1. mycological research 110 (2006) 1257–1270
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/mycres
Diverse tulasnelloid fungi form mycorrhizas with epiphytic
orchids in an Andean cloud forest
´
Juan Pablo SUAREZa,b,*, Michael WEIßb, Andrea ABELEb, Sigisfredo GARNICAb,
Franz OBERWINKLERb, Ingrid KOTTKEb
a
´cnica Particular de Loja, San Cayetano Alto s/n C.P. 11 01 608, Loja, Ecuador
Centro de Biologıa Celular y Molecular, Universidad Te
´
b
Eberhard-Karls-Universita Tubingen, Botanisches Institut, Spezielle Botanik und Mykologie, Auf der Morgenstelle 1,
¨t ¨
D-72076 Tubingen, Germany
¨
article info abstract
Article history: The mycorrhizal state of epiphytic orchids has been controversially discussed, and the
Received 3 May 2006 state and mycobionts of the pleurothallid orchids, occurring abundantly and with a high
Received in revised form number of species on stems of trees in the Andean cloud forest, were unknown. Root sam-
7 August 2006 ples of 77 adult individuals of the epiphytic orchids Stelis hallii, S. superbiens, S. concinna and
Accepted 12 August 2006 Pleurothallis lilijae were collected in a tropical mountain rainforest of southern Ecuador. Ul-
Published online 31 October 2006 trastructural evidence of symbiotic interaction was combined with molecular sequencing
Corresponding Editor: of fungi directly from the mycorrhizas and isolation of mycobionts. Ultrastructural analy-
John W. G. Cairney ses displayed vital orchid mycorrhizas formed by fungi with an imperforate parenthesome
and cell wall slime bodies typical for the genus Tulasnella. Three different Tulasnella isolates
Keywords: were obtained in pure culture. Phylogenetic analysis of nuclear rDNA sequences from cod-
Heterobasidiomycetes ing regions of the ribosomal large subunit (nucLSU) and the 5.8S subunit, including parts of
Molecular phylogeny the internal transcribed spacers, obtained directly from the roots and from the fungal iso-
Pleurothallidinae lates, yielded seven distinct Tulasnella clades. Tulasnella mycobionts in Stelis concinna were
Southern Ecuador restricted to two Tulasnella sequence types while the other orchids were associated with up
Tropical mountain rain forest to six Tulasnella sequence types. All Tulasnella sequences are new to science and distinct
Ultrastructure from known sequences of mycobionts of terrestrial orchids. The results indicate that tulas-
nelloid fungi, adapted to the conditions on tree stems, might be important for orchid
growth and maintenance in the Andean cloud forest.
ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction Williamson 1972), but a high infection rate was reported
from canopy-dwelling orchid species in Florida (Benzing
Although most land plants are associated with symbiotic 1982). Different degrees of infection including non-infected
fungi forming mycorrhizas or mycorrhiza-like associations, roots were observed in epiphytic orchids in Ecuador (Ber-
many epiphytes live without such associations, e.g. mosses, mudes & Benzing 1989). Goh et al. (1992) found high fungal col-
many liverworts, bromeliads, and ferns (Kottke 2002). Find- onization in the epiphytic orchid Dendrobium crumenatum from
ings on the mycorrhizal state of epiphytic orchids were con- a natural stand in Singapore, but only low or no mycorrhiza-
troversial. Only sporadic fungal colonization was found tion in orchids from nurseries. Rivas et al. (1998) and Pereira
in a number of epiphytic Malaysian orchids (Hadley & et al. (2005) reported intense colonization of epiphytic orchids
* Corresponding author.
E-mail address: jpsuarez@utpl.edu.ec
0953-7562/$ – see front matter ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.mycres.2006.08.004

2. 1258 ´
J. P. Suarez et al.
in Costa Rica and Brazil, respectively. All investigators stated minimized. The genera Stelis and Pleurothallis belong to
that fungal colonization was restricted to roots attaching to subtribe Pleurothallidinae, the largest subtribe in the tribe
the substrate; aerial roots were not colonized. Epidendreae of Orchidaceae (Luer 1986a,b), which is widely dis-
Identification of root-associated fungi was mostly achieved tributed in tropical America. These two genera include 485
by isolation on sterile media (Rasmussen 2002). However, the Pleurothallis and 465 Stelis species reported until now for Ecua-
distinction between endophytic fungi inhabiting only the ve- dor (L. Endara, pers. comm.). Many of these epiphytes are en-
lamen or the root surface and reliably mycorrhiza-forming demic species of Ecuadorian tropical forests. Only a few of
fungi colonizing the cortical tissue was mostly unclear (Cur- them are in culture so far. The rapid loss of habitats requires
rah et al. 1997; Pridgeon 1987). Xylaria (Ascomycota) was fre- an understanding of the symbiotic relationships in order
quently isolated (Bayman et al. 1997, Tremblay et al. 1998), to support conservation efforts for these orchids. According
but was never proven experimentally or demonstrated by ul- to Hamilton et al. (1995) approximately 90 % of the Northern
trastructure to form mycorrhizas with orchids. Fungal isola- Andean forests have been already destroyed. Consequently,
tion from pelotons as a more selective approach has been the orchids and their fungi might be lost in the near future
successfully attempted in terrestrial orchids (Warcup & Talbot if not taken into culture. As the mycorrhizal state and myco-
1967, 1971, 1980, Bougoure et al. 2005). In cases where sexual bionts of the epiphytic Pleurothallidinae were unknown, no
stages could be achieved, the isolated fungi were determined advice could be given to laboratories interested in orchid cul-
as basidiomycetes belonging to the Sebacinales, Tulasnellales turing or to local forest management aiming to rehabilitate
or Ceratobasidiales (Warcup 1981; Warcup & Talbot 1967, the tropical mountain forest with its epiphytic orchid diversity
1971, 1980). Tulasnella anamorphs (Epulorhiza) were isolated, (see: http://www.bergregenwald.de of which this work is a
e.g. from epiphytic Epidendrum conopseum in Florida (Zettler part). We therefore started with light- and transmission elec-
et al. 1998), epiphytic Epidendrum rigidum, Polystachia concreta tron microscopic investigation of the selected orchid species,
(Pereira et al. 2003), and terrestrial Oeceoclades maculata from and continued with DNA isolation and sequencing of the
Brazil (Pereira et al. 2005). DNA sequencing supported the most frequently observed fungal group, the Tulasnellales. In
presence of Tulasnella, Sebacinales and Ceratobasidium in parallel, isolation of mycelia was carried out, yielding several
Cypripedium spp. from the temperate Northern Hemisphere Tulasnella isolates. We were especially interested to see
(Shefferson et al. 2005). Molecular tools were also used to iden- whether the Tulasnellales present as mycobionts of epiphytic
tify fungal isolates obtained from pelotons (Bougoure et al. orchids in the tropical mountain rain forest were distinct
2005) or by direct DNA isolation from pelotons (Kristiansen from those described for other habitats of the Northern Hemi-
et al. 2001). A taxon distantly related to Laccaria, an ectomycor- sphere and Australia. This knowledge would help to decide if
rhiza-forming fungus, was found in Dactylorhiza majalis local or ubiquitous fungal isolates were appropriate for culti-
(Kristiansen et al. 2001) in addition to Tulasnella. Ectomycor- vation of the local orchids, and would support evaluation of
rhiza-forming mycobionts were also proven for non- loss of local fungi for rehabilitation of orchids in the tropical
photosynthetic orchids by DNA isolations and sequencing mountain forest area.
directly from mycorrhizas (Taylor & Bruns 1997, 1999; Taylor
et al. 2003, Bidartondo et al. 2004, Selosse et al. 2004, Julou et al.
2005), thus widening the previous knowledge on orchid Materials and methods
mycobionts.
Selosse et al. (2004) confirmed their molecular finding of Tu- Study site
ber spp. (Ascomycota) as orchid mycobionts by ultrastructural
demonstration of ascomycetous hyphae in the cortical cells of The study site is located on the eastern slope of the Cordillera El
the orchid roots. Ascomycetes can be discerned from basidio- Consuelo in the northern Andes of southern Ecuador. The area
mycetes by ultrastructure of the cell wall and the septal pore, ´
of about 1000 ha belongs to the Reserva Biologica San Francisco
and different groups of basidiomycetes can be distinguished and borders the Podocarpus National Park in the north, half
by the parenthesomes covering the dolipores (Wells & Ban- way between Loja and Zamora, Zamora-Chinchipe Province
doni 2001); tulasnelloid fungi display characteristic slime bod- ´ ´
(3 58 S, 79 04 W). The tropical mountain rainforest covers the
ies in the cell walls (Bauer 2004). In spite of these diagnostic steep slopes between 1850 and 2700 m a.s.l. Characteristic
possibilities transmission electron microscopy has rarely and most frequent trees are Melastomataceae, Rubiaceae, Laura-
been used in orchid studies addressing fungal identity. How- ceae and Euphorbiaceae reaching a height of 25 m. Crown density
ever, the previous work is encouraging (Currah Sherburne as measured by a spherical densitometer is 94 % on average,
1992; Andersen 1996) and minimizes errors resulting from only 7.5 % were open canopy (Homeier 2004).
contamination during isolation of fungi or DNA directly from The richness and abundance of epiphytes is due to the
mycorrhiza samples. In our study of the orchid mycobionts semi- to sub-humid climate with rainfall during ten months
of four epiphytic, pleurothallid orchid species in the Andean and even more frequent fog combined with moderate temper-
cloud forest of south Ecuador, we therefore combined ultra- atures (Richter 2003). Mean annual precipitation at 1950 m
structural studies with DNA sequencing and isolation. a.s.l. is 2200 mm, annual mean temperature is 15,5 C (14,4-
Stelis concinna, S. hallii, S. superbiens, and Pleurothallis lilijae 17,5 C). Precipitation increases with higher elevation and
Foldats were selected because of the abundance and frequent reaches 4000 mm at 2600 m asl. Air humidity in two months
flowering of these small orchids in the tropical mountain is 96 % on average and does not fall below 70 % during the
rainforest of the study area. Thus severe violations of the drier season (Noske 2004). The high air humidity is especially
¨
orchid populations in this highly endangered forest could be important for stem epiphytes.

3. Diverse tulasnelloid fungi form mycorrhizas 1259
Sampling Fungal isolation
Sampling was carried out at small paths at an altitudinal gra- Isolation of fungi was initiated the day of sampling. Colonized
dient between 1850 and 2100 m a.s.l. Stelis hallii was found in root pieces were surface-sterilized. Roots were rinsed in
the forest covering the steep slopes between 1800 and distilled water with some drops of liquid soap, immersed in
1900 m a.s.l., while S. superbiens and Pleurothallis lilijae were ethanol (70 %) for 30 s, immersed in Ajax chloro 20 % (house-
collected in the forest covering the mountain ridge between hold bleach, sodium hypochlorite 5.25 %) for 10 min and
1900 and 2100 m a.s.l. Stelis concinna was restricted to the up- finally rinsed in sterile distilled water. The velamen was
per part of the mountain ridge where the forest was less then removed using a stereo microscope, a thin blade and for-
dense, with only 92 % crown density, and exposition to fre- ceps. Five square sections of 1-3 mm thickness were cut by
quent and heavy winds. hand from the middle part of the root and transferred to
Roots were collected continuously during three years a plate with MYP media (malt extract 7 g, peptone 1 g, and
from 2003 until 2005 from a total of 77 flowering individuals, agar agar 15 g lÀ1) or MMNC media (modified Melin-Norkrans;
22 of S. hallii, 17 of S. superbiens, 25 of S. concinna, and 13 of Kottke et al. 1987; NaCl 0,025 g, KH2PO4 0,5 g, (NH4)2HPO4 0,25 g,
Pleurothallis lilijae. All selected plants were epiphytes on CaCl2 0,05 g, MgSO4 Â 7H2O 0,15 g, FeCl3 (1 %) 1 ml, thiamin
trunks or branches of standing trees at 50 cm to 200 cm 1 ml, malt extract 5 g, glucose 10 g, caseinhydrolysate 1 g,
above the forest floor. Distances between trees with flower- agar 20 g, riboflavin 1 ml of 0.01 % solution, trace elements
ing orchids varied between 50 cm and several metres (up to ´
10 ml according to Fortin and Piche 1979). No antibiotics
20 m). Identification of trees was not taken into consider- were added.
ation. Roots of one flowering individual orchid per tree
stem were collected. One to four roots per plant individual
were packed in aluminum foil to prevent desiccation DNA extraction, PCR and sequencing
and transported to the laboratory the same day. As pre-
investigation had shown that mycorrhizal fungi colonized Portions of 1-2 cm length of well colonized roots of which the
only roots in contact with the stems, best when also covered velamen was removed were collected in cups the same day or
by mosses or a minute humus layer, later on only such roots dried and kept on silica gel for later DNA isolations. DNA was
were selected. Root samples were processed the day of extracted from the fresh or dried mycorrhizal tissue and from
collection as pre-investigation had revealed a fast loss of fungal mycelium of our own isolates using a Plant Mini Kit
vitality in the symbiotic fungi. Vouchers of the orchid (Qiagen, Hilden, Germany). A first attempt to PCR amplify ge-
specimens were deposited in the Herbarium of UTPL, Loja, nomic DNA was carried out from mycorrhizal tissue using
Ecuador, including flowers fixed in ethanol. Vouchers of universal fungal primer combinations ITS1F/ITS4, ITS1F/NL4,
the mycorrhizas were embedded in resin and deposited in NLMW1/LR5, NLMW1/TW14 and ITS1F/TW14 (details con-
the Herbarium of Tubingen University (TUB).
¨ cerning the primers used are given in the Electronic Appendix
A). Several PCR products were obtained and sequenced. DNA
isolated from fungal cultures was amplified using the primer
Light and transmission electron microscopy combination ITS1F/NL4 or ITS1/NL4. Nested PCR was con-
ducted to specifically amplify DNA from tulasnelloid fungi,
Light microscopy was used to select material with fungal coils. as the ultrastructural analysis had revealed these fungi fre-
Transversal sections were cut from the middle part of each quently in the cortical tissue of all the orchid species under in-
root sample by hand using a razor blade. Sections were vestigation. The first amplification was carried out with the
stained by Methyl blue 0.05 % solution (C. I. 42780, Merck) in primer combination ITS1F/TW14 or ITS1/TW14 and the sec-
lactic acid for 10 min on microscopic slides. The samples ond, using template obtained in the first PCR in dilutions of
were examined in fresh lactic acid at 100- to 1000-fold magni- 10À1, 10À2 and 10À3, with the primer combinations ITS1/
fication (Leitz SM-LUX or Zeiss Axioskop 2). ITS4-Tul for the internal transcribed spacers (ITS1, 5.8S nu-
Root pieces of 1 cm length of all the samples displaying clear ribosomal gene and ITS2) and NLMW1/LR5, ITS4-TulR/
high frequency of vital looking hyphal coils, 56 in total and LR5 and 5.8S-Tul/NL4 for the 5’ part of the nuclear large sub-
at least ten of each species, were fixed in 2.5 % glutaralde- unit ribosomal DNA (nucLSU). Primers ITS4-Tul and ITS4-
hyde-formaldehyde in Sørensen buffer (Karnovsky 1965), TulR target a Tulasnella-specific sequence at the 3’ end of
post-fixed in 1 % osmium tetroxide for 1 h, dehydrated in an ITS2. The Tulasnella-specific primer 5.8S-Tul (5’-TCATTCGAT
acetone series and flat embedded in Spurr’s resin low viscos- GAAGACCGTTGC-3’) designed for this study targets a specific
ity, longer pot-life formulation (Spurr 1969). Semithin sections sequence at the 5’ end of the 5.8S rDNA.
were cut from the embedded samples, stained with 0.6 % neo- PCR conditions were as follows: initial denaturation at
fuchsin crystal-violet, mounted in Entellan, and observed in 94 C for 3 min; 35 cycles, each cycle consisting of one step
the light microscope. 20 samples with apparently vital of denaturation at 94 C for 30 s; annealing depending of the
hyphae, originating from different plant individuals, were primer combinations for 45 s and extension at 72 C for
selected for ultrathin cutting. Sections were mounted on For- 1 min; a final extension at 72 C for 7 min was performed to
mvar-coated copper grids and stained with 1 % uranyl acetate finish the PCR. The PCR reaction volume was 50 ml, with con-
(40 min) and lead citrate (12 min). Sections were examined us- centrations of 1.5 mM MgCl2, 200 mM of each dNTP (Life Tech-
ing transmission electron microscopes Zeiss TEM 902 or Zeiss nologies, Eggenstein, Germany), 0.5 mM of each of the primers
TEM109. (MWG-Biotech, Ebersberg, Germany), 1U Taq polymerase (Life

4. 1260 ´
J. P. Suarez et al.
Technologies, Eggenstein, Germany), with an amplification MrModeltest, version 2.2 (Nylander 2004) involving four
buffer (Life Technologies, Eggenstein, Germany). incrementally heated Markov chains over four million gener-
In every PCR a control including PCR mix without DNA ations and using random starting trees. Trees were sampled
template was included. Success of the PCR amplifications every 100 generations resulting in a total of 40000 trees from
was tested in 0.7 % agarose, stained in a solution of ethidium which the last 24000 were used to compute a 50 % majority
bromide 0.5 mg mlÀ1. PCR products were purified using the rule consensus tree. Each analysis was repeated to check
QIAquick protocol (Qiagen). Cycle sequencing was conducted the reproducibility of the results (Huelsenbeck et al. 2002).
using BigDye version 3.1 chemistry, and sequencing was An accumulation curve of clades vs number of collected indi-
done on an ABI 3100 Genetic Analyzer (Applied Biosystems, viduals from the four orchid species was computed with Esti-
Foster City, CA). Both strands of DNA were sequenced. mateS (Version 7.5, R. K. Colwell, unpubl.).
Sequence editing was performed using Sequencher version We determined the proportional differences between se-
4.5 (Gene Codes, Ann Arbor, MI). The sequences obtained quences within each clade of the nucLSU D1/D2 in order to de-
in this study are available from GenBank under accession fine sequence types. We compared the number of Tulasnella
numbers DQ178029-DQ178118 (Table 1). sequence types within single and between different orchid
We also included in this study sequence data from Tulas- species. The proportional differences between sequences
nella reference strains kindly provided by the National Insti- were pooled into five tables (Electronic Appendix B).
tute of Agrobiological Sciences (NIAS), Japan, which were
previously isolated from Australian orchids and determined
by J. H. Warcup (Warcup Talbot 1967, 1971). Results
Phylogenetic analyses Microscopical and ultrastructural features of the mycorrhizas
We used BLAST (Altschul et al. 1997) against the NCBI nucleo- Fungal pelotons were present in nearly all cross-sections of
tide database (GenBank; http://www.ncbi.nlm.nih.gov/) to de- roots sampled directly from the tree bark. No fungal pelotons
tect published sequences with a high similarity to the nucLSU were observed in aerial roots. This observation was confirmed
sequences obtained from the Ecuadorian epiphytic orchids. by sampling roots of another 65 epiphytic Stelis and Pleurothal-
For thorough phylogenetic analysis of the Tulasnella se- lis orchids, indicating that the roots became colonized only
quences we analyzed nucLSU and ITS-5.8S alignments includ- where the fungi contacted the bark or the thin humus layer.
ing the closest BLAST matches together with the sequences Pelotons were distributed throughout the cortex, with no dif-
from the Warcup Tulasnella reference isolates (see above) ference between cortical layers. Vital, blue staining and col-
and other sequences from Tulasnellaceae and related groups lapsed, slightly yellow coloured pelotons were visible in the
retrieved from GenBank. same cells suggesting that cells became re-infected several
Sequences were aligned using the G-INS-i or L-INS-i strat- times. According to the light microscopical observations,
egy as implemented in MAFFT v5.667 (Katoh et al. 2005). Due many fungal pelotons were found collapsed after the plants
to the heterogeneity of the Tulasnella sequences we had to ex- had been kept one night in the laboratory. Abundant hyphae
clude considerable portions of the nucLSU sequences for phy- colonized the velamen.
logenetic analysis. Even the 5.8S ribosomal region, considered TEM observations confirmed the known fungal-root inter-
as universally conserved, exhibited a remarkable heterogene- action in orchid mycorrhizas. Hyphae of more or less equal di-
ity as was already mentioned by Bidartondo et al. (2003). As ameter were surrounded by the plant plasma membrane, the
expected, the ITS1 and ITS2 rDNA could not be aligned over plant vacuole forming small compartments or a network of
the whole data set. Therefore, we used the 5.8S region to cal- small vacuoles (Fig 1). Degenerating hyphae were attached
culate phylogenetic trees of a wider phylogenetic spectrum to collapsed pelotons (Fig 2). Alive hyphae contained abundant
and produced several other phylogenetic analyses including glycogen granules (Figs 1, 3 and 5). The hyphae formed septa,
subsets of related sequences, for which we used portions of clamps were not observed. (Fig 3). The septa showed dolipores
the ITS1 and ITS2 regions in addition to the 5.8S sequences. with imperforate, dish-shaped parenthesomes with slightly
The alignments used can be obtained from TreeBASE (http:// recurved margins (Fig 6). These tulasnelloid parenthesomes
www.treebase.org/) under accession number S1629. were observed in all the 20 mycorrhizas analyzed by TEM. Oc-
Neighbour-joining (NJ) and a Bayesian likelihood approach casionally, the hyphal walls were split into two layers and a fi-
were used to estimate the phylogenetic relationships. The brillar or slimy mass appeared between the two layers (Figs 4
neighbour-joining analysis was performed in PAUP* (Swofford and 5, arrows). This phenomenon became very prominent in
2002) using the BIONJ modification of the NJ algorithm to ageing cultures and the slime was then strongly osmiophilic
accomplish the observed high genetic variability in the se- (not shown). The combination of this type of parenthesomes
quences used (Gascuel 1997). DNA substitution models and in- and the ‘‘slime bodies’’ in the cell walls was confirmed for
dividual model parameters were estimated using the Akaike all the investigated mycorrhizas and in the Tulasnella isolates.
information criterion (AIC) as implemented in Modeltest, ver- The recurved ends of the parenthesome were only detected by
sion 3.7 (Posada Crandall 1998). For the Bayesian approach serial sectioning, since the appearance of the parenthesomes
based on Markov chain Monte Carlo (MCMC) we used varied among the sections and may appear flattened or bowed
MrBayes, version 3.0b4 (Huelsenbeck Ronquist 2001). Each in a steeper angle. In three samples we additionally found flat,
dataset was analyzed using the DNA substitution models esti- imperforate parenthesomes, indicating sebacinoid fungi (Wil-
mated using the Akaike information criterion (AIC) in liams Thiol 1989; not shown). In one sample a dome-shaped

5. Diverse tulasnelloid fungi form mycorrhizas 1261
Table 1 – List of sampled individuals from which tulasnelloid sequences were obtained. Letters and numbers behind the
species names correspond to species, orchid individual, and root (superscript). Superscript b marks a second sequence
obtained from the same root sample. Clades A-G correspond to the MCMC phylogenetic analysis. The two rDNA regions
from the same root listed in each line originate from a single PCR amplicon
Orchid species nucLSU nrDNA ITS-5.8S
clade GenBank accession no. clade GenBank accession no.
Pleurothallis lilijae C2.1.1 A DQ178035 A DQ178099
Pleurothallis lilijae C2.1.2 E DQ178067
Pleurothallis lilijae C2.1.3 A DQ178040 A DQ178100
Pleurothallis lilijae C2.15 E DQ178080
Pleurothallis lilijae C2.17 A DQ178102
Pleurothallis lilijae C2.21 E DQ178079
Pleurothallis lilijae C2.1MN A DQ178034 A DQ178098
Pleurothallis lilijae C2.5MN7 F DQ178047 F DQ178069
Pleurothallis lilijae C2.MN1 D DQ178063 D DQ178116
Pleurothallis lilijae C2.MN5 F DQ178049 F DQ178070
Pleurothallis lilijae C2MN2 E DQ178068 E DQ178081
Pleurothallis lilijae C2MN6 B DQ178045
Stelis concinna 7.6 A DQ178108
Stelis concinna 7.7 A DQ178106
Stelis concinna 7.8 A DQ178091
Stelis concinna 7.13.2 A DQ178109
Stelis concinna 7.13.3 A DQ178107
Stelis concinna 7.13.4 A DQ178110
Stelis concinna 7.14.2 A DQ178112
Stelis concinna 7.18.3 A DQ178043 A DQ178095
Stelis concinna 7.18.4 E DQ178082
Stelis concinna 7.19.1 A DQ178094
Stelis concinna 7.19.3 A DQ178032 A DQ178093
Stelis concinna 7.20.1 A DQ178030 A DQ178096
Stelis concinna 7.20.2 A DQ178042 A DQ178088
Stelis concinna 7.20.3 A DQ178033 A DQ178090
Stelis concinna 7.20.4 A DQ178041 A DQ178089
Stelis concinna 7.21.1 E DQ178075
Stelis concinna 7.21.2 E DQ178076
Stelis concinna 9.2 A DQ178111
Stelis concinna 9.3 culture G DQ178029 G DQ178029
Stelis concinna 9.6 A DQ178084
Stelis concinna 9.7 A DQ178092
Stelis concinna 9.8 A DQ178038 A DQ178097
Stelis concinna 9.9 A DQ178031 A DQ178086
Stelis hallii 1.1 B DQ178044 B DQ178113
Stelis hallii 1.2 E DQ178065
Stelis hallii 1.2b D DQ178051
Stelis hallii 1.4 G DQ178118
Stelis hallii 1.6 B DQ178114
Stelis hallii 1.7 D DQ178050
Stelis hallii 1.8 A DQ178085
Stelis hallii 1.11 culture E DQ178066 E DQ178066
Stelis hallii 1.15 D DQ178057
Stelis hallii 1.16 D DQ178055
Stelis hallii 1.17 A DQ178103
Stelis hallii 1.18 A DQ178037 A DQ178104
Stelis hallii 1.18b D DQ178053
Stelis hallii 1.19 D DQ178059 E DQ178073
Stelis hallii 1.19b E DQ178071
Stelis hallii 1.21 E DQ178072
Stelis hallii 1.21b E DQ178077
Stelis hallii 1.23 D DQ178060
Stelis superbiens C3.5.2 culture A DQ178036 A DQ178036
Stelis superbiens C3.5.3 E DQ178083
Stelis superbiens C3.5.4 E DQ178078
Stelis superbiens C3.9.2 A DQ178039 A DQ178087
Stelis superbiens C3 MN3 D DQ178058 D DQ178117
Stelis superbiens C3.MN4 C DQ178046 C DQ178115
(continued on next page)

6. 1262 ´
J. P. Suarez et al.
Table 1 (continued)
Orchid species nucLSU nrDNA ITS-5.8S
clade GenBank accession no. clade GenBank accession no.
Stelis superbiens C3.1MN D DQ178056
Stelis superbiens C3.2MN D DQ178052 A DQ178105
Stelis superbiens C3.3MN D DQ178054
Stelis superbiens C3.4MN D DQ178061 A DQ178101
Stelis superbiens C3.4MN4 D DQ178062
Stelis superbiens C3.5MN5 E DQ178064 E DQ178074
Stelis superbiens C3.5MN5b F DQ178048
parenthesome was found that displayed coarse perforations growth rates contrasting with the relative fast growth rate
and might thus putatively be assigned to Ceratobasidium (Cur- observed in the fungi isolated. Ascomycetes closest to Crypto-
rah Sherburne 1992; not shown). No simple-pored ascomy- sporiopsis were the most frequently isolated fungi.
cetes were found in the cortical tissue of the 20 investigated
samples, although they were present in the velamen (not Molecular identification and phylogenetic analysis
shown). of mycorrhiza-associated fungi
Fungal isolation and molecular identification of isolates The combinations of universal fungal primers yielded PCR
products preliminarily identified by BLAST searches as closest
Fungal growth was observed in only 44 plates out of 108 used to Cryptosporiopsis, Fusarium, Trichoderma (Ascomycota) and
for fungal isolation, each one containing five root pieces. Four Bjerkandera, Antrodiella (Basidiomycota). Tulasnella sequences
fungal cultures were obtained from a total of 13 plates of Stelis were infrequently obtained, only the primer combination
hallii, 15 from 36 plates of Stelis superbiens, 22 from 55 plates of NLMW1/LR5 yielding few PCR products. Primer combinations
Stelis concinna, and three from four plates of Pleurothallis lilijae including ITS1F and ITS4 failed to amplify Tulasnella DNA as
mycorrhizas. A preliminary molecular identification of the was already reported by Bidartondo et al. (2003). Sequences
fungal isolates was carried out by BLAST searches against of Sebacinales, basidiomycetes involved in a broad range of
the GenBank nucleotide database retrieving the most similar mycorrhizal associations (Weiß et al. 2004), were also
available sequences (data not shown). The isolated fungi detected, but at lower frequence than Tulasnellales sequences.
were mainly ascomycetes closest to Xylaria, Hypoxylon and No cultures of Sebacinales were obtained from root samples
Cryptosporiopsis and less often basidiomycetes closest to Bjer- (Kottke et al. 2007).
kandera, Polyporus and Tulasnella. Three cultures were identi- The total number of investigated roots was 134, consider-
fied as Tulasnella. These Tulasnella cultures exhibited slow ing that three or four roots were collected from each of the
77 orchid individuals. Tulasnelloid fungi were detected in 84
samples (63 %), including the PCR products obtained by the
tulasnelloid specific primer combinations without successfull
Fig 1 – Ultrastructure of the cortical tissue of Stelis concinna
root displaying alive hyphae (h) of equal diameter in Fig 2 – Degenerating hyphae adjoining collapsed hyphae
active host cell (c). (v) Small compartments of orchid cell (ch) in an active cortical cell of Stelis concinna root.
vacuoles. Bar [ 1 mm. Bar [ 1 mm.

7. Diverse tulasnelloid fungi form mycorrhizas 1263
Fig 3 – Branched hypha displaying septa (arrow) without
Fig 5 – Hypha in cortical root tissue of Stelis concinna
clamp formation in root cortical tissue of Stelis concinna.
displaying fibrillar slime between cell wall layers
Bar [ 1 mm.
(arrowhead) and a doliporus with imperforate, slightly
dish-shaped parenthesomes (arrow). Bar [ 0.5 mm.
sequencing. The nested PCR conducted in order to selectively
amplify Tulasnella DNA using the primer combination ITS1/ 5.8S overview tree (Electronic Appendix C) in such a way
TW14 in the first amplification and the primer combinations that we obtained best consistency with the rooted LSU tree
ITS1/ITS4-Tul for the ITS-5.8S region and ITS4-TulR/LR5 or (Fig 7). Portions of ITS1 and ITS2 were added to the 5.8S align-
5.8S-Tul/NL4 for a part of the LSU region, respectively, in the ment where phylogenetic analysis was restricted to suitable
second PCR yielded PCR products for the majority of samples. subsets of sequences detected in the 5.8S analysis, finally
PCR success was higher with DNA extracted from fresh root resulting in an increase of phylogenetic resolution for these
samples and lower with DNA from dried roots. subsets (Figs 8 and 9).
The phylogenetic analyses of nucLSU and ITS-5.8S se- Our analysis of proportional differences between se-
quences yielded consistent results. Seven clades, which in quences within each clade of the nucLSU D1/D2 yielded 13
the following we refer to as clades A to G, were retrieved Tulasnella sequence types. We treated sequences as belonging
from the analyses of both ribosomal regions (Fig 7 and Elec-
tronic Appendix C). BIONJ (trees not shown) and MCMC
yielded similar groupings of Tulasnella clades. Only small var-
iations were present in the clade support values. As men-
tioned above, the 5.8S tree (Electronic Appendix C) was less
resolved than the nucLSU tree. However, the unexpected het-
erogeneity displayed by the 5.8S data set made it difficult to
find a suitable outgroup sequence. Therefore, we rooted the
Fig 6 – Close-up of a median section through the doliporus.
The parenthesomes consist of two electron-dense
Fig 4 – Square section of active hypha of Tulasnella, dis- membranes bordering an internal electron transparent
playing mitochondria (m), glycogen rosettes, and fibrillar zone and show slightly recurved borders (arrows).
slime between cell wall layers (arrowheads). Bar [ 1 mm. Bar [ 0,3 mm.

8. 1264 ´
J. P. Suarez et al.
Fig 7 – Phylogenetic placement of Tulasnella sequences from Stelis hallii, Stelis superbiens, Stelis concinna and Pleurothallis
lilijae inferred by MCMC analysis of nuclear rDNA coding for the 5’ terminal domain of the large ribosomal subunit (nucLSU).
Numbers on branches designate neighbor-joining bootstrap values / MCMC estimates of posterior probabilities (only values
exceeding 50 % are shown). Note that genetic distances cannot be directly correlated to branch lengths in the tree, since
highly diverse alignment regions were excluded for tree construction. The tree was rooted with Multiclavula mucida
AF287875.

9. Fig 8 – Phylogenetic placement of Tulasnella sequences, clades A-C, from Stelis hallii, Stelis superbiens, Stelis concinna and
Pleurothallis lilijae inferred by MCMC analysis of nuclear ITS-5.8S rDNA. Numbers on branches designate neighbor-joining
bootstrap values / MCMC estimates of posterior probabilities (only values exceeding 50 % are shown). Note that genetic dis-
tances cannot be directly correlated to branch lengths in the tree, since highly diverse alignment regions were excluded for
tree construction. The tree was rooted with Tulasnella sequences from clade D from the analysis of 5.8S rDNA (Electronic
Appendix C).

10. 1266 ´
J. P. Suarez et al.
Fig 9 – Phylogenetic placement of Tulasnella sequences, clades E and F, from Stelis hallii, Stelis superbiens, Stelis concinna
and Pleurothallis lilijae inferred by MCMC analysis of nuclear ITS-5.8S. Numbers on branches designate neighbor-joining
bootstrap values / MCMC estimates of posterior probabilities (only values exceeding 50 % are shown). Note that genetic
distances cannot be directly correlated to branch lengths in the tree, since highly diverse alignment regions were
excluded for tree construction. The tree was rooted with the Tulasnella sequence from the Warcup isolate
T. violea DQ520097.

11. Diverse tulasnelloid fungi form mycorrhizas 1267
to the same sequence type when proportional differences recurved margins consist of two electron-dense membranes
were 1 %. Clades D and E comprised four sequence types, bordering an internal electron transparent zone as was al-
while the other clades consisted only of one sequence type ready shown by Andersen (1996) for Rhizoctonia repens N.Ber-
each (Fig 7, Electronic Appendix B). A direct comparison be- nard ¼ Epulorhiza repens (syn.), the anamorph of Tulasnella
tween 5.8S-ITS and nucLSU phylogenies was hampered by deliquescens (syn. T. calospora sensu Warcup Talbot 1967 fide
the fact that publicly available sequence data were restricted Roberts 1999). The combination of this parenthesome type
to either one or the other of these two DNA regions for the and the slime bodies in the cell walls was previously described
vast majority of specimens studied so far (Table 1). from mycorrhiza-like associations of the liverwort Aneura pin-
Mycobionts of the same sequence type were shared among guis housing Tulasnella (Ligrone et al. 1993), and apparently is
orchid species, e.g. sequence types 2, 6, and 12. Tulasnella se- the best structural indication of Tulasnella (Bauer 2004). The
quences from all four studied orchid species were present in ascomycetes that we isolated from the roots were not found
sequence type 1 (Fig 7). Tulasnellas belonging to different se- in the cortical tissue of the orchids, but colonized only the ve-
quence types were detected in mycorrhizas from the same lamen. Therefore these ascomycetes cannot be considered as
plant and even from the same root piece (Table 1). Six different mycorrhiza-forming fungi. Without specific primers Tulasnella
Tulasnella sequence types were found in mycorrhizas of S. hallii is nearly undetectable (Bidartondo et al. 2003). The use of both
and S. superbiens and five in P. lilijae, while only two sequence Tulasnella-specific primers ITS4-Tul and 5.8S-Tul that target
types were detected in mycorrhizas of S. concinna (Fig 7). ITS2 and 5.8S, respectively, increased the success of Tulasnella
amplification, but a complete dataset of all tulasnelloid fungi
associated with the investigated orchids can currently not be
Discussion guaranteed. The accumulation curve of clades vs. number of
collected individuals was not saturated (Electronic Appendix D).
The importance of orchid mycorrhizal fungi and their role in Tulasnella was confirmed as a genus with a range of orchid
orchid seed germination are known since Bernard’s observa- mycorrhiza forming species (Kristiansen et al. 2004; Ma et al.
tions (Bernard 1909). In natural conditions, the protocorm 2003; McCormick et al. 2004; Shefferson et al. 2005; Warcup
does not develop further unless it receives an exogenous car- 1971, 1981, 1985; Warcup Talbot 1967, 1971, 1980). Tulasnella
bohydrate supply from a suitable mycorrhizal fungus (Smith calospora was proposed by Hadley (1970) as an universal orchid
Read 1997). Although not demonstrated in nature so far, symbiont considering the capacity to establish in vitro symbi-
this dependence will also account for germination of epiphytic otic associations with a broad range of orchid species. There
orchids. Our finding of constant colonization of roots in con- are, however, taxonomic problems concerning the species
tact with the bark supports the view of Benzing (1982) that concept in Tulasnella (Roberts 1999). The T. calospora strains
the role of the fungi may be crucial also for the adult epiphytic (T. deliquescens fide Roberts 1999) isolated by Warcup from ter-
orchids. The most important benefit for the orchid may be to restrial Australian orchids (Warcup Talbot 1967) belong to
retain the fungus in order to assure further seed germination. two separate clades according to our nucLSU tree (Fig 7). The
Saprotrophic capabilities, documented for several Tulasnella strains are even more clearly distinguished in the 5.8S-ITS
species (Roberts 1999), could explain the growth of Tulasnella tree (Fig 8), clustering with Tulasnella’s detected in terrestrial
on tree bark, including the capacity of fruiting close to colo- orchids from a wide range of localities, such as Spathaglottis
nized roots (J.P.S., pers. obs.), and promotion of seed germina- plicata from Singapore and Epipactis gigantea from California.
tion. We observed decline of vitality of hyphae after only one Our molecular analyses indicate that T. calospora is likely to
night of plant storage in the laboratory, independent of comprise several distinct species. Taxonomic problems ac-
whether or not the samples were cooled. The decline of vital- count also for T. violea and T. asymmetrica, as T. violea accession
ity was first indicated by loss of stainability of the pelotons, AY293216 appears close to T. pruinosa AF518662 in the nucLSU
which appeared yellow instead of blue in the light microscope. phylogenetic tree, while the Warcup T. violea isolate from the
TEM revealed very rare occurrences of vital pelotons, but orchid Thelymitra sp. clustered separately; the Warcup
abundant collapsed hyphae in this material. No isolates of T. asymmetrica strains (T. pinicola fide Roberts 1999) isolated
Tulasnellales were obtained from the stored samples and PCR from the orchid Thelymitra sp. fall into two groups (Fig 7). On
amplification success was very low, although only fully tur- the other hand Tulasnella’s with quite different trophic strat-
gescent roots were processed. It is rather unlikely that the egies are displayed as closely related in the nucLSU tree, e.g.
fast decline of hyphal vitality was linked to carbon shortage the mycobiont of the myco-heterotrophic liverwort Cryptothal-
as plenty of starch grains were visible in the root tissue and lus mirabilis (AY192482) that also forms ectomycorrhizas with
large amounts of glycogen were found in the vital hyphae. Dis- Pinus and Betula (Bidartondo et al. 2003), and T. irregularis
ruption of the extraradical mycelium or increase of plant (AY243519), a Warcup isolate from Dendrobium dicuphum, an
defense reaction could be involved (Hadley 1982). So far, the Australian orchid. Attempts are currently undertaken to col-
reason for this fast decline is unclear but it might erroneously lect and describe Tulasnella’s occurring on the bark of the
imply the impression of low hyphal colonization rate. sampled trees in our research area and to induce basidia for-
Our combination of ultrastructural research and DNA se- mation in the isolate cultures. Morphological description
quencing revealed that Tulasnella species were regularly asso- and determination combined with sequence typing appeared
ciated with the epiphytic Stelis and Pleurothallis species. We as promising tools to further clarify relationships between
successfully isolated three of the associated Tulasnella’s, these poorly studied mycobionts.
identity proven by DNA sequences and septal porus type. Irrespective of the taxonomic problems, available se-
The flat bell-shaped, imperforate parenthesomes with slightly quence data from nucLSU and ITS-5.8S made it possible to

12. 1268 ´
J. P. Suarez et al.
compare the seven Tulasnella clades of symbionts of epiphytic strains by the National Institute of Agrobiological Sciences
orchids studied here with named Tulasnella species and my- (NIAS), Japan, is also acknowledged.
corrhizal Tulasnella’s mostly from terrestrial orchids. Tulas-
nella’s of the studied epiphytic orchid species were distinct
Supplementary data
from so far known Tulasnellas associated with terrestrial or-
chids. Tulasnelloid fungi associated with the terrestrial tem-
Supplementary data associated with this article can be found,
perate orchids Cypripedium spp. (subfamily Cypripedioideae)
in the online version, at 10.1016/j.mycres.2006.08.004.
and Dactylorhiza majalis (AY634130) (subfamily Orchidoideae)
were displayed in a basal position with respect to the Tulas-
nella sequence types from Stelis and Pleurothallis in the nucLSU references
phylogeny (Fig 7). In the 5.8S nrDNA phylogeny, the tulasnel-
loid fungi associated with Dactylorhiza majalis (AY634130),
Orchis purpurea (AJ549121), Spathaglottis plicata (AJ313457), Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W,
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