PLEASE SCROLL DOWN FOR ARTICLE

This article was downloaded by: [Suárez, Juan Pablo]On: 13 December 2010Access details: Access Details: [subscription number 930987564]Publisher Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

MycologyPublication details, including instructions for authors and subscription information:http://www.informaworld.com/smpp/title~content=t913878484

Orchids keep the ascomycetes outside: a highly diverse group ofascomycetes colonizing the velamen of epiphytic orchids from a tropicalmountain rainforest in Southern EcuadorPaulo Herreraa; Juan Pablo Suáreza; Ingrid Kottkea

a Centro de Biología Celular y Molecular, Universidad Técnica Particular de Loja, Loja, Ecuador

Online publication date: 10 December 2010

To cite this Article Herrera, Paulo , Suárez, Juan Pablo and Kottke, Ingrid(2010) 'Orchids keep the ascomycetes outside: ahighly diverse group of ascomycetes colonizing the velamen of epiphytic orchids from a tropical mountain rainforest inSouthern Ecuador', Mycology, 1: 4, 262 — 268To link to this Article: DOI: 10.1080/21501203.2010.526645URL: http://dx.doi.org/10.1080/21501203.2010.526645

Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf

This article may be used for research, teaching and private study purposes. Any substantial orsystematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply ordistribution in any form to anyone is expressly forbidden.

The publisher does not give any warranty express or implied or make any representation that the contentswill be complete or accurate or up to date. The accuracy of any instructions, formulae and drug dosesshould be independently verified with primary sources. The publisher shall not be liable for any loss,actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directlyor indirectly in connection with or arising out of the use of this material.

MycologyVol. 1, No. 4, December 2010, 262–268

ISSN 2150-1203 print/ISSN 2150-1211 online© 2010 Mycological Society of ChinaDOI: 10.1080/21501203.2010.526645http://www.informaworld.com

TMYCOrchids keep the ascomycetes outside: a highly diverse group of ascomycetes colonizing the velamen of epiphytic orchids from a tropical mountain rainforest in Southern EcuadorMycologyPaulo Herrera*, Juan Pablo Suárez and Ingrid Kottke

Centro de Biología Celular y Molecular, Universidad Técnica Particular de Loja, San Cayetano Alto s/n C.P. 11 01 608, Loja, Ecuador

(Received 3 September 2010; final version received 10 September 2010)

Orchid mycorrhizal detection, based only on fungal isolation from roots, is biased due to difficulties in isolating the respec-tive fungi. Previous investigations have shown that mostly ascomycetes, housed in the sheltered compartment of the vela-men covering the roots of epiphytic orchids, are isolated on agar plates. Roots of 83 individual epiphytic orchid of Stelishallii, S. superbiens, S. concinna, Stelis sp. and Pleurothallis lilijae were sampled in the Reserva Biológica San Francisco,Southern Ecuador. The velamen was partly removed and root tissue macerated and transferred to Petri dishes containingcorn meal agar, malt extract agar and Melin Norkrans modified medium. DNA was extracted from the cultures and the5.8S-ITS region and partial nrLSU sequenced. Phylogenetic analysis revealed members of 12 orders of ascomycetes associ-ated with the roots of these orchids, a much higher diversity than previously known. Twelve isolates were related to theHelotiales. Our results have uncovered some of the fungal diversity within the velamen of epiphytic orchids; fungi invading,most likely, from tree bark or humus accumulation close to the roots.

Keywords: ascomycetes; epiphytic orchids; velamen; Helotiales; tropical mountain rain forest

IntroductionRoots of epiphytic orchids are cover by a specialized,multi-seriate epidermis, known as the velamen (Esau1964). At maturity, the velamen consists of non-livingcells, often bearing secondary wall thickening and pores.The principal roles of this tissue are water storage and thereduction of water loss from the cortex (Esau 1964; Benzinget al. 1982). However, the velamen is colonized by a largevariety of organisms, especially fungi and bacteria (Benzinget al. 1982; Bayman and Otero 2006). Light and electronmicroscopy has revealed abundant fungal colonization ofthe velamen, but only members of the Basidiomycotahave, so far, been observed to penetrate the passage cellsof the exodermis, forming pelotons in the cortical cells ofepiphytic orchids (Kottke and Suárez 2009; Kottke et al.2010). Ascomycota have not been detected in healthy rootcortical cells of tropical, epiphytic orchids, although a rep-resentative number of roots, sampled in the tropical moun-tain rainforest of Southern Ecuador, were investigated bytransmission electron microscopy (TEM) (Suárez et al.2006, 2008; Kottke et al. 2010). To date, only Tuber sp.,as a member of the Ascomycota, has been recorded as amycorrhizal fungus of the terrestrial orchid Epipactismicrophylla from France using TEM and DNA sequenc-ing (Selosse et al. 2004). However, non-mycorrhizal fungihave been identified from the roots of various terrestrial

and epiphytic orchids, mostly during the attempt to findmycorrhizal fungi (Bayman et al. 1997; Currah et al. 1997;Tremblay et al. 1998; Taylor et al. 2003; Suárez et al.2006; Tao et al. 2008; Yuan et al. 2009).

This paper focuses on fungal isolates from epiphyticorchid roots, sampled in the tropical mountain rainforestof Southern Ecuador, Northern Andes, and identified bymolecular tools.

Materials and methodsStudy site and samplingThe study site was located on the eastern slope of the Cor-dillera El Consuelo in the Northern Andes of southernEcuador. The area of ∼1000 ha belongs to the ReservaBiológica San Francisco and borders the PodocarpusNational Park in the north, half way between Loja andZamora in Zamora-Chinchipe Province (3°58´ S, 79°04´W). Sampling was carried out along small paths at an alti-tudinal gradient between 1850 and 2100 m a.s.l. Threeroots per plant were collected between August and October2005 from a total of 83 flowering individuals, 28 of Stelishallii Lindl., 26 of Stelis superbiens Lindl., 11 of Stelisconcinna Lind., 14 of Pleurothallis lilijae Foldats and fourof Stelis sp. All selected plants were epiphytes on trunks orbranches of standing trees.

*Corresponding author. Email: piherrera@utpl.edu.ec

Downloaded By: [Suárez, Juan Pablo] At: 17:48 13 December 2010

Mycology 263

Fungal isolationCollected roots were transferred to the laboratory on theday of sampling. Three colonized root pieces per plantwere surface-sterilized. Roots were rinsed in distilledwater with a few drops of liquid soap, immersed in 70%ethanol for 30 s, then immersed in 20% “Ajax Cloro”(household bleach, sodium hypochlorite 5.25%) for10 min and finally rinsed in sterile distilled water. Thevelamen was partly removed and discarded. The remain-ing root tissue with still adhering parts of the velamen wasmacerated in a 1.5-ml micro-tube containing some dropsof sterile distilled water. From each sample, three drops ofmacerated tissue were placed on plates of corn meal agar(CMA; C1176, Sigma), malt extract agar (MEA; M6907,Sigma) and Melin Norkrans modified medium (MMNC;Suárez et al. 2006). Petri dishes were incubated at 28°C inthe dark. The fungal isolates were transferred to potatodextrose agar (PDA) after the colony had reached 1 cm indiameter. Pure cultures are preserved in the Centro deBiología Celular y Molecular of Universidad Técnica Par-ticular de Loja.

DNA isolation and PCR and sequencingGenomic DNA was recovered from fresh fungal culturesusing a Plant Mini Kit (Qiagen, Hilden, Germany), accord-ing to the manufacturer’s instructions. The nrDNA 5.8S,including the internal transcribed spacers ITS1 and ITS2and a partial region of nrDNA 28S, were amplified with aset of universal primer combinations ITS1 (5′-TCC GTAGGT GAA CCT GCG G-3′; White et al. 1990) and NL4(5′-GGT CCG TGT TTC AAG ACG G-3′; White et al.1990) and fungal primer combination ITS1F (5′-CTTGGT CAT TTA GAG GAA GTA A-3′; Gardes and Bruns1993) and LR5 (5′-TCC TGA GGG AAA CTT CG-3′;Vilgalys and Hester 1990).

PCR conditions were as follows: initial denaturizing at94°C for 3 min; 35 cycles, each cycle consisting of onestep of denaturizing at 94°C for 30 s; annealing, dependingon the primer combinations for 45 s and extension at 72°Cfor 1 min; a final extension at 72°C for 7 min was per-formed to finish the PCR. A control including PCR mixwithout DNA template was included in every PCR. Suc-cess of the PCR amplifications were tested in 0.7% agar-ose stained in a solution of 0.5 μg ml–1 ethidium bromide.PCR products were purified using the QIAquick protocol(Qiagen).

ITS-5.8S and partial 28S sequences of nuclear ribos-omal RNA genes of our isolates were sequenced usingprimers ITS1, NL4, NL1 and ITS4. Sequence editing wasperformed using Sequencher version 4.5 (Gene Codes,Ann Arbor, MI, USA). The sequences obtained in thisstudy are available from GenBank under accession num-bers HQ207016–HQ207070.

Phylogenetic analysesBLAST (Altschul et al. 1997) was used against the NCBInucleotide database (GenBank; http://www.ncbi.nlm.nih.gov/)to detect published sequences of high similarity. A multi-ple analysis method (Lee 2001) was employed andsequences were aligned using POA software (Lee et al.2002) and MAFFT v5.667 (Katoh et al. 2005) under theG-INS-i option. A partial nrDNA 28S region of 562 bpwas considered for phylogenetic purposes without exclu-sions. Bayesian analysis, based on Markov chain MonteCarlo (MCMC), was implemented in MrBayes versionv3.1.2 (Huelsenbeck and Ronquist 2001), including tworuns, each involving four incrementally heated Markovchains over four million generations and using randomstarting trees. Trees were sampled every 100 generations,resulting in a total of 40,000 trees, from which the last30,000 were used to compute a 50% majority rule consen-sus tree. Each dataset was analyzed using the DNA substi-tution model GTR+I+G (Swofford et al. 1996). Additionally,a maximum likelihood calculation was implemented inRAxML version 7.0.4 software (Stamatakis 2006) usingthe GTRMIX algorithm. We used Saccharomyces cerevi-siae (EU649673) as the outgroup. Relationships amongthe 12 sequences of Helotiales were examined in a furtheranalysis considering the nrDNA region ITS1-5.8S-ITS2.Xylaria sp. (FN392317) was used as the outgroup. Align-ments and phylogenetic analyses were performed asdescribed above.

ResultsNinety pure cultures were obtained after 249 isolationattempts. Sixty isolates were obtained from S. superbiens,12 from S. hallii, five from Stelis sp., three from S.concinna, and 10 from P. lilijae; 56% of isolates grew inCMA, 20% in MEA and 24% in MMNC.

DNA sequences were obtained from 58 of the 90 iso-lates. Phylogenetic analyses of the partial region ofnrDNA 28S placed 55 isolates in 12 different orders ofAscomycota (Figure 1) and three into Basidiomycota(Polyporals, not shown). The most frequent ascomycetesisolates belonged to Hypocreales (16), Helotiales (12) andXylariales (10) (Table 1). One isolate was located inBoliniales, another in Chaetosphaeriales. Two isolateswere similar to Capnodiales, four to Pleosporales, two toBotryosphaeriales, and one to Thelebolales. One isolateclustered with Rhytismatales, three isolates with Eurotiales,and two isolates were similar to Chaetothyriales (Table 1).Members of several ascomycete orders were identifiedfrom more than one orchid species (Table 1).

Phylogenetic analysis of the Heliotales sequencesshows three well-supported groups (Figure 2). Nine iso-lates form a group allied to Pezicula and Cryptosporiopsis.The second group with one of our isolates is related to

Downloaded By: [Suárez, Juan Pablo] At: 17:48 13 December 2010

264 P. Herrera et al.

Figure 1. (A and B) Phylogenetic placement of ascomycete sequences obtained from the velamen of Stelis hallii, Stelis superbiens, Stelisconcinna, Pleurothallis lilijae and Stelis sp. Tree inferred from nuclear rDNA coding for the 5′-terminal domain of the large ribosomalsubunit (nucLSU) using a maximum likelihood analysis implemented in RaxML with the GTRMIX algorithm. Numbers on branchesdesignate Bayesian MCMC estimates of posterior probabilities and ML values (only values exceeding 50% are shown). The tree wasrooted with Saccharomyces cerevisiae EU649673.

Downloaded By: [Suárez, Juan Pablo] At: 17:48 13 December 2010

Mycology 265

Figure 1. (Continued).

Downloaded By: [Suárez, Juan Pablo] At: 17:48 13 December 2010

266 P. Herrera et al.

Hyalodendriella. Two isolates form a sister group toHyphodiscus. Sequences AY394684 and AY394687 ofHymenoscyphus = Rhizoscyphus ericae aggregate, previ-ously obtained from ectomycorrhizae of Graffenriedaemarginata (Melastomataceae) at RBSF (Haug et al.2004) clustered in a separated clade from our presentsequences for Helotiales. None of these sequences wasidentical to those published in GenBank.

DiscussionAnalysis of the 28S nrDNA gene revealed a broad system-atic coverage of the ascomycete orders associated with theroots of the five pleurothallid orchids. Previously, the mostcommon ascomycetes isolated from the roots of epiphyticorchids have been circumscribed to only six orders: Xylar-iales, Phyllachorales, Diaporthales, Hypocreales, Botry-osphaeriales and Pleosporales (Richardson and Currah1995; Bayman et al. 1997; Tremblay et al. 1998; Yuanet al. 2009). We have recorded members related to 12orders: Xylariales, Boliniales, Chaetosphaeriales, Hypoc-reales, Capnodiales, Pleosporales, Botryosphaeriales, Cha-etothyriales, Eurotiales, Thelebolales, Helotiales, andRhytismatales. As sampling efforts increase and identifi-cation is improved by molecular techniques, the diversityof fungi in orchid roots may be much higher than previ-ously thought (Kageyama et al. 2008), which may accountfor the highly diverse epiphytic orchids with large vela-men in the tropical mountain rainforests of the NorthernAndes.

The Order Helotiales includes an ecologically diversegroup of plant pathogens, wood, debris and soil saprobes,plant endophytes, and mutualistic ericoid mycorrhizal(ERM) and ectomycorrhizal (ECM) fungi (Vrålstad et al.2002; Haug et al. 2004). Our isolates belonging to theHelotiales were isolated from four of the five orchid

species and were not closely related to the mycorrhiza-forming Heliotiales. This result is of special interestbecause we found mycorrhiza-forming Helotiales in thisrainforest associated with G. emarginata (Haug et al.2004) and Ericaceae (Setaro 2009, unpublished data) andspeculated on the potential symbiosis with orchids. Theresult, however, substantiates our TEM observations thatonly Basidiomycota from the Tulasnellales, Sebacinales,Ceratobasidiales (Heterobasidiomycetes) and the recentlydetected Atractiellales (Pucciniomycotina) are present incortical tissue and form mycorrhizae with the respectiveepiphytic orchids (Suárez et al. 2006, 2008; Kottke et al.2010). TEM observations also excluded the isolated Poly-porales as a mycorrhizal fungi of the investigated orchids.As orchid mycobionts, members of the Homobasidiomyc-etes appeared to be restricted to partial or fully mycoheter-otrophic orchids of the northern hemisphere and East Asia(for references, see Yukava et al. 2009; Kottke and Suárez2009; Selosse et al. 2010).

We only sampled roots in close contact with the barkof standing trees where mosses or debris had accumulated,because mycorrhizae are only formed under these circum-stances. In our opinion, saprobic ascomycetes associatedwith the velamen may indirectly improve nutrient accessof the orchids by decomposing the substrate outside theroots from where they most likely invaded the velamen.Experiments to substantiate this assumption could be ofinterest for the orchid cultivation.

A large diversity of ascomycetes was recorded, indi-cating that moderate surface sterilization and partialremoval of the velamen did not eliminate all fungi fromthe root surface. This fact should be kept in mind whensearching for orchid mycobionts via isolation from surfacesterilized tissue. Many fungi colonize only the nonlivingcells of the velamen and should neither be consideredendophytes, as these, by definition, colonize healthy tissue

Table 1. Identified isolates from five epiphytic orchid species: Stelis hallii, S. superbiens, S. concinna, Stelis sp. and Pleurothallislilijae. Numbers in parenthesis indicate the number of sampled individuals.

Orchid species

Orders of ascomycetes

Stelis superbiens (26)

Stelis hallii (28)

Stelis concinna (11)

Pleurothallis lilijae (14) Stelis sp. (4)

Total isolates (n)

Xylariales 6 3 1 – – 10Boliniales – – 1 – – 1Chaetosphaeriales 1 – – – – 1Hypocreales 12 – – 2 2 16Capnodiales 1 1 – – – 2Pleosporales 4 – – – – 4Botryosporales 2 – – – – 2Chaetothyriales – – – 2 – 2Eurotiales – – 1 – 2 3Thelebolales 1 – – – – 1Helotiales 7 1 – 3 1 12Rhytismatales 1 – – – – 1Total (n) 35 5 3 7 5 55

Downloaded By: [Suárez, Juan Pablo] At: 17:48 13 December 2010

Mycology 267

Figure 2. Phylogenetic placement of Helotialean fungal isolate sequences from the velamen of Stelis hallii, Stelis superbiens, Stelisconcinna, Pleurothallis lilijae and Stelis sp. Tree inferred from the ITS-5.8S region using a maximum likelihood analysis implementedin RaxML with the GTRMIX algorithm. Numbers on branches designate Bayesian MCMC estimates of posterior probabilities and MLvalues (only values exceeding 50% are shown). The tree was rooted with Xylaria sp. FN392317.

Downloaded By: [Suárez, Juan Pablo] At: 17:48 13 December 2010

268 P. Herrera et al.

without causing disease (Arnold et al. 2000), nor mycor-rhizal fungi. The absence of true orchid mycobionts fromour isolation trials may also indicate that other unculturablefungi are present in the tissue.

AcknowledgementsThis research was funded by the Deutsche Forschungsgemein-schaft (DFG RU 816). We thank Nature and Culture International(NCI) for providing research facilities at RBSF.

ReferencesAltschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller

W, Lipman DJ. 1997. Gapped BLAST and PSI-Blast: a newgeneration of protein database search programs. NucleicAcids Res. 25:3389–3402.

Arnold A, Maynard Z, Gilbert G, Coley P, Kursar T. 2000. Are trop-ical fungal endophytes hyperdiverse Ecol Lett. 3:267–274.

Bayman P, Lebrón LL, Tremblay RL, Lodge DJ. 1997. Variationin endophytic fungi from roots and leaves of Lepanthes(Orchidaceae). New Phytologist 135:143–149.

Bayman P, Otero JT. 2006. Microbial endophytes of orchidroots. In: Schulz BJE, Boyle C, Sieber TN, editors. Micro-bial root endophytes. New York: Springer; p. 153–178.

Benzing DH, Ott DW, Friedman WE. 1982. Roots of Sobralia-macrantha (Orchidaceae): structure and function of the vela-men–exodermis complex. Am J Bot. 69 (4):608–614.

Currah RS, Zelmer CD, Hambleton S, Richardson KA. 1997.Fungi from orchid mycorrhizas. In: Arditti J, Pridgeon A,editors. Orchid biology: reviews and perspectives, vol. VII.Dordrecht: Kluwer; p 117–170.

Esau K. 1964. Anatomy of seed plants. New York: Wiley.Gardes M, Bruns TD. 1993. ITS primers with enhanced specificity

for basidiomycetes: application to the identification of mycor-rhizae and rusts. Mol Ecol. 2:113–118.

Haug I, Lempe J, Homeier J, Weiss M, Setaro S, Oberwinkler F,Kottke I. 2004. Graffenrieda emarginata (Melastomataceae)forms mycorrhizas with Glomeromycota and with a memberof the Hymenoscyphus ericae aggregate in the organic soil ofa neotropical mountain rain forest. Can J Bot. 82:340–356.

Huelsenbeck JP, Ronquist FR. 2001. MrBayes: Bayesian infer-ence of phylogenetic trees. Bioinformatics 17:754–755.

Kageyama SA, Mandyam KG, Jumpponen A. 2008. Diversity,function and potential applications of the root-associatedendophytes. In: Varma A, editor. Mycorrhiza. State of theArt, Genetics and Molecular Biology, Eco-Function, Bio-technology, Eco-Physiology, Structure and Systematics.New York: Springer; 128 pp.

Katoh K, Kuma K, Toh H, Miyata T. 2005. MAFFT version 5:improvement in accuracy of multiple sequence alignment.Nucleic Acids Res. 33:511–518.

Kottke I, Suárez JP. 2009. Mutualistic, root-inhabiting fungi oforchids identification and functional types. In: Pridgeon AM,Suarez JP. editors. Proceedings of the Second Scientific Con-ference on Andean Orchids. Universidad Técnica Particular deLoja, Loja, Ecuador, ISBN 978-9942-00-502-1; pp 84–99.

Kottke I, Suárez JP, Herrera P, Cruz D, Bauer R, Haug I, GarnicaS. 2010. Atractiellomycetes belonging to the ‘rust’ lineage(Pucciniomycotina) form mycorrhizae with terrestrial and epi-phytic neotropical orchids. Proc R Soc B 277:1289–1298.

Lee M. 2001. Unalignable sequences and molecular evolution.Trends Ecol Evol. 16:681–685.

Lee C, Grasso C, Sharlow M. 2002. Multiple sequencealignment using partial order graphs. Bioinformatics 18:452–464.

Richardson KA, Currah RS. 1995. The fungal community associ-ated with the roots of some rainforest epiphytes of CostaRica. Selbyana 16:49–73.

Selosse M-A, Faccio A, Scappaticci G, Bonfante P. 2004. Chlo-rophyllous and achlorophylous specimens of Epipactismicrophylla (Neottieae, Orchidaceae) are associated withectomycorrhizal septomycetes, including truffles. MicrobEcol. 47:416–426.

Selosse M-A, Martos F, Perry BA, Padamsee M, Roy M, PaillerT. 2010. Saprotrophic fungal mycorrhizal symbionts inachlorophyllous orchids. Plant Signal Behav. 5:1–5.

Stamatakis A. 2006. RAxML-VI-HPC: Maximum likelihood-based phylogenetic analyses with thousands of taxa andmixed models. Bioinformatics 22:2688–2690.

Suárez JP, Weiss M, Abele A, Garnica S, Oberwinkler F, KottkeI. 2006. Diverse tulasnelloid fungi form mycorrhizas withepiphytic orchids in an Andean cloud forest. Mycol Res.110:1257–1270.

Suárez JP, Weiss M, Abele A, Oberwinkler F, Kottke I. 2008.Members of Sebacinales subgroup B form mycorrhizae withepiphytic orchids in a neotropical mountain rain forest.Mycol Prog. 7:75–85.

Swofford DL, Olsen GJ, Waddell PJ, Hillis DM. 1996. Phyloge-netic inference. In: DM Hillis, C Moritz, BK Mable, editors.Molecular Systematics. Sunderland (MA): Sinauer Associ-ates; p. 407–514.

Tao G, Liu ZY, Hyde KD, Liu XZ, Yu ZN. 2008. Whole rDNAanalysis reveals novel and endophytic fungi in Bletillaochra-cea (Orchidaceae). Fungal Diversity 33:101–122.

Taylor DL, Bruns TD, Szaro TM, Hodges SA. 2003. Divergencein mycorrhizal specialization with Hexalectris spicata(Orchidaceae), a non-photosynthetic desert orchid. Am JBot. 90:1168–1179.

Tremblay RL, Zimmerman J, Lebrón LL, Bayman P, Sastre I,Axelrod F, Alers-García J. 1998. Host specificity and lowreproductive success in the rare endemic Puerto Rican orchidLepanthes caritensis. Biol Conserv. 85:297–304

Vilgalys R, Hester M. 1990. Rapid genetic identification andmapping of enzymatically amplified ribosomal DNAfrom several Cryptococcus species. J Bacteriol. 172:4238–4246.

Vrålstad T, Myre E, Schumacher T. 2002. Molecular diversityand phylogenetic affinities of root-associated ascomycetes ofthe Helotiales in burnt and metal polluted habitats. New Phy-tologist 155:131–148.

White TJ, Bruns TD, Lee SB, Taylor JW. 1990. Amplificationand direct sequencing of fungal ribosomal RNA genes forphylogenetics. In: Innis MA, Gelfand H, Sninsky JS, WhiteTJ, editors. PCR Protocols and applications: A LaboratoryManual. San Diego: Academic Press; pp. 315–322.

Yuan Z, Chen Y, Yang Y. 2009. Diverse non-mycorrhizal fungalendophytes inhabiting an epiphytic, medicinal orchid (Dend-robium nobile): estimation and characterization. World JMicrobiol Biotechnol. 25:295–303.

Yukawa T, Ogura-Tsujita Y, Shefferson RP, Yokoyama J. 2009.Mycorrhizal diversity in Apostasia (Orchidaceae) indicatesthe origin and evolution of orchid mycorrhiza. Am J Bot. 96:1997–2009.

Downloaded By: [Suárez, Juan Pablo] At: 17:48 13 December 2010