PROCEEDINGS OF THE SECOND SCIENTIFIC CONFERENCE ON ANDEAN ORCHIDS MUTUALISTIC, ROOT-INHABITING FUNGI...

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PROCEEDINGS OF THE SECOND SCIENTIFIC CONFERENCE ON ANDEAN ORCHIDS

This digital version has been licensed under the 3.0 Ecuador Creative Commons: Attribution - Noncommercial - No Deriva-tive Works. | http://www.creativecommons.org/licences/by-nc-nd/3.0/ec/

Kottke I, Suárez JP (2009) Mutualistic, root-inhabiting fungi of orchids identification and functional types.In: Pridgeon A M, Suarez JP (eds) Proceedings of the Second Scientific Conference on Andean Orchids.

Universidad Técnica Particular de Loja, Loja, Ecuador, pp 84-99.

MUTUALISTIC, ROOT-INHABITING FUNGI OF ORCHIDSIDENTIFICATION AND FUNCTIONAL TYPES

Ingrid Kottke1 and Juan Pablo Suárez Chacón2

1 Eberhard-Karls-Universität Tübingen, Spezielle Botanik, Mykologie und Botanischer Garten, Auf der Mor-genstelle 1, D-72076 Tübingen, Germany

2 Centro de Biología Celular y Molecular, Universidad Técnica Particular de Loja, San Cayetano Alto s/n C.P. 1101608, Loja, Ecuador

ABSTRACT

Orchids depend on fungi for germination and protocorm development and maintain mycor-rhizas in their adult forms. Mycorrhizal fungi are therefore a driving force in orchid evolution and speciation. However, reliable data on fungal identities are crucial. Information from molecular phylo-genetics and transmission electron microscopy congruently revealed distinct fungal groups as orchid mycobionts. Sebacinales Group B, Tulasnellales, and Ceratobasidiales were found associated with ter-restrial orchids in open grasslands and arbuscular mycorrhizal forests and with epiphytes, whereas Sebacinales Group A, Thelephorales, Russulales, some Euagaricales, and Tuberales form mycorrhizas with terrestrial orchids in ectomycorrhizal forests. Enzyme and isotope analyses revealed that the for-mer obtain carbon from rotten organic material to nourish the protocorm, whereas the latter take car-bon from ectomycorrhizas of woody plants to supply nutrients to protocorms and adult heterotrophic or mixotrophic plants. Mycobionts of terrestrial orchids are of narrower host range than previously expected. The few investigations on mycobionts of epiphytic orchids indicate sharing of hosts. DNA-based fungal identities of mycobionts from tropical terrestrial and epiphytic orchids, host range, and inclusion of data from several ecological parameters are still needed to determine whether association strategies differ between epiphytic and terrestrial orchids or between temperate and tropical habitats.

METHODS FOR IDENTIFICATION OF ORCHID MYCOBIONTS

Orchid mycobionts were previously only known by isolation of the fungi from orchid roots or protocorms, description of hyphal features of the anamorphic state (Currah et al., 1997; Roberts, 1999), and occasional induction of the teleomorphs (Warcup and Talbot, 1966, 1967a,b, 1980; Cur-rah et al., 1987; Warcup, 1988). Re-infection experiments claimed separation of the true mycobiont isolates from endophytes of the velamen and other contaminants (Bernard, 1909; Brundrett, 2006;

85This digital version has been licensed under the 3.0 Ecuador Creative Commons: Attribution - Noncommercial - No Deriva-tive Works. | http://www.creativecommons.org/licences/by-nc-nd/3.0/ec/

Bonnardeaux et al., 2007). Transmission electron microscopical studies of the isolates and in situ hyphae yielded distinctive features to discern and confirm the main systematic groups of orchid my-cobionts (Currah and Sherburne, 1992; Andersen, 1996; Gleason and McGee, 2001; Selosse et al., 2004, Suarez et al., 2006, 2008). Ultrastructural details are also needed to demonstrate that the fungi are forming pelotons (coils) inside vital protocorm and root cortical cells (Peterson and Massicotte, 2004). DNA–sequencing of the mycobionts directly from the mycorrhizas combined with molecular phylogenetics have yielded an unexpected richness of fungal genotypes including fungal groups previ-ously unknown as orchid mycobionts.

ORCHIDS DEPEND ON MYCOBIONTS

Orchids need fungi supplying carbohydrates and soil nutrients to promote growth of the non-photosynthetic protocorm stage (Bernard, 1909; Smith and Read, 1997). Similar mycobionts are ha-bitually harbored in root cortical cells of adult green and non-photosynthetic orchids (Fig. 1a). These fungi colonize not only the velamen and epidermal cells where many other fungi also find shelter (Fig. 1b) but penetrate the root exodermis via living passage cells (Fig. 1d ) and form coils (pelotons) in root cortical cells (Fig. 1c). Hyphae soon collapse and become encased by root cell wall material (Fig. 1c; Peterson et al., 1996). This type of interaction was found in both orchid protocorms and roots independent of orchid and mycobiont species involved (Dörr and Kollmann, 1969; Peterson and Cur-rah, 1990; Rasmussen, 1990; Selosse et al., 2004, Suarez et al., 2006, 2008; Abadie et al., 2006).

Plant access to carbon, nitrogen, and phosphorus via the fungal mycelium was demonstrated (Smith, 1967; Alexander et al., 1984; Alexander and Hadley, 1985; Gebauer and Meyer, 2003; Trudell et al., 2003; Bidartondo et al., 2004; Cameron et al., 2006, 2007), and bidirectional transfer of carbon between the green-leaved orchid Goodyera repens (L.) R.Br. and its mycobiont Ceratobasidium cornige-rum was proven in Petri dish microcosms, supporting the view of a true mutualistic interaction (Cam-eron et al., 2006). With respect to carbon acquisition, two functionally distinct groups of fungi were found associated with the orchids: mycobionts with saprophytic capabilities and mycobionts with ectomycorrhizal associations. Isolates of saprophytic orchid mycobionts displayed enzyme activities for degrading cellulose and other complex organic compounds (Hadley, 1969; Midgley et al., 2006), indicating access to carbon from humus, although this has so far not been demonstrated in nature. Ectomycorrhizal fungi supply carbon obtained from woody plant roots to the achlorophyllous, my-coheterotrophic or green, mixotrophic orchids (McKendrick et al., 2000a; Gebauer and Meyer, 2003; Trudell et al., 2003).

MUTUALISTIC, ROOT-INHABITING FUNGI OF ORCHIDS IDENTIFICATION AND FUNCTIONAL TYPES

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ORCHID MYCOBIONTS AS IDENTIFIED BY MOLECULAR PHYLOGENYAND ULTRASTRUCTURAL CHARACTERS

For many years, only few and unspecific fungi, so-called Rhizoctonia species, isolated from or-chid mycorrhizas were considered as orchid mycobionts (Hadley, 1970). However, isolation of fungal DNA directly from the mycorrhizas followed by sequencing yielded an unexpected richness of geno-types of not only previously known orchid mycobiont groups but also unexpected ectomycorrhizal fungi. Obviously, many of the mycobionts do not grow on artificial media. Claims for re-infection to prove the mycobiont nature of the fungi can, therefore, not be strictly met. Isolation is also biased by large numbers of fungi, mostly ascomycetes that only colonize the velamen, epidermal cells or root hairs (Williamson and Hadley, 1970; Bayman and Otero, 2006).

Sequencing of mycobionts directly from the mycorrhizas and ultrastructural studies of hy-phae in orchid root cortical cells proved three groups of hymenomycetous heterobasidiomycetes -- the Sebacinales, Tulasnellales, and Ceratobasidiales -- and several groups of hymenomycetous homobasi-diomycetes, especially Thelephorales, Russulales, and Tuberales of Ascomycota as orchid mycobionts (Tables 1-4). Heterobasidiomycetes had already been indicated by teleomorphs induced in cultures by Warcup and Talbot (1966, 1967a,b, 1980) and Warcup (1988). However, molecular phylogenet-ics revealed that Sebacinales fall into two distinct groups, A and B (Weiss et al., 2004). Autotrophic orchids were found associated with Sebacinales group B, related to Sebacina vermifera sensu Warcup and Oberwinkler, whereas mycoheterotrophic or mixotrophic orchids growing with ectomycorrhiza forming trees are associated with Sebacinales group A, related to S. incrustans and S. epigaea (Table 1). Tulasnella calospora and T. violea sensu Warcup came out as polyphyletic, and related genotypes were found with numerous orchids (Table 2). Ceratobasidiales were also found with diverse orchids, but in situ proofs are rare (Gleason and McGee, 2001), and most authors worked on fungal isolates only (Table 3). Taxonomy within the three heterobasidomycete groups is unresolved, and taxon names cannot yet be provided. Although earlier findings pointed to a liaison of orchids with ectomycorrhiza-forming fungi (Warcup 1991; Zelmer and Currah, 1995), DNA-sequencing of orchid mycobionts directly from the mycorrhizas revealed that this is a widespread phenomenon of orchids in ectomycor-rhizal forests (Table 4). These orchids are either heterotrophic or mixotrophic, although the latter was not proven for every species so far (Table. 4).

Ultrastructural hyphal characters allow unambiguous distinctions among the four basidiomy-cete groups and the ascomycetes in root cortical cells and fungal isolates. Sebacinales are characterized by fine hyphae with electron-dense cell walls and dolipores with imperforate, straight or dish-shaped pore caps (Fig. 2a; Andersen 1996; Gleason and McGee, 2001; Setaro et al., 2006; Suarez et al., 2008). Tulasnellales possess dolipores with imperforate, dish-shaped pore caps with recurved ends (Fig. 2b) and slime bodies in the cell walls (Fig. 2c), becoming electron-dense during aging (Fig. 2d; Ander-sen, 1996; Gleason and McGee, 2001; Suarez et al., 2006). Ceratobasidiales are distinct by having dolipores with dome-shaped pore caps that contain large perforations (Fig. 2e; Gleason and McGee, 2001), whereas the homobasidiomycetes have dolipores with dome-shaped but multi-perforate pore caps (Fig. 2f, Abadie et al., 2006). Ascomycete mycobionts possess simple cell wall pori accompanied by Woronin bodies and mostly electron-translucent cell walls (Fig. 2g; Setaro et al., 2006). The only ascomycetes so far proven as mycobionts of orchids in situ are members of the ectomycorrhizal genus Tuber (Selosse et al., 2004). However, further taxonomic resolution cannot be obtained using only ultrastructural characters.


Figure 1. Light micrographs of sections through mycorrhizal roots: a. Patchy distribution of fungal pelotons (p) in root cortical cells (CC) of Elleanthus sp. Outermost root layers comprise the velamen (V). Scale bar = 50 µm; b. Dense fungal colonization (h) of the two-layered velamen (V) and fungal pelotons (p) of Tulasnella sp. occur in the root cor-tical cells (CC), but hyphae are not present in the thick-walled exodermal cells (Ex) of Elleanthus sp. Scale bar = 50 µm; c. Pelotons (p) of fine, sebacinoid fungus and collapsed hyphae (ch) in root cortical cells (CC) of Sobralia rosea Poepp. & Endl. Scale bar = 10 µm; d. Hypha (h) traversing living, thin-walled passage cell (PC) of the exodermis of Elleanthus sp. Shown also are velamen (V) and tilosomes (T), outer wall excrescences of the passage cell. Scale bar = 10 µm.


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Sebacinales Group A

Orchid species Geographic area Material and methods References

Cephalanthera damasonium (Mill.) Druceheterotrophic and mixotrophic

France ITS from mycorrhizas, BLAST Julou et al., 2004

Cephalanthera longifolia (L.) Fritsch heterotrophic and mixotrophic

Estonia ITS-RFLPs and sequences from mycorrhizas, BLAST

Abadie et al., 2006

Cypripedium fasciculatum Kellog ex S.Watson mixotrophic

USA nucLSU, mtLSU RFLPs and sequences from mycorrhizas, molec. phylogeny

Shefferson et al., 2005, 2007; Suarez et al., 2008

Epipactis atrorubens (Hoffm.) Bessermixotrophic

Germany ITS, mtLSU from mycorrhizas, BLAST Bidartondo et al., 2004

Epipactis helleborine (L.) Crantzmixotrophic

Germany ITS, mtLSU from mycorrhizas, molec. phylogeny

Bidartondo et al., 2004;Suarez et al., 2008

Epipactis microphylla (Ehrh.) Sw.heterotrophic and mixotrophic

France ITS, nrLSU from mycorrhizas, BLAST Selosse et al., 2004

Hexalectris revoluta CorrellH. spicata (Walter) Barnhartheterotrophic

USA ITS RFLPs, ITS/nrLSU sequences from mycorrhizas, molec. phylogeny

Taylor et al., 2003

Neottia nidus-avis (L.) Rich.heterotrophic

France, Germany, UK ITS, mtLSU sequences from mycorrhizas, molec. phylogeny

McKendrick et al., 2002; Selosse et al., 2002:Bidartondo et al. 2004

Sebacinales Group B

Caladenia carnea R.Br.C. falcata (Nicholls) M.A.Clem.

Australia ITS-RFLPs, nucLSU sequences from isolates, molec. phylogeny

Bougoure et al. 2005:Suarez et al., 2008

Caladenia catenata (Sm.) Druce Australia Warcup’s isolates; nucLSU Warcup 1988, Weiss et al. 2004

Cypripedium californicum A.Gray C. parviflorum Salisb.

USA nucLSU, mtLSU RFLPs and sequences from mycorrhizas, molec. phylogeny

Shefferson et al. 2005, 2007;Suarez et al. 2008

Cyrtostylis reniformis R.Br. Australia Warcup’s isolates; nucLSU, molec. phylogeny Warcup 1988; Weiss et al. 2004

Epipactis palustris (L.) Crantz Germany ITS, mtLSU from mycorrhizas, molec. phylogeny

Bidartondo et al. 2004: Suarez et al. 2008

Eriochilus scaber Lindl. Australia Warcup’s isolates; nucLSU, molec. phylogeny Warcup 1988; Weiss et al. 2004

Glossodia minor R.Br. Australia Warcup’s isolates; nucLSU, molec. phylogeny Warcup 1988; Weiss et al. 2004

Microtis media R.Br. Australia ITS from isolates, BLAST Bonnardeux et al. 2007

Microtis unifolia (G.Forst.) Rchb.f. Australia Warcup’s isolates, nucLSU, molec. phylogeny Warcup 1988; Weiss et al. 2004

Pleurothallis lilijae Foldats, epiphytic Ecuador nucLSU/ITS from mycorrhizas, molec. phylogeny

Suarez et al. 2008

Stelis concinna Lindl., S. superbiens Lindl.,S. hallii Lindl., epiphytic

Ecuador nucLSU/ITS from mycorrhizas, molec. phylogeny

Suarez et al. 2008

Table 1. Sebacinales Group A and Group B identified by molecular techniques in orchid species from diverse regions.


Orchid species Geographic area Material and methods Reference

Acianthus exsertus R.Br. Australia RFLPs and of ITS/nrLSU from isolates, molec. phylogeny

Bougoure et al., 2005;Suarez et al., 2006

Anacamptis laxiflora (Lam.) R.M. Bateman, Pridgeon & M.W.Chase [as Orchis laxiflora]Anacamptis morio (L.) R.M.Bateman, Pridgeon & M.W.Chase [as Orchis morio]

Hungary ITS from isolates, molec. phylogeny Illyes, unpublished; Suarez et al., 2006

Arundina graminifolia (D.Don.) Hochr. Singapore ITS from isolates, molec. phylogeny Ma et al., 2003; Suarez et al., 2006

Cephalanthera austiniae (A.Gray) A.Heller heterotrophic USA Pelotons from protocorms and mycorrhizas, isolates, ITS, mtLSU, nrLSU, molec. phylogeny

McCormick et al., 2004

Cypripedium acaule Aiton, C. arietinum R.Br., C. calceolus L., C. californicum A.Gray, C. candidium Muhl. ex Willd., C. debile Rchb.f., C. fasciculatum Kellogg ex S.Watson, C. guttatum Sw., C. japonicum Thunb., C. macranthos Sw., C. montanum Douglas ex Lindl., C. parviflorum Salisb., C. reginae Walter

Alaska, Estonia, Japan,USA, Taiwan, Russia

nucLSU, mtLSU RFLPs and sequences from mycorrhizas, molec. phylogeny

Shefferson et al., 2005, 2007

Cypripedium fasciculatum Kellogg ex S.Watsonmixotrophic

USA ITS, nrLSU, SSU, RFLPs and sequences from mycorrhizas, molec. phylogeny

Whitridge and Southworth, 2004, Suarez et al., 2006

Dactylorhiza majalis (Rchb.) P.F.Hunt & Summerh. DenmarkGermany

mtLSU, nrLSU from mycorrhizas, molec. phylogeny

Kristiansen et al., 2001, Bidartondo et al., 2004; Suarez et al., 2006

Dendrobium crumenatum Sw. Singapore ITS from isolates, molec. phylogeny Ma et al., 2003; Suarez et al., 2006

Disa bracteata Sw. Australia ITS from isolates, BLAST Bonnardeux et al., 2007

Epipactis atrorubens (Hoffm.) Besser, E. gigantea Douglas ex Hook., E. palustris (L.) Crantz


Bidartondo et al., 2004;Suarez et al., 2006

Goodyera pubescens (Willd.) R.Br. USA ITS, mtLSU, nrLSU from mycorrhizas, molec. phylogeny


Liparis lilliifolia (L.) Rich. ex Lindl. USA Protocorms, adults, isolates ITS, mtLSU, nrLSU, molec. phylogeny


Liparis loeselii (L.) Rich. Hungary ITS from isolates, molec. phylogeny Illyes et al., 2005; Suarez et al., 2006

Microtis parviflora R.Br. Australia RFLPs and of ITS/nrLSU from isolates, molec. phylogeny

Bougoure et al., 2005;Suarez et al., 2006

Neuwiedia veratrifolia Blume Borneo mtLSUfrom single pelotons andisolates, molec. phylogeny

Kristiansen et al., 2004

Table 2. Tulasnellales identified by molecular techniques in orchid species from diverse regions


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MYCORRHIZAS FOR ORCHID EVOLUTION AND SPECIATION

Co-evolution on a gross level between orchids and their mycobionts is not indicated by the cur-rent results (Fig. 3). Although Sebacinales are considered the most basal group of hymenomycetous basidiomycetes (Weiss and Oberwinkler, 2001), these fungi were not confirmed in orchid subfamilies Apostasioideae and Vanilloideae and only rarely found in subfamily Cypripedioideae, all considered basal orchid groups. The switch to ectomycorrhizal fungi occurred independently several times during orchid evolution (Fig. 3; Molvray et al., 2000), linked with gradual change from autotrophy to mixot-rophy and finally mycoheterotrophy (Bidartondo et al., 2004). Association with ectomycorrhizal fungi allowed the orchids to enter the shadiest ectomycorrhizal forests in temperate climate as epiparasites of trees. All investigated orchids associate with several fungi, mostly from diverse groups, and overlap-ping of associations may be common (Fig. 3). However, the taxonomic level of fungal genotypes is still a matter of debate, and therefore specific species-to-species associations or sharing of individual fungal species are hard to prove. There are currently only a few molecular studies on mycobionts from large numbers of related orchid species sampled in a multitude of locations (Fig. 3). These studies of terrestrial orchids (Taylor et al., 2002, 2004; Shefferson et al., 2007) found a narrow range of as-sociations, whereas molecular phylogenetic investigation of Tulasnellales and Sebacinales B associated with epiphytic orchids in an Andean cloud forest found predominant sharing of mycobionts (Suarez et al., 2006, 2008). The findings need confirmation from other epiphytic and terrestrial orchids to determine whether association strategies differ between epiphytic and terrestrial orchids or between temperate and tropical habitats.

Platanthera chlorantha (Custer) Rchb. Germany ITS, mtLSU from mycorrhizas, molec. phylogeny

Bidartondo et al., 2004

Pleurothallis lilijae Foldats, epiphytic Ecuador nrLSU/ITS from mycorhizas, molec. phylogeny

Suarez et al., 2006, 2008

Spathoglottis plicata Blume Singapore ITS from isolates, molec. phylogeny Ma et al., 2003; Suarez et al., 2006

Stelis concinna Lindl., S.superbiens Lindl., S. hallii Lindl., epiphytic

Ecuador nrLSU/ITS from mycorhizas, molec. phylogeny

Suarez et al., 2006, 2008

Thelymitra pauciflora R.Br. Australia RFLPs and of ITS/nrLSU from isolates, FASTA

Bougoure et al., 2005

Tipularia discolor (Pursh) Nutt. USA Protocorms, adults, isolates ITS, mtLSU, nrLSU, molec. phylogeny


Vanda sp. Singapore ITS from isolates, molec. phylogeny Ma et al., 2003; Suarez et al., 2006

Vanilla planifolia Jacks. ex Andrews, V. poitaei Rchb.f. Puerto Rico ITS, mtLSU from isolates and mycorrhizas, molec. phylogeny

Porras-Alfaro and Bayman, 2007


Figure 2. Transmission electron micrographs of orchid mycobionts. a. Sebacinoid hypha in root cortical cell of So-bralia rosea with imperforate, slightly dish-shaped pore caps (Pa) with straight margins, covering the dolipore. Scale bar = 1 µm; b. Tulasnelloid hypha in root cortical cell of Elleanthus sp. with imperforate pore caps (Pa), dish-shaped with recurved margins. Scale bar =1 µm; c. Square section of tulasnelloid hyphae (h) with slime bodies (Sb) in cell walls (arrowhead) in root cortical cell of Elleanthus sp. Scale bar = 1 µm; d. Tulasnelloid hypha displaying several layers of slime bodies (arrows) in the cell walls. Scale bar = 1 µm; e. Hypha of Ceratobasidiales in root cortical cell of Stelis sp. displaying dome-shaped pore caps (Pa) with several holes. Scale bar = 1 µm; f. Homobasidiomycete in ectomycorrhiza displaying regular perforate pore caps (Pa). Scale bar = 0.5 µm; g. Ascomycete hypha with simple pore and Woronin bodies (Wb). Scale bar = 1 µm.


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Figure 3. Distribution of mycobionts in subfamilies of Orchidaceae as determined by molecular techniques. Color of orchid name indicates dominant fungal group; colored lines indicate subordinate fungi associated. Heterotrophic and mixotrophic state of orchids is given when proven by isotope measurements. Homobasidiomycetous ectomy-corrhiza-forming fungi (bECMF).


Orchid species Geographic area Material and methods Reference

Campylocentrum fasciola (Lindl.) Cogn.C. filiforme (Sw.) Cogn.

Puerto Rico ITS from isolates, molec. phylogeny Otero et al., 2002


France ITS from mycorrhizas, BLAST Julou et al., 2004

Cephalanthera longifolia (L.) Fritschheterotrophic and mixotrophic


Abadie et al., 2006

Chamaegastrodia shikokiana Makino & Maek. heterotrophic

Japan ITS from isolates, molec. phylogeny Yagame et al., 2008

Cypripedium californicum A.Gray USA nucLSU, mtLSU RFLPs and sequences from mycorrhizas, molec. phylogeny


Dactylorhiza majalis (Rchb.) P.F.Hunt & Summerh., Epipactis helleborine (L.) Crantz, E. palustris (L.) Crantz, Platanthera chlorantha Custer (Rchb.)



Hexalectris spicata (Walter) Barnhart, H. spicata var. arizonica (S.Watson) Catling & V.S.Engelmycoheterotroph

USA ITS RFLPs,nrLSU sequences from isolates, molec. phylogeny

Taylor et al., 2003

Ionopsis satyrioides (Sw.) Rchb.f.I. utricularioides (Sw.) Lindl.

Puerto Rico ITS from isolates, molec. phylogeny Otero et al., 2002, 2004, 2007

Neuwiedia veratrifolia Blume Borneo mtLSU, single pelotons from mycorrhizas and isolates, molec. phylogeny

Kristiansen et al., 2004

Oeceoclades maculata (Lindl.) Lindl. Puerto Rico ITS from isolates, molec. phylogeny Otero et al., 2002

Oncidium altissimum (Jacq.) Sw. Puerto Rico ITS from isolates, molec. phylogeny Otero et al., 2002

Psychilis monensis Sauleda Puerto Rico ITS from isolates, molec. phylogeny Otero et al., 2002

Pterostylis nutans R.Br. Australia ITS from isolates, molec. phylogeny Irwin et al., 2007

Pterostylis sanguinea D.L.Jones & M.A.Clem. Australia ITS from isolates, molec. phylogeny Bonnardeux et al., 2007

Pterostylis nutans R.Br.P. obtusa R.Br.

Australia Isolates, ITS-RFLPs, ITS/LSU sequences, FASTA

Bougoure et al., 2005

Sarcochilus falcatus R.Br.S. olivaceus Lindl.

Australia TEM porus of isolates Gleason and McGee, 2001

Tolumnia variegata (Sw.) Braem Puerto Rico ITS from isolates, molec. phylogeny Otero et al., 2002, 2004

Vanilla planifolia Jacks. ex Andrews, V. poitaei Rchb.f., V. aphylla Blume, V. phaeantha Rchb.f.

Puerto Rico, Cuba, Costa Rica

nrITS, mtLSU from isolates and mycorrhizas, molec. phylogeny

Porras-Alfaro & Bayman, 2007

Table 3. Ceratobasidiales identified by molecular techniques or ultrastructure in orchid species from diverse regions.


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Fungal group or species

Orchid species Geographic area

Material and methods Reference

Thelephoraceae Cephalanthera austiniae (A.Gray) A.Hellerheterotrophic

USA ITS, mtLSU, nrLSU RFLPs and sequences from isolates and mycorrhizas, molec. phylogeny

Taylor and Bruns, 1997; McCormick et al., 2004

Cortinariaceae, Hymenogaster, Inocybe, Thelephoraceae


Germany, France

ITS, mtLSU from mycorrhizas, molec. phylogeny

Bidartondo et al., 2004; Julou et al., 2004

Thelephoraceae, Cortniariaceae, Amphinema, Wilcoxina

Cephalanthera longifolia (L.) Fritschheterotrophic and mixotrophic


Abadie et al., 2006

Tomentella Cephalanthera rubra (L.) Rich.mixotrophic



Russulaceae Corallorhiza maculata (Raf.) Raf., C. mertensiana Bong., heterotrophic

USA ITS, mtLSU, nrLSU - RFLPs and sequences from mycorrhizas, molec. phylogeny

Taylor and Bruns, 1997, 1999Taylor et al., 2003

Thelephoraceae Corallorhiza trifida Châtelheterotrophic

Scotland ITS from mycorrhizas, BLAST McKendrick et al., 2000b

Thelephoraceae Cypripedium candidium Muhl. ex Willd., C. montanum Douglas ex Lindl.

USA nucLSU, mtLSU RFLPs and sequences from mycorrhizas , molec. phylogeny


Russula laurocerasii Cypripedium acaule Aiton USA nucLSU, mtLSU RFLPs and sequences from mycorrhizas, molec. phylogeny

Shefferson et al., 2007

Russula, Lactarius, Suillus

Cypripedium fasciculatum Kellogg ex S.Watsonmixotrophic

USA ITS, nrLSU, SSU, RFLPs and sequences from mycorrhizas, molec. phylogeny

Whitridge and Southworth, 2005

Russula Cypripedium parviflorum Salisb. USA nucLSU, mtLSU RFLPs and sequences from mycorrhizas, molec. phylogeny


Hygrocybe, Cantharellus

Cypripedium reginae Walter USA nucLSU, mtLSU RFLPs and sequences from mycorrhizas , molec. phylogeny

Shefferson et al., 2007

GymnomycesRussula

Dipodium hamiltonianum F.M.Baileyheterotrophic

Australia ITS from mycorrhizas, BLAST Dearnaley and Le Brocque, 2006

Russula Dipodium variegatum M.A.Clem.heterotrophic

Australia ITS, from mycorrhizas, BLAST Bougoure and Dearnaley, 2005

Table 4. Ectomycorrhiza-forming homobasidiomycetes identified by molecular techniques in orchid species from diverse regions.


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Inocybe, Tuber Epipactis atrorubens (Hoffm.) Bessermixotrophic

Germany ITS, mtLSU from mycorrhizas , molec. phylogeny


Wilcoxina Epipactis distans Arv.-Touv.mixotrophic



Tuber Epipactis helleborine (L.) Crantzmixotrophic



Cortinariaceae, Hymenosgastraceae, Thelephoraceae, Russula foetens Fr., Tuber spp.

Epipactis microphylla (Ehrh.) Sw.heterotrophic and mixotrophic

France ITS, nrLSU from mycorrhizas, BLAST

Selosse et al., 2004

Russula delica Fr., R. chloroides Kalchbr., R. brevipes Peck

Limodorum abortivum (L.) Sw., L. brulloi Bartolo & Pulv., L. trabutianum Batt., mixotrophic

Italy ITS-RFLPs and sequences from mycorrhizas, molec. phylogeny

Girlanda et al., 2006


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