84 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, Su��rez 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 T��cnica Particular de Loja, Loja, Ecuador, pp 84-99. MUTUALISTIC, ROOT-INHABITING FUNGI OF ORCHIDS IDENTIFICATION AND FUNCTIONAL TYPES Ingrid Kottke1 and Juan Pablo Su��rez Chac��n2 1 Eberhard-Karls-Universit��t T��bingen, Spezielle Botanik, Mykologie und Botanischer Garten, Auf der Mor- genstelle 1, D-72076 T��bingen, Germany 2 Centro de Biolog��a Celular y Molecular, Universidad T��cnica 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

85 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/ 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 (D��rr 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

86 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/ ORCHID MYCOBIONTS AS IDENTIFIED BY MOLECULAR PHYLOGENY AND 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.