae or establish endophytic colonization. Yet, we demonstrate that, irrespectively of the environmental variations, the level of specialization significantly varies according to the fungal lineages, probably reflecting the different evolutionary origins of these plant-fungus symbioses. Frequent fungal sharing between plants questions the roles of the different fungi in community functioning and highlights the importance of considering networks of interactions rather than isolated hosts.

4 citations

Journal ArticleDOI
TL;DR: In this article , the mycobiome of the achlorophyllous gametophytes of Ophioderma pendulum using a high-throughput metabarcoding approach was examined.
Abstract: Abstract Premise Several ferns and lycophytes produce subterranean gametophytes, including the Ophioglossaceae, Psilotaceae, and some members of the Schizaeaceae, Gleicheniaceae, and Lycopodiaceae. Despite the surge in plant‐microbiome research, which has been particularly boosted by high‐throughput sequencing techniques, the microbiomes of these inconspicuous fern gametophytes have rarely been examined. The subterranean gametophytes are peculiar due to their achlorophyllous nature, which makes them rely on fungi to obtain nutrients. Furthermore, the factors that shape the fungal communities (mycobiomes) of fern gametophytes have not been examined in depth. Methods and Results We present a workflow to study the mycobiome of the achlorophyllous gametophytes of Ophioderma pendulum using a high‐throughput metabarcoding approach. Simultaneously, each gametophyte was investigated microscopically to detect fungal structures. Two primer sets of the nuclear ITS sequence targeting general fungi were applied, in addition to a primer set that specifically targets the nuclear small subunit ribosomal rDNA region of arbuscular mycorrhizal fungi. Both the microscopic and metabarcoding approaches revealed many diverse fungi inhabiting a single gametophyte of O. pendulum. Discussion This study provides methodological details and discusses precautions for the mycobiome investigation of achlorophyllous gametophytes. This research is also the first to uncover the mycobiome assembly of an achlorophyllous gametophyte of an epiphytic fern.

4 citations

Journal ArticleDOI
TL;DR: In this article , the effects of compatible and incompatible fungi on seed germination, protocorm formation, seedling development, and colonization patterns in Dendrobium officinale were compared.
Abstract: Orchids require specific mycorrhizal associations for seed germination. During symbiotic germination, the seed coat is the first point of fungal attachment, and whether the seed coat plays a role in the identification of compatible and incompatible fungi is unclear. Here, we compared the effects of compatible and incompatible fungi on seed germination, protocorm formation, seedling development, and colonization patterns in Dendrobium officinale; additionally, two experimental approaches, seeds pretreated with NaClO to change the permeability of the seed coat and fungi incubated with in vitro-produced protocorms, were used to assess the role of seed coat played during symbiotic seed germination.The two compatible fungi, Tulasnella sp. TPYD-2 and Serendipita indica PI could quickly promote D. officinale seed germination to the seedling stage. Sixty-two days after incubation, 67.8 ± 5.23% of seeds developed into seedlings with two leaves in the PI treatment, which was significantly higher than that in the TPYD-2 treatment (37.1 ± 3.55%), and massive pelotons formed inside the basal cells of the protocorm or seedlings in both compatible fungi treatments. In contrast, the incompatible fungus Tulasnella sp. FDd1 did not promote seed germination up to seedlings at 62 days after incubation, and only a few pelotons were occasionally observed inside the protocorms. NaClO seed pretreatment improved seed germination under all three fungal treatments but did not improve seed colonization or promote seedling formation by incompatible fungi. Without the seed coat barrier, the colonization of in vitro-produced protocorms by TPYD-2 and PI was slowed, postponing protocorm development and seedling formation compared to those in intact seeds incubated with the same fungi. Moreover, the incompatible fungus FDd1 was still unable to colonize in vitro-produced protocorms and promote seedling formation.Compatible fungi could quickly promote seed germination up to the seedling stage accompanied by hyphal colonization of seeds and formation of many pelotons inside cells, while incompatible fungi could not continuously colonize seeds and form enough protocorms to support D. officinale seedling development. The improvement of seed germination by seed pretreatment may result from improving the seed coat hydrophilicity and permeability, but seed pretreatment cannot change the compatibility of a fungus with an orchid. Without a seed coat, the incompatible fungus FDd1 still cannot colonize in vitro-produced protocorms or support seedling development. These results suggest that seed coats are not involved in symbiotic germination in D. officinale.

3 citations

References
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Journal ArticleDOI
TL;DR: The combination of morphological and molecular methods provides a practical approach to the characterization of the mycorrhizal status of epiphytic bromeliad Vriesea werkleana, and perhaps other tropicalEpiphytes.
Abstract: Arbuscular mycorrhizal fungi influence the growth, morphology, and fitness of a variety of plant species, but little is known of the arbuscular mycorrhizal (AM) fungal associations of plant species in forest canopies. Plant species' associations with AM fungi are most often elucidated by examining the roots for fungal structures; however, morphological data may provide a limited resolution on a plant's mycorrhizal status. We combined a traditional staining technique with a molecular marker (the 18S ribosomal gene) to determine whether or not a variety of epiphytic bromeliads form arbuscular mycorrhizal fungal associations. Using these methods we show that the epiphytic bromeliad Vriesea werkleana forms arbuscular mycorrhizal fungal associations with members of the genus Glomus. AM fungal sequences of this plant species formed three distinct clades nested within a larger Glomus clade; two of the clades did not group with any previously sequenced lineage of Glomus. Novel clades may represent novel species. Although Vriesea werkleana is associated with multiple AM fungal species, each individual plant is colonized by a single lineage. The combination of morphological and molecular methods provides a practical approach to the characterization of the mycorrhizal status of epiphytic bromeliads, and perhaps other tropical epiphytes.

21 citations

Journal ArticleDOI
TL;DR: It was found that the fatty acid palmitic acid alone caused an increase in growth rate equal to that of the pine-root exudate, and it was shown that certain cytokinins, especially isopentenylaminopurine, could act as substitutes for the total root exudates.
Abstract: The object of this investigation was the promotion by root exudates of the growth rate of ectomycorrhizal fungi, discovered by Elias Melin in 1954. Eight ectomycorrhizal and ten non-mycorrhizal species were used as test fungi in the experiments. Different species often reacted differently: none of the eight isolated strains of Suillus luteus were promoted by pineroot exudate, whereas the growth rates of all seven strains of S. granulatus were increased. Among the other ectomycorrhizal species, S. variegatus, Laccaria bicolor, Pisolithus tinctorius and Thelephora terrestris, each represented by only one, two or three strains, usually reacted to the pine root exudate with an increased growth rate; S. bovinus and Paxillus involutus did not respond at all. Hitherto, studies of root-exudate effects on fungi have been based exclusively on the responses of ectomycorrhizal species; in the present study saprotrophic fungi were also used as test organisms. Seven out of ten saprotrophic species reacted with markedly accelerated growth when exposed to a pineroot exudate. Melin's assumption that a constituent of the root exudate, the “M-factor”, could replace the exudate growth-promoting activity was verified. By means of TLC fraction it was found that the fatty acid palmitic acid alone caused an increase in growth rate equal to that of the pine-root exudate. In line with previously published data by Gogala (1970), we also showed that certain cytokinins, especially isopentenylaminopurine, could act as substitutes for the total root exudate. Thus both palmitic acid and isopentenylaminopurine are able to function as M-factors equivalent to a root exudate.

20 citations

Journal ArticleDOI
TL;DR: This study provides the first evidence of an endophytic fungal community in both the seeds and seedlings of two epiphytic bromeliads species that subsequently benefit plant growth.

20 citations

Journal ArticleDOI
TL;DR: It is indicated that habitat management can shape OrM fungal communities, and the spatial distribution of these fungi appears to be weakly structured outside the orchid patches.
Abstract: Orchid mycorrhizal (OrM) fungi play a crucial role in the ontogeny of orchids, yet little is known about how the structure of OrM fungal communities varies with space and environmental factors. Previous studies suggest that within orchid patches, the distance to adult orchids may affect the abundance of OrM fungi. Many orchid species grow in species-rich temperate semi-natural grasslands, the persistence of which depends on moderate physical disturbances, such as grazing and mowing. The aim of this study was to test whether the diversity, structure and composition of OrM fungal community are influenced by the orchid patches and management intensity in semi-natural grasslands. We detected putative OrM fungi from 0 to 32 m away from the patches of host orchid species (Orchis militaris and Platanthera chlorantha) in 21 semi-natural calcareous grasslands using pyrosequencing. In addition, we assessed different ecological conditions in semi-natural grasslands but primarily focused on the effect of grazing intensity on OrM fungal communities in soil. We found that investigated orchid species were mostly associated with Ceratobasidiaceae and Tulasnellaceae and, to a lesser extent, with Sebacinales. Of all the examined factors, the intensity of grazing explained the largest proportion of variation in OrM fungal as well as total fungal community composition in soil. Spatial analyses showed limited evidence for spatial clustering of OrM fungi and their dependence on host orchids. Our results indicate that habitat management can shape OrM fungal communities, and the spatial distribution of these fungi appears to be weakly structured outside the orchid patches.

19 citations

Journal ArticleDOI
TL;DR: Generalist mycobionts may be relevant for the preservation of hyperdiverse orchid communities in the tropics and a significantly nested network architecture indicated the existence of a core of generalist Tulasnellaceae OTUs interacting with both rare and common orchids.

17 citations

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What help does a tree give to the orchids?

The paper does not explicitly mention the help that a tree gives to orchids. The paper focuses on the spatial structure of fungal communities on tree bark and their relationship with epiphytic plants, including orchids.

Is the tree affected by the presence of the orchids?

The paper does not directly address the question of whether the tree is affected by the presence of orchids. The paper focuses on the spatial structure of fungal communities on tree bark and their relationship with epiphytic plants, specifically orchids.