Neodiversity 7: 1–10 www.neodiversity.org published online,17th July 2014

EPULORHIZA AMONILIOIDES SP. NOV.: A NEW ANAMORPHIC SPECIES OF ORCHID MYCORRHIZA FROM BRAZIL

P.R.M. ALMEIDA1, C. VAN DEN BERG1, A. GÓES­NETO1

1Programa de Pós­Graduação em Botânica, Universidade Estadual de Feira de Santana, Av. Transnordestina s.n., 44036­900, Feira de Santana, Babhia, Brazil. e­mail: [email protected]

ABSTRACT

In this study, a new anamorphic species of orchid mycorrhizal fungus is described and illustrated based on both morphological and molecular characteristics. Mycorrhizal fungal strains were isolated and selected from root samples of three species of rupicolous, epiphytic and terrestrial tropical orchids that are native to Brazil: Encyclia dichroma, E. ghillanyi and Brassavola tuberculata. Epulorhiza amonilioides sp. nov. is distinguished from the other species of Epulorhiza by the absence of monilioid cells in pure culture. Phylogenetic analyses based on 5.8S ITS rDNA and mtLSU indicated that E. amonilioides is phylogenetically different from other fungal lineages of Tulasnella previously identified on tropical and temperate orchid and Sebacinaceae species. E. amonilioides forms a different clade that groups with some anamorphic mycorrhizal fungi corresponding to Tulasnella irregularis. E. amonilioides is strictly related to T. irregularis, its putative teleomorph. E. amonilioides is the seventh species described for the genus and is the second from Brazil.

polyphenol oxidase production (Currah and The genera Epulorhiza, Ceratorhiza and Zelmer 1992; Currah et al. 1987; Zelmer Moniliopsis (Basidiomycota) were proposed and Currah 1995; Currah et al. 1997; by Moore as anamorphic fungi that form a Pereira et al. 2003). Although there are mycorrhizal association with orchids (Moore features that can be easily detected for the 1987). Their segregation from Rhizoctonia identification of the genus, infraspecific was based on the morphology of the delimitation remains difficult (Rasmussen dolipore septum and on anamorph­ 1995, 2002). This is due mainly to the teleomorph connections. Epulorhiza was difficulty of defining diagnostic features defined as bearing a dolipore septum with a and the absence of stable morphological non­perforated parenthosome, with Tulasnella features in pure cultures (Andersen 1990). J.Schröt. as its teleomorph (Moore 1987, To combine speed and efficiency in the 1996). identification of mycorrhizal fungi at genus The circumscription of Epulorhiza was level, most current studies have focused on also marked by morphological features of the direct identification, by PCR and isolates in pure culture, which distinguish it sequencing, of the ITS (Internal Transcribed from the other genera of rhizoctonioid fungi Spacer) region of the rDNA in root samples (Currah and Zelmer 1992). These features (Shefferson et al. 2005, 2007; Bougoure et are (i) slow growth, (ii) the margin or al. 2005; Kristiansen et al. 2004; Mc practically all the colony being submerged, Cormick et al. 2004; Suárez et al. 2006; (iii) monilioid cells being oval or comp­ Shimura et al. 2009; Nontachaiyapoom et letely spherical, and (iv) the absence of 1 ALMEIDA ET. AL.­ NEW EPULORHIZA FROM BRAZIL al. 2010; Roche et al. 2010). This method areas in the interior of Bahia; (ii) Encyclia has enabled great progress in the study of ghillanyi Pabst, rupicolous plants that grow natural populations, though it does not allow on rock surfaces in semi­arid areas in the the examination of isolate morphology for a interior of Bahia; and (iii) Encyclia comparative analysis of morphological and dichroma (Lindl.) Schltr., which grows in molecular data (Almeida et al. 2007). This humid sandy areas along the Bahia coast may be reflected by the current situation of and are always in patches of vegetation, Epulorhiza, which has only six described together with species of Myrtaceae, species (Currah and Zelmer 1992; Currah et Euphorbiaceae and Cactaceae, in Restinga al. 1987; Zelmer and Currah 1995; Currah et (strand vegetation amidst Atlantic Forest). al. 1997; Pereira et al. 2003); one of them, Here, we describe and illustrate these E. epiphytica, was described from Brazil by isolates as a new species of Epulorhiza, Pereira, Rollemberg & Kasuya (Pereira et based on both morphological and molecular al. 2003). features. While analyzing isolates of orchid species from the northeastern region of MATERIALS AND METHODS Brazil, especially from the Bahia state, we Study area, isolation and detected some anamorphs that not only had morphological characterization of the different morphological features in pure fungi — Mycorrhizal fungi were isolated culture but also showed ITS rDNA and from plants of nine populations (four mtLSU sequences that were distinct from populations of Encyclia dichroma, four of E. established sequences in GenBank. These ghillanyi and one of Brassavola tuberculata) isolates were obtained from three species of distributed from south to northeast Bahia orchids: (i) Brassavola tuberculata Hook., (Table 1). epiphytic or rupicoulous plants in semi­arid Mycorrhizal fungi were directly isolated

Table 1. Locations of the 10 plant populations and the number of E. amonilioides isolates analyzed.

2 ALMEIDA ET. AL.­ NEW EPULORHIZA FROM BRAZIL from pelotons using a modified version of cycles of 95°C for 30 sec, 53°C for 45 sec, the protocol of Warcup and Talbot (1967). 72°C for 1 min, and a final extension at The isolates were cultured in six different 72°C for 10 min. Amplicons were purified culture media and incubated for 60 days at by digestion with exonuclease and alkaline 28°C in darkness to evaluate morphology in phosphatase (ExoSAP­IT, GE Healthcare), pure culture and to allow the formation of and sequenced in an ABI 3130XL monilioid cells and sclerotia. The media automated sequencer (Applied Biosystems). were potato dextrose agar (PDA; Difco), The new sequences were deposited in cornmeal agar (CMA; Difco), malt extract GenBank under the numbers JF907598 ­ agar, tap water agar, coconut milk agar JF907605 and treebase ID 11445. (Zelmer and Currah 1995), and oat meal Phylogenetic analysis — agar (30 g oat meal, 15 g agar, 1000 ml Chromatograms were edited with water). PREGAP4 and GAP4 (Staden et al. 1998). The growth rate was determined by Sequence similarity was verified using averaging three independent radial measure­ BLASTn (Zhang et al. 2000) with available ments obtained every 48 hours, in PDA at sequences in GenBank (http://www.ncbi. 28°C in darkness, over two weeks. The nlm.nih.gov/genbank/), and was subsequ­ number of nuclei per cell was observed in ently aligned with ClustalX (Thompson et young hyphae stained with HCl­Giemsa al. 1997). Ambiguous aligned regions with (Sakena 1961). Observations of micro­ high proportion of gaps were excluded. morphological features were carried out To enable wider phylogenetic analysis, with both light microscopy (Zeiss) and only the 5.8S nuclear rDNA region was scanning electron microscopy (SEM; LEO used, which allowed the inclusion of most 1430VP). Enzymatic assays to evaluate Epulorhiza/Tulasnella and Sebacina sequences in cellulase and polyphenol oxidase were GenBank. This approach allowed us to performed according to Smith (1977) and evaluate the degree of divergence between Davidson et al. (1938), respectively. The our sequences and all other sequences pure cultures were deposited in the CCMB obtained from mycorrhizal fungi of both (Culture Collection of Microorganisms of tropical and temperate orchids. These other Bahia): CCMB 513­519. sequences were also included in the Genomic DNA extraction, ITS rDNA sequence analysis of both genes of E. amplification and amplicon sequencing epiphytica, to evaluate their positioning — Isolates were cultivated in broth malt relative to GenBank sequences of different extract for 15 days at 28°C in darkness, and lineages. DNA was extracted according to the CTAB Maximum Likelihood (ML) phylogenetic protocol of Doyle and Doyle (1987) with analysis was carried out online using some modifications. ITS and mtLSU RAxML version 7.0.3 (Stamatakis et al. regions of the isolates were amplified using 2006), in the CIPRES web interfaceportal primers ITS5­ITS4 (White et al. 1990) and (http://www.phylo.org/sub_sections/portal) ML5­ML6 (Bruns et al. 1998). PCR was using the GTRMIX DNA substitution model performed in a final volume of 25 µL and with 1000 bootstrap iterations (Felsenstein cycled as follows. For the ITS rDNA region, 1985). We selected Thanatephorus the thermal profile was 94°C for 4 min, 35 cucumeris AF354097 and Waitea circinata cycles of 94°C for 1 min, 50°C for 1 min AD001658 as external sequences for the and 72°C for 3 min, and a final extension at analyses of 5.8S ITS rDNA and mtLSU, 72°C for 7 min. For the mtLSU region, the respectively, because they are taxa of thermal profile was 95°C for 3 min, 35 Ceratobasidiaceae, which is phylogenetically

3 ALMEIDA ET. AL.­ NEW EPULORHIZA FROM BRAZIL closer to Tulasnellaceae than other Basidio­ activity for polyphenol oxidase and weak mycota (Moncalvo et al. 2006). cellulolytic activity. ADDITIONAL ISOLATES: Colonies in RESULTS PDA, isolated from Encyclia ghillanyi Epulorhiza amonilioides P.R.M.Almeida, Pabst, collected on rocky surfaces in the Van den Berg & A.Góes­Neto, sp. nov. municipalities of Itiruçu, Santa Terezinha (Figure 1). Mycobank: MB 561179 and Ruy Barbosa, State of Bahia, Brazil, July, 17th 2007; deposited as CCMB 515­ Coloniae in agaro PDA et agaro CMA 519. lentissime crescentes, eburneae vel pallido­ eburneae, grumosae vel laeves cum mycelio DISCUSSION aerio parvo margine submersa integra vel Taxonomic conclusions based on diffusa. In agaro PDA odor dulcis. Hyphae molecular data — Epulorhiza amonilioides hyalinae ordinatim septatae, 2.5 µm differs from all other Epulorhiza species in diametro, cellulis hyphalibus binucleatis. that it does not produce monilioid cells in Cellulae monilioides absentes. Sclerotium culture. This is particularly rare in 0.5­1 mm diametro, sine cellulis monilioidibus, Epulorhiza, because all species and even indifferentiatum ubi submersum et cum most strains produce monilioid cells, an cortice pallido et medula obscura super important feature that is common among agaro. Auctus 0.004­0.1mm in horas prope Rhizoctonia s. l. (Saksena and Vaartaja 1960, 28C. Oxidasis polyphenolis negativa, 1961; Currah and Zelmer 1992; Currah et al. tenuiter cellulolytica. 1997; Roberts 1999; Rasmussen 1995, 2002). Although E. amonilioides does not In PDA and CMA, colonies with slow produce monilioid cells, all other growth in culture, cream to pale cream in morphological features in culture are shared color, with clotted to flat aspect and scarce with the species and strains of this genus. aerial mycelia with submerged margin entire These features include very slow growth or diffuse. Hyaline hyphae regularly septate, rate, production of sclerotia and regularly 2.5 µm in diameter and possessing septated hyphae with right­angle branches binucleate cells. Absence of monilioid cells. (Currah and Zelmer 1992; Currah et al. In PDA, sweet scent. Sclerotia 0.5­1 mm in 1987; Zelmer and Currah 1995; Currah et al. diameter, without formation of monilioid 1997; Pereira et al. 2003), which are cells, undifferentiated when submerged and considered to be of low taxonomic value for with cortex and dark medulla on agar. the group (Andersen 1990). However, the ETYMOLOGY: amonilioides (Latin) absence of monilioid cells has persisted refers to the abscence of monilioid cells. during four years of observation in same HOLOTYPE: Colonies in PDA, isolated conditions of pure culture (PDA), and can from Encyclia dichroma (Lindl.) Schltr., be considered a diagnostic feature for this collected in sandy dunes (Restinga, Coastal new species. Furthermore, in vitro symbiotic Atlantic Forest) next to the airport in the germination experiments were performed municipality of Salvador, State of Bahia, with E. amonilioides, and we verified its Brazil, January, 20th 2007, 12° 55' 17.53'' S, ability to germinate seeds of Cattleya 38° 19' 27.47'' W; deposited as CCMB 513­ sincorana (Schltr.) Van den Berg (data not 514 . shown). This species of epiphytic orchid GROWTH RATE: In PDA at 28°C, grows on rocky surfaces and as an epiphyte 0.004­0.1 mm/h. on Vellozia (Veloziaceae) in Bahia, Brazil. ENZYMATIC ASSAY: Absence of We observed great variation in the 4 ALMEIDA ET. AL.­ NEW EPULORHIZA FROM BRAZIL

macromorphological features of cultured municipalities of Salvador and Maracás, strains of E. amonilioides isolated from which are close to the extreme limits of the populations of Encyclia dichroma and E. geographical distribution of these plants in ghillanyi in different geographic areas of Bahia. The morphology of these strains on Bahia (data not shown). E. amonilioides PDA ranged from pale cream­colored strains with contrasting morphological colonies of flat aspect that did not produce features were found in the plants from the sclerotia to cream­colored colonies with

Figure 1. Epulorhiza amonilioides. A – C show colonies with distinct macromorphological features. A, a strain isolated from E. dichroma in the population of SA (on sandy dunes in the municipality of Salvador). B, a strain isolated from B. tuberculata. C, a strain isolated from E. ghillanyi in the population of MA (on inselbergs in the municipality of Maracás). D, sclerotia formed on agar, observed in colonies isolated from E. ghillanyi in the population of MA. E, SEM image of root cortex tissue of E. dichroma, showing colonization through the formation of pelotons (indicated by arrows). F, SEM image of E. amonilioides mycelia, without the formation of monilioid cells.

5 ALMEIDA ET. AL.­ NEW EPULORHIZA FROM BRAZIL clotted aspect that produced sclerotia this group (Warcup and Talbot, 1967; (Figure 1). Therefore, as previously pointed McCormick et al. 2004; Suárez et al. 2006). out by Andersen (1990), there are Finally, clade D is formed by T. violea considerable morphological variations (Quél.) Bourdot & Galzin and T. pruinosa within a single species of Epulorhiza, even Bourdot & Galzin, which have already been when the analyses are performed on a established in previous phylogenetic studies geographical scale (as in our study). as distinct species from all other Tulasnella In the topologies of Figures 2 and 3, E. strains (McCormick et al. 2004; Suárez et amonilioides forms a monophyletic group al. 2006; Roche et al. 2010). distinct from all previously identified The mtLSU data also agreed with the Tulasnella strains isolated from tropical and 5.8S rDNA data, as E. amonilioides forms a temperate orchids. Moreover, none of the monophyletic group with T. irregularis Epulorhiza/Tulasnella taxa groups with any (Figure 3). Furthermore, by analyzing Sebacinaceae (Selosse et al. 2002; Ma et al. mtLSU data separately, some species of 2003; Shefferson et al. 2005, 2007; Tulasnella, specifically T. violea and T. Bougoure et al. 2005; Kristiansen et al. calospora, seem to be polyphyletic. These 2004; McCormick et al. 2004; Porras­ findings have already been pointed out by Alfaro & Bayman 2007; Suárez et al. 2006; Roche et al. (2010), who suggested the Shimura et al. 2009; Illyes et al. 2009; possibility of sequence misidentification in Nontachaiyapoom et al. 2010; Roche et al. GenBank. 2010). The 5.8S rDNA and mtLSU data Based on all the morphological and demonstrate the phylogenetic relationships molecular data in the present study, T. among the groups of Tulasnella, and place irregularis is probably the teleomorph of E. the different isolates into distinct groups. amonilioides. Sequence analysis shows high From the 5.8S rDNA data, four groups were identity (99­100%) between E. amonilioides retrieved (Figure 2). The first, clade A, with and T. irregularis for both the ITS rDNA significant statistical support from and mtLSU sequences (Figures 2 and 3). bootstrapping, includes E. amonilioides, Furthermore, the sequences obtained from orchid mycorrhizal fungi from Thailand E. amonilioides were identical between (Nontachaiyapoom et al. 2010), and other isolates from the same plant and even mycorrhizal fungi isolated from orchids in between isolates from the three different temperate areas (Illyes et al. 2009). These studied plant hosts. mycorrhizal fungi are more strictly related Although Nontachaiyapoom et al. (2010) to Tulasnella irregularis Warcup & P.H.B. found some orchid mycorrhizal fungi in Talbot (Warcup and Talbot 1980). Second, Thailand that are phylogenetically related to clade B groups a vast number of E. amonilioides/T. irregularis, these fungi mycorrhizal fungi identified in studies with have not yet been described as new orchids from both temperate and tropical anamorphs. Moreover, E. amonilioides has areas (Ma et al. 2003; McCormick et al. features that contrast with Thai isolates 2004; Porras­Alfaro and Bayman 2007). In [according to the descriptions of our study, this clade belongs to the group of Nontachaiyapoom et al. (2010)]. E. E. epiphytica/Tulasnella danica Hauerslev. amonilioides is more strictly related to T. Clade C, the third group, also includes irregularis (Figures 2 and 3) than the mycorrhizal fungi isolated and identified in anamorphs described in Nontachaiyapoom temperate and tropical orchids; however, all et al. (2010). Thus, E. amonilioides is the the mycorrhizal fungi that represent T. seventh species described for the genus calospora (Boud.) Juel strains are part of (Currah and Zelmer 1992; Currah et al.

6 ALMEIDA ET. AL.­ NEW EPULORHIZA FROM BRAZIL

Figure 2. Maximum likelihood tree of Tulasnella strains using the 5.8S ITS nuclear rDNA region as the data set. Numbers next to the branches represent bootstrap values. Only bootstrap values greater than or equal to 50% are shown.

7 ALMEIDA ET. AL.­ NEW EPULORHIZA FROM BRAZIL

Figure 3. Maximum likelihood tree of Tulasnella strains using the mtLSU region as the data set. Numbers next to the branches represent bootstrap values. Only bootstrap values greater than or equal to 50% are shown. 8 ALMEIDA ET. AL.­ NEW EPULORHIZA FROM BRAZIL

1987; Zelmer and Currah 1995; Currah et their reaction on gallic or tannic acid medium. al. 1997; Pereira et al. 2003), and the Journal of Agricultural Research 57: 683­695. second from Brazil. Doyle, J.J. & Doyle, J.L. 1987. A rapid isolation procedure for small quantities of fresh tissue. Phytochemical Bulletin 19: 11­15. ACKNOWLEDGMENTS Illyes, Z.; Halasz, K.; Rundnoy, S.; This study was carried out with the Ouanphanivanh, N.; Garay, T.; Bratek, Z. support of the Conselho Nacional de 2009. Changes in the diversity of the Desenvolvimento Científico e Tecnológico mycorrhizal fungi of orchids as a function of – CNPq (grant 481715/2010­0), Fundação the water supply of the habitat. Journal of Applied Botany and Food Quality 83(1): 28­ de Amparo da Pesquisa do Estado da Bahia 36. – FAPESB (grant PNX0014/2009). CvdB Felsenstein, J. 1985. Confidence limits on and AGN thank CNPq for scholarships (PQ­ phylogenies: an approach using the bootstrap. 1C and PQ2, respectively). Evolution 39: 783­91 Kristiansen, K.A.; Freudenstein, J.V.; Rasmussen, F.N.; Rasmussen, H.N. 2004. Molecular LITERATURE CITED identification of mycorrhizal fungi in Almeida, P.R.M.; van den Berg, C.; Góes­Neto, Neuwiedia veratrifolia (Orchidaceae). A. 2007. Morphological and molecular Molecular Phylogenetics and Evolution 33: characterization of species of Tulasnella 251­258. (Homobasidiomycetes) associated with Neotropical Ma, M.; Tan, T.K.; Wong, S.M. 2003. plants of Laeliinae (Orchidaceae) occuring in Identification and molecular phylogeny of Brazil. Lankesteriana 7(1­2): 22­27. Epulorhiza isolates from tropical orchids. Andersen, T.F. 1990. A study of hyphal Mycological Research 107(9):1041­1049. morphology in the form genus Rhizoctonia. McCormick, M.K.; Whigham, D.F.; O’Neill, J. Mycotaxon 37: 25­46. 2004. Mycorrhizal diversity in photosynthetic Bougoure, J.J.; Bougoure, D.S.; Cairney, W.G.; terrestrial orchids. New Phytologist 163: 425­ Dearnaley, J.D.W. 2005. ITS – RFLP and 438. sequence analysis of endophytes from Moore, R.T. 1996. The dolipore/parenthesome Acianthus, Caladenia and Pterostylis septum in modern taxonomy. In: Sneh B, (Orchidaceae) in southeastern Queensland. Jabaji­Hare S, Neate S, Dijist G (ed) Mycological Research 109(4): 452­460. Rhizoctonia species: taxonomy, molecular Bruns, T.D.; Szaro, T.M.; Gardes, M.; Cullings, biology, ecology, pathology, and disease K.M.; Pan, J.J.; Taylor, D.L.; Horton, T.R.; control. Dordrecht, the Netherlands: Kluwer Kretzer, A.; Garbelotto, M.; Li, Y. 1998. A Academic Publishers, pp 13­35. sequence data for the identification of Moore, R.T. 1987. The genera of Rhizoctonia­ ectomycorrhizal basidiomycetes by like fungi: Ascorhizoctonia, Ceratorhiza gen. phylogenetic analysis. Molecular Ecology 7: nov., Epulorhiza gen. nov., Moniliopsis, and 257­272. Rhizoctonia. Mycotaxon 29: 91­99. Currah, R.S.; Sigler, L; Hambleton S. 1987a. Moncalvo, J­M.; Nilsson, R.H.; Koster, B.; New records and new taxa of fungi from the Dunham, S.M.; Bernauer, T.; Matheny, P.B.; mycorrhiza of terrestrial orchids of Alberta. McLenon, T.; Margaritescu, S.; Weiß, M.; Canadian Journal of Botany 65: 2473­2482. Garnica, S.; Danell, E.; Langer, G.; Langer, E.; Currah, R.S. & Zelmer, C.D. 1992. A key and Larsson, E.; Larsson, K­H.; Vilgalys, R. 2006. notes for the genera of fungi with orchids and a The cantharelloid clade: dealing with new species in the genus Epulorhiza. Reports incongruent gene trees and phylogenetic of the Tottori Mycological Institute 30: 43­59. reconstruction methods. Mycologia 98: 937­ Currah, R.S.; Zettler, L.W.; McInnis, T.M. 1997a. 948. Epulorhiza inquilina sp. nov. from Platanthera Nontachaiyapoom, S.; Sasirat, S.; Manoch, L. (Orchidaceae) and a Key to Epulorhiza 2010. Isolation and identification of Species. Mycotaxon 61: 338­342. Rhizoctonia­like fungi from roots of three Davison, R.W.; Campbell, W.A.; Blaisedell, D.j. orchid genera, Paphiopedilum, Dendrobium, 1938. Differentiation of wood­decay fungi by 9 ALMEIDA ET. AL.­ NEW EPULORHIZA FROM BRAZIL

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