Diversity and host specificity of endophytic Rhizoctonia-like fungi from tropical orchids

American Journal of Botany89(11):1852–1858.2002.

D IVERSITY AND HOST SPECIFICITY OF ENDOPHYTIC

R HIZOCTONIA-LIKE FUNGI FROM TROPICAL ORCHIDS1

J.T UPAC O TERO,2J AMES D.A CKERMAN,AND P AUL B AYMAN

Department of Biology,University of Puerto Rico,R?′o Piedras,P.O.Box23360,San Juan,Puerto Rico00931-3360USA All orchids have an obligate relationship with mycorrhizal symbionts.Most orchid mycorrhizal fungi are classi?ed in the form-genus Rhizoctonia.This group includes anamorphs of Tulasnella,Ceratobasidium,and Thanatephorus.Rhizoctonia can be classi?ed according to the number of nuclei in young cells(multi-,bi-,and uninucleate).From nine Puerto Rican orchids we isolated108 Rhizoctonia-like fungi.Our isolates were either bi-or uninucleate,the?rst report of uninucleate Rhizoctonia-like fungi as orchid endophytes.We sequenced the internal transcribed spacer(ITS)region of nuclear ribosomal DNA from26isolates and identi?ed four fungal lineages,all related to Ceratobasidium spp.from temperate regions.Most orchid species hosted more than one lineage,dem-onstrating considerable variation in mycorrhizal associations even among related orchid species.The uninucleate condition was not a good phylogenetic character in mycorrhizal fungi from Puerto Rico.All four lineages were represented by fungi from Tolumnia variegata,but only one lineage included fungi from Ionopsis utricularioides.Tropical epiphytic orchids appear to vary in degree of speci?city in their mycorrhizal interactions more than previously thought.

Key words:Ceratobasidium;orchid mycorrhizae;Puerto Rico;Rhizoctonia;speci?city.

All orchids have an obligate relationship with mycorrhizal symbionts during seed germination,with most of the symbi-onts being Rhizoctonia-like fungi(Arditti,1992).Understand-ing the mycorrhizal symbiosis is of great importance,as avail-ability of the fungal symbionts may play a key role in deter-mining orchid distribution and diversity.Furthermore,most studies of orchid mycorrhizal interactions have concentrated on terrestrial orchids from temperate regions,whereas the ma-jority of orchid species are epiphytes in tropical regions. Studies to date regarding the speci?city of orchid mycor-rhizal relationships have drawn con?icting conclusions.Two distinct approaches have been taken in the investigation of speci?city of orchid mycorrhizal relationships:(1)in vitro seed germination experiments and(2)taxonomic comparisons of fungi present in orchid roots of mature plants.One germina-tion study of epiphytic orchids suggesting speci?city(Cle-ments,1987)is balanced by another that suggests more gen-eralist interactions(Zettler,Burkhead,and Marshall,1999). Answering the question of speci?city depends on a thorough sampling and detailed taxonomy of orchid mycorrhizal fungi. The systematics of orchid mycorrhizal fungi has been stud-ied using both morphological(Warcup and Talbot,1966,1971; Currah,Hambleton,and Smreciu,1988;Rasmussen,1995), and molecular characters(Taylor and Bruns,1997,1999;Kris-tiansen et al.,2001).Rhizoctonia-like fungi include the ana-morphic(asexual)genera Ceratorhiza,Epulorhiza,Moniliop-sis,and Rhizoctonia(Moore,1988)of a variety of teleomorphs (sexual stages of Ceratobasidium,Thanatephorus,Tulasnella, and Sebacina;Warcup and Talbot,1966,1971).Some of these are well known as plant pathogens of a wide variety of crops

1Manuscript received5February2002;revision accepted16May2002. The authors thank P.Pabo′n,A.Carrillo,J.Garc?′a,and L.Fidalgo for as-sistance in the laboratory,J.E.Garc?′a-Arrara′s for use of facilities,N.S. Flanagan for support and comments on the manuscript,and A.Alegr?′a,P. Silverstone-Sopkin,and L.G.Naranjo of Universidad del Valle,Cali,Colom-bia for their inspiration.This research was supported from a grant from the Organization for Tropical Studies and a NSF-EPSCoR scholarship(EPS-9874782)to J.T.Otero and funds from NASA-IRA and NIH-RCMI to U.P.R. DNA sequencing was made possible by grant NSF DEB-9806792to W.O. McMillan.

2Author for reprint requests(e-mail:is975785@https://www.360docs.net/doc/f411659077.html,).(Sneh,Burpee,and Ogoshi,1991).Rhizoctonia are character-ized by right-angle branching,a constriction at the branch point,and a septum in the branch hypha near its point of origin.Frequently,they have chains of in?ated hyphae,known as monilioid cells(Sneh,Burpee,and Ogoshi,1991).Sexual stages are rarely encountered in the?eld or laboratory.Con-sequently,the broad vegetative criteria for identi?cation have resulted in paraphyletic taxonomy,with various unrelated fun-gi being grouped together.

One morphological feature that has helped in the classi?-cation of Rhizoctonia is the number of nuclei present in the young cells(Sneh,Burpee,and Ogoshi,1991).Multi-,bi-and uninucleate cells have been observed.The important plant pathogen species complex Rhizoctonia solani(teleomorphs: Thanatephorus,Ceratobasidiales)possesses multinucleate cells.Binucleate cells have been seen in fungi corresponding to the genus Ceratobasidium(Ceratobasidiales)and Tulasnella (Tulasnellales).Uninucleate strains occur in anamorphs of Ceratobasidium(Hietala,Vahala,and Hantula,2001),but have rarely been reported(Hietala,1997).

An additional way to classify these fungi is by anastomosis groups(AG).When two isolates belong to the same AG,their hyphae are able to fuse.Rhizoctonia solani has13AGs.The binucleate Rhizoctonia spp.include21AGs(Sneh,Burpee, and Ogoshi,1991),and the uninucleate Rhizoctonia spp.in-clude only one AG to date(Hietala,Sen,and Lilja,1994;Sen, Hietala,and Zelmer,1999).

The Rhizoctonia solani(multinucleate)AGs known to be associated with orchids are AG-6and AG-12(Carling,1996; Carling et al.,1999;but see Masuhara,Katsuya,and Yama-guchi,1993).Nevertheless,the most common group of orchid mycorrhizal fungi is binucleate Rhizoctonia(Currah et al., 1997).Uninucleate Rhizoctonia have not been previously re-ported from orchid roots.

Molecular systematics has substantially advanced the tax-onomy of Rhizoctonia spp.(Vilgalys and Gonza′lez,1990; Cubeta and Vilgalys,1997;Kuninaga et al.,1997;Salazar et al.,2000;Gonza′lez et al.,2001)and orchid mycorrhizae(Tay-lor and Bruns,1997,1999;Pope and Carter,2001;Kristiansen et al.,2001),mostly based on nuclear ribosomal internal tran-

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O TERO ET AL .—T ROPICAL ORCHID MYCORRHIZAE T ABLE 1.

Study sites.

Site

Latitude

Longitude

Elevation (m)

Vegetation

Holdridge life zone

Cambalache Cayey

Charco Azul Dorado N.Dorado S.Guajataca Guanica Maricao Mona

Sabana Seca San Cristobal Tortuguero 18?30?N 18?05?N 18?05?N 18?20?N 18?15?N 18?25?N 18?00?N 18?15?N 18?05?N 18?25?N 18?15?N 18?27?N 66?20?W 66?00?W 66?05?W 66?35?W 67?00?W 67?00?W 67?15?W 67?00?W 67?55?W 66?10?W 66?10?W 66?30?W 20–40800–850600–6505–1020–40350–40020–50800–85020–4030–40600–6405–10Secondary forest Secondary forest Mature forest Secondary forest Secondary forest Mature forest Mature forest Mature forest Mature forest Secondary forest

Pasture with Psidium guajava Pasture with Randia aculeata Subtropical moist forest Subtropical wet forest Subtropical moist forest Subtropical moist forest Subtropical moist forest Subtropical moist forest Subtropical dry forest Subtropical wet forest Subtropical dry forest Subtropical moist forest Subtropical moist forest Subtropical moist forest

T ABLE 2.Host orchid species studied.

Species Subfamily/subtribe a No.popu-lations sampled No.plants sampled

Distribution b

Campylocentrum fasciola

Campylocentrum ?liforme Erythrodes plantaginea Epidendroideae/Angraecinae Epidendroideae/Angraecinae Orchidoideae/Goodyerinae 111221Greater Antilles and Central and South America Cuba,Hispaniola and Puerto Rico West Indies

Ionopsis satyrioides Epidendroideae/Oncidiinae 12Grater Antilles,Martinique,Panama and northern South America

Ionopsis utricularioides Epidendroideae/Oncidiinae 412Central America,Florida (USA),Galapagos Islands,Mexi-co,tropical South America and West Indies

Oeceoclades maculata Epidendroideae/Eulophiinae 12Tropical Africa,Florida,Panama,South America and West Indies

Oncidium altissimum Psychilis monensis Tolumnia variegata

Epidendroideae/Oncidiinae Epidendroideae/Laeliinae Epidendroideae/Oncidiinae

116

1220Lesser Antilles and Puerto Rico Mona Island Greater Antilles

a Based on Cameron et al.(1999)and Freudenstein and Chase (2001).b

Based on Ackerman (1995).

scribed spacer (ITS)sequences.Pope and Carter (2001)stud-ied ITS sequences of Rhizoctonia solani AGs including orchid mycorrhizal fungi from Australia.Kristiansen et al.(2001)am-pli?ed rDNA from terrestrial orchids of Europe and North America and sequenced a tropical mycorrhizal fungus from Asia.However,the phylogenetic relationships of most tropical orchid mycorrhizal fungi remain unknown.

In the present study we addressed the following questions:(1)What are the phylogenetic placement and diversity of Puerto Rican Rhizoctonia -like endophytes relative to other Rhizoctonia ?Based on the literature we expected to ?nd a variety of fungi related to Ceratobasidium ,Thanatephorus ,and Tulasnella .(2)What is the level of speci?city of epiphytic orchids for their Rhizoctonia -like fungi?If there is speci?city,then we expect that the fungi isolated from a single orchid species will belong to a single fungal clade.And ?nally,(3)what is the phylogenetic relationship between uni-and binu-cleate Rhizoctonia -like fungi associated with orchids?We ex-pected consistent differences in morphology and phylogeny between uni-and binucleate Rhizoctonia -like fungi from Puerto Rico.

To answer these questions we sampled 44plants from nine tropical orchid species belonging to seven genera and ?ve sub-tribes.The orchids were collected from a number of ecologi-cally diverse sites on Puerto Rico and Mona islands.We used ITS sequence data together with morphological data to esti-mate relationships among fungi.

MATERIALS AND METHODS

Study sites and orchid hosts —We isolated Rhizoctonia -like fungi from nine orchid species collected from 11localities in Puerto Rico (Table 1).The or-chids were Campylocentrum fasciola (Lindl.)Cogn.,Campylocentrum ?lifor-me (Sw.)Cogniaux,Erythrodes plantaginea (L.)Fawcett &Randle,Ionopsis satyrioides (Sw.)Reichenbach f.,Ionopsis utricularioides (Sw.)Lindl.,Oec-eoclades maculata (Lindl.)Lindl.,Oncidium altissimum (Jacq.)Sw.;Psychilis monensis Sauleda,and Tolumnia variegata (Sw.)Braem (Table 2).All but the Erythrodes ,Oeceoclades ,and Oncidium were epiphytes.

Fungal isolation —Plants were collected sporadically from January 1998to April 2001.We took special care not to damage their root systems.The next day,undamaged roots were cut from the plants and surface-sterilized with consecutive washes of 70%ethanol (1min),2.5%sodium hypochlorite (30s),and a ?nal rinse of 70%ethanol (1min)(Bayman et al.,1997).Because our epiphytic plants did not have dense mycorrhizal infections,it was not possible to see pelotons without staining.We therefore isolated endophytic fungi and chose Rhizoctonia -like isolates for further analysis because they are known to be mycorrhizal in other tropical orchids (Currah et al.,1997).Three 0.5-cm pieces from each root were cultured in potato dextrose agar (PDA)with 50ppm each streptomycin,tetracycline,and penicillin.We inoculated four roots per plant.Samples were incubated at 27?C.After a light microscopy examination,Rhizoctonia -like fungi were transferred to pure culture in PDA.Rhizoctonia -like fungi were identi?ed using the concept of Sneh,Burpee,and Ogoshi (1991).A total of 108Rhizoctonia -like isolates were found.No perfect stages were observed on PDA or water agar (Sneh,Burpee,and Ogoshi,1991).Of these isolates,we selected 26for sequencing representing all or-chids species sampled.All isolates from orchids with less than three total

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OTANY Fig.1.Maximum likelihood tree of the ITS sequences of Ceratobasidium fungi.Numbers in the branches represent the bootstrapping percentage that supports the branch.Numbers in parentheses are the number of nuclei of each isolate.

isolates were included.Among isolates from T.variegata and I.utriculariodes (the most heavily sampled species)isolates were chosen based on morpho-logical diversity on PDA.

DNA isolation and ITS ampli?cation —The DNA of 26of the Rhizoctonia -like isolates was extracted following the procedure of Lee and Taylor (1990).The polymerase chain reaction (PCR)was performed using the primers ITS-1and ITS-4(White et al.,1990).The PCR products were cleaned using Qiaquick columns (Qiagen,Valencia,California,USA),according to the man-ufacturer’s instructions,and sequenced in both directions.Each 5-?L sequenc-ing reaction consisted of 1?L ABI PRISM BigDye Terminator Cycle Se-quencing Ready Reaction Mix (Applied Biosystems,Foster City,California,USA),1?L 5?dilution buffer (40mmol/L Tris HCl,pH 9,1mmol/L MgCl 2),1?mol/L of primer,and 2.5?mol/L template DNA.The sequencing cycle consisted of 95?C for 10s,50?C for 5s,and 60?C for 4min,for 40cycles.The sequencing product was precipitated in 60%isopropanol and re-suspended in 1.5?L of loading buffer (5:1de-ionized formamide:25mmol/L EDTA (pH 8.0),0.05%mass/volume blue dextran Amresco,Solon,Ohio,USA).Reactions were denatured at 90?C for 3min,loaded onto 6%ther-mopage acrylamide gels,and run on the ABI PRISM 377Sequencer (Applied Biosystems,Foster City,California,USA)for 4h.

We edited both strands of the sequences using the Sequencher 3.0program (Gene Codes Corporation,Ann Arbor,Michigan,USA),and the resulting consensus sequences were aligned with sequences published in GenBank.In order to align positions resulting from insertions and deletions (indels),se-quence alignment was performed using SOAP v1.05a (Lo ¨ytynoja and Mil-inkovitch,2001).Twenty-four Clustal W (Thomson,Higgins,and Gibbons,1994)alignments were produced,varying the gap opening penalty between 15and 25in steps of 2,and the gap extension penalty between 8and 14also in steps of 2.The resulting consensus alignments were assessed by eye.All isolates from O.maculata and some from P.monensis were excluded from the phylogenetic analysis because (1)we had no evidence that they were mycorrhizal fungi,(2)the sequences were not related to any known mycor-rhizal fungi,and (3)the sequences were so divergent that they precluded unambiguous alignment.The remaining sequences were subjected to a Mod-eltest 3.06to determine the mode of evolution to be used for phylogenetic analysis (Posada and Crandall,1998).We also included sequences of other uni-and binucleate Ceratobasidium gleaned from GenBank (https://www.360docs.net/doc/f411659077.html,/v89/)using a BLAST search with a minimum score.The optimal distance model for our data was an HKW85distance.A neighbor joining (NJ)and a heuristic search with maximum likelihood (ML)method were done with PAUP (Swofford,1998)and MacClade (Maddison and Mad-dison,1992)using multinucleate Rhizoctonia fungi as the outgroup to con-struct a phylogram.Because the data generated the same topology using both methods,we present the ML tree with bootstrap values of 1000replications from the NJ tree.Finally,a homology distance using a corrected p was con-structed to produce a similarity matrix.

Morphological measurements —Fungal growth rate was measured from the average of two perpendicular diameters of the colony taken every 24h.Hy-phal length and width and monilioid cell length and width were measured in 20cells for each culture using light microscopy at 400?after staining with toluidine blue (Goh,Sim,and Lim,1992).The number of nuclei per cell was determined in 20young cells by ?uorescence microscopy at 500?after stain-ing with 1%Hoescht 33342solution for 10min.Each set of measurements was repeated in three different subcultures.

Statistical analysis —Student’s t tests were used to test signi?cance of dif-ferences in growth and morphological data.This was done to compare uni-and binucleate fungi and to compare uninucleate fungi isolated from T.var-iegata with those from I.utricularioides .All analyses were performed using the Minitab 11statistical software (Minitab,State College,Pennsylvania,USA)after ensuring data were normally distributed.The proportion of uni-and binucleate fungi isolated from different orchid species was compared by Fisher’s exact test.

RESULTS

Variation in isolation of Rhizoctonia-like fungi —Success in isolation of Rhizoctonia -like fungi varied among the nine orchid species.Of the 528cultures attempted we isolated 108Rhizoctonia -like fungi.Isolations were most successful with Ionopsis satyrioides (8of 24cultures:33%success),I.utri-cularioides (32of 144:22%),and Tolumnia variegata (58of 240:24%).Success rate for the other species varied from 4to 13%.We attempted to isolate fungi from other orchids (e.g.,Epidendrum spp.,Lepanthes spp.)without success.A great variety of other endophytic fungi were isolated from orchid roots including Xylaria spp.,Pestalotia spp.,Colletotrichum spp.,and many unidenti?ed fungi (data not shown).Phylogenetic analysis and diversity —Most of the orchid endophytes we studied were closely related to each other and formed a well-supported group within Ceratobasidium (Fig.1).All endophytes were closely related to different anasto-mosis groups of Ceratobasidium spp.

Two main clades were found in the phylogenetic analysis.One,the ABC clade,included most isolates from epiphytic

November2002]1855

O TERO ET AL.—T ROPICAL ORCHID MYCORRHIZAE

T ABLE3.Minimum percentage of homology within and among clades

of the ITS-1and ITS-2using uncorrected p distance.A,B,C,and

D?the Puerto Rican orchid Rhizoctonia endophyte clades;Uni-F

?uninucleate Rhizoctonia from Finland(Hietala,Vahala,and Han-

tula,2001);R.solani?multinucleate(Kuninaga et al.,1997).

Clade A B C D Uni-F R.solani

ITS-1

A

B

C

D

Uni-F R.solani 97.71

88.26

88.70

76.14

75.70

70.98

96.69

92.37

79.81

79.58

70.96

95.68

77.87

78.19

69.91

92.71

80.68

70.35

99.26

71.0869.74

ITS-2

A

B

C

D

Uni-F R.solani 99.07

92.59

95.83

84.17

84.68

80.85

95.83

93.98

81.82

80.98

81.56

99.52

84.00

84.63

82.66

95.38

80.38

77.28

99.11

76.8687.71

T ABLE4.Number of uni-and binucleate Rhizoctonia-like fungi iso-

lates isolated from roots of Puerto Rican orchids by host species.

Host

Cultures

attempted Uninucleate Binucleate Total

Campylocentrum fasciola

Campylocentrum?liforme

Erythrodes plantaginea

Ionopsis satyrioides

Ionopsis utricularioides

Oeceoclades maculata

Oncidium altissimum

Psychilis monensis

Tolumnia variegata

24

24

12

24

144

24

12

12

240

2

29

?a

35

2

2

1

6

3

3

1

?

23

2

2

1

8

32

3

1

1

58

Total5046641108

a Psychilis monensis is excluded from the uninucleate and binucleate

columns because of culture contamination prior to examination for nu-

clei.

orchids from Puerto Rico;the other,D clade,included an iso-late from Oncidium altissimum and one from Tolumnia var-iegata(Fig.1).The ABC clade was supported by a bootstrap-ping index of99%and the D clade by100%.The ABC clade included three well-supported subclades of endophytic Rhi-zoctonia-like fungi from Puerto Rican orchids(A,B,and C); bootstrapping values for each subclade were100,94,and94%, respectively(Fig.1).The ABC clade was monophyletic and included22of the26isolated fungi from the orchid species Campylocentrum fasciola,C.?liforme,Ionopsis satyrioides,I. utricularioides,and Tolumnia variegata.This ABC clade in-cluded a sequence of Ceratobasidium sp.AG-Q(AF354095, isolated from soil in Japan;Gonza′lez et al.,2001)(Fig.1). The D clade grouped with Ceratobasidium AG-H(AF354089, isolated from soil in Japan;Gonza′lez et al.,2001).

The two remaining isolates were jto048(from Psychilis mo-nensis of Mona Island),and jto109(from the terrestrial orchid E.plantaginea).The sequence from isolate jto109grouped with Ceratobasidium sp.AG-A,AG-Bo and Rhizoctonia AG-A.The sequence from jto048had no close matches.

Four groups of isolates had identical ITS sequences when the informative alignable bases were considered(jto075and jto091from I.satyroides;jto024and jto032,isolated from I. utricularioides;jto043and jto047from I.utricularioides;and jto071,jto072,jto115,jto118,and jto124,isolated from I.sa-tyroides,C.fasciola,and C.?liforme;Fig.1).In all cases the fungi with identical sequences came from the same study site; in one case(jto071and jto072)the isolates came from the same root and might have been a single colony.

The minimum homology among clades A,B,C,and D was always greater than92.7%(Table3).The homology among uninucleate Rhizoctonia from Finland and C.bicorne was 99.3%for ITS-1and99.1%for ITS-2.On the other hand,the homology among the R.solani samples was lower,69.7%for ITS-1and87.7%for ITS-2.

The most intensively sampled orchid species differed in the level of speci?city with their endophytic fungi.Five plants of T.variegata from four sites produced?ve isolates that be-longed to four different subclades(A,B,C,and D).On the other hand,four plants of I.utricularioides from two sites produced seven isolates that belonged to just one subclade(B).

Uni-vs.binucleate Rhizoctonia—Of108Rhizoctonia-like fungi,66(62.2%)were uninucleate(Table4).One isolate had both uni-and binucleate cells.There were no consistent ge-netic differences between uni-and binucleate fungi isolated from Puerto Rican orchids(Fig.1).Subclades A,B,and C had a mix of uni-and binucleate fungi.The only clade com-posed entirely of binucleate fungi was clade D,which had only two samples.Tropical,mycorrhizal Rhizoctonia that were uni-nucleate belonged to a distinct clade from the pathogenic uni-nucleate fungi from Finland and Norway(Hietala,Vahala,and Hantula,2001).

All three of the orchid species from which more than three fungi were isolated had at least one uninucleate Rhizoctonia-like fungus.In Tolumnia variegata,the most extensively sam-pled orchid species,60.3%of Rhizoctonia-like fungi were uni-nucleate.Ionopsis utricularioides had the highest proportion of uninucleate Rhizoctonia-like fungi with90.6%.The pro-portion of uninucleate Rhizoctonia-like fungi isolated from I. utricularioides was signi?cantly different from T.variegata (?2?9.20;df?1;P?0.002),I.satyroides(Fisher’s exact test,df?1,P?0.0005)and O.maculata(Fisher’s exact test, df?1,P?0.003).

The proportion of uninucleate Rhizoctonia-like fungi varied among sites(?2?40.6;df?5;P?0.0001).In the South Dorado site97.1%of fungi were uninucleate.Other well-rep-resented sites were San Cristo′bal,Tortuguero,and Cambalache with50.0,44.4,and55.5%,respectively.All fungi from Sa-bana Seca were uninucleate,but sample size was small(N?5).Differences among sites were not solely due to distribution of species:for isolates from T.variegata alone,there was a signi?cant difference among sites in the proportion of uninu-cleate Rhizoctonia-like fungi(?2?18.5;df?2;P?0.001). Morphological data were signi?cantly different between uni-and binucleate fungi for two of?ve parameters.There were differences in growth rate(measured centimetres per day) (uninucleate:mean?1.85;binucleate:mean?1.27;t?7.53, df?48,P?0.001)and width of monilioid cells(uninucleate: mean?11.3?m;binucleate:mean?10.9?m;t?2.10,df ?82,P?0.04),but not in hyphal length(uninucleate:mean ?45.0?m;binucleate:mean?47.4?m;t?1.22,df?73, P?0.23),hyphal width(uninucleate:mean?5.8?m;bi-nucleate:mean?5.8?m;t?0.03,df?65,P?0.98)and monilioid cell length(uninucleate:mean?21.6?m;binucle-ate:mean?21.4?m;t?0.23,df?62,P?0.82). Morphological differences among fungi from different or-

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chid hosts were signi?cant for growth rate(F

2,291?10.66,P

K0.001),but not for hyphal length(F

2,291?0.38,P?0.68)

or hyphal width(F

2,291?

0.47,P?0.63).

DISCUSSION

Phylogenetic relationships—This is the?rst time that phy-logenetic relationships of Rhizoctonia-like fungi from tropical orchids have been studied using molecular methods.The phy-logenetic tree(Fig.1)suggests that the Puerto Rican Rhizoc-tonia-like endophytes of orchids are anamorphs of the genus Ceratobasidium.Most of the endophytic Rhizoctonia-like fun-gi from this study formed a monophyletic group(clade ABC, Fig.1).This clade also included Ceratobasidium AG-Q,iso-lated from soil in Japan.Ceratobasidium AG-H,also from Japanese soil,was the closest known relative to clade D.Cer-atobasidium AG-D from Japan and Ceratobasidium CAG-1 from USA were related to clades ABC and D.The teleo-morphs of AG-Q and AG-D are Ceratobasidium cornigerum (Bourd.)Rogers and C.gramineum(Ikata&T.Matsuura)On-iki,Ogoshi&Araki,respectively(Sneh,Burpee,and Ogoshi, 1991),suggesting that orchid endophytes belonging to clades ABC and D include at least two species.Unfortunately,we were not able to induce sexual structures under laboratory con-ditions,a common problem in this group(Vilgalys and Cubeta, 1994).

The two terrestrial orchids,Oeceoclades and Erythrodes, had fungi very different than epiphytic orchids.Isolates from Oeceoclades were too divergent to be included in our phylo-genetic analysis.The fungus isolated from E.plantaginea was closely related to three isolates of Ceratobasidium AG-A, which are separate from clades ABC and D. Ceratobasidium spp.are known as pathogens of turfgrasses and cereals(Currah et al.,1997)and have been reported as orchid endophytes in Australia,North America,and tropical Asia(Currah et al.,1997).Ceratorhiza,the anamorphic genus of Ceratobasidium(Currah,1991),is one of the most common endophytes isolated from temperate orchids(Zelmer and Cur-rah,1995;Currah et al.,1997),but has only once been re-ported from Neotropical orchids(Richardson,Currah,and Hambleton,1993).

There have been few attempts to isolate and identify Rhi-zoctonia-like fungi from neotropical orchids.Ceratorhiza goodyerae-repentis was reported in Campylocentrum macu-latum(Lindl.)Rolfe and Rodriguezia compacta Schltr.from Costa Rica(Richardson,Currah,and Hambleton,1993;Rich-ardson and Currah,1995).In Puerto Rico multinucleate Rhi-zoctonia-like fungi were isolated from Lepanthes spp.(Bay-man et al.,1997)and from bark of trees with orchid epiphytes (Tremblay et al.,1998).Endophytic fungal communities from tropical orchid roots are very rich and include both Rhizoc-tonia-like and non-Rhizoctonia-like fungi(Richardson,Cur-rah,and Hambleton,1993;Richardson and Currah,1995;Bay-man et al.,1997)yet little is known of their role in orchid biology.To choose only Rhizoctonia-like endophytes for my-corrhizal studies may miss fungi critical for orchid establish-ment.An alternative is to isolate pelotons for culturing(Had-ley,1970;Rasmussen,1995)or DNA ampli?cation(Kristian-sen et al.,2001).Unfortunately,many epiphytic tropical or-chids do not have the massive mycorrhizal infections of terrestrial orchids,making it dif?cult to isolate pelotons. There is evidence that our endophytic Rhizoctonia-like fungi are potential mycorrhizal symbionts(in the sense of Masuhara and Katsuya,1994).Morphology of pelotons in adult roots of Campylocentrum spp.,Ionopsis spp.,and Tolumnia variegata match the morphology of cultured Rhizoctonia-like fungi.Ad-ditionally,symbiotic germination experiments in vitro with seeds from I.utricularioides and T.variegata resulted in sig-ni?cantly enhanced development of orchid seedlings(J.T. Otero,P.Bayman and J.D.Ackerman,unpublished data). How divergent are subclades A,B,C,and D?A possible way to answer this question is to compare the levels of genetic variability within each clade and subclade with those of related fungal species.Percentage homology of ITS sequences among and within anastomosis groups of Rhizoctonia solani has been determined(Kuninaga et al.,1997).The minimum ITS-1and ITS-2sequence homology within A,B,and C subclades(Table 3)was higher than that reported for R.solani AG-1,but similar to R.solani AG-3and AG-4(Kuninaga et al.,1997).The minimum homology among endophytic subclades A,B,C,and D was higher than that among R.solani AG groups.A,B,C, and D are distinct entities that have similar levels of variation in the ITS regions as anastomosis groups of R.solani.How-ever,these comparisons are approximate because the algorithm used by Kuninaga et al.(1997)employed different weightings than the one used here.ITS-1was more variable than ITS-2 among Puerto Rican clades and also in R.solani(Kuninaga et al.,1997).

Uninucleate Rhizoctonia-like endophytes—The uninucle-ate condition has been reported in Rhizoctonia quercus(Bur-pee,Sanders,and Cole,1980)and in a Rhizoctonia pathogenic on wheat(Hall,1986).The only well-known species of uni-nucleate Rhizoctonia-like fungi occurs as a seedling pathogen of conifer nurseries in Finland and Norway(Hietala,Sen,and Lilja,1994).The reproductive structures of these fungi pro-duced under laboratory conditions were typical of Ceratobas-idium and?t the morphological concept of Ceratobasidium bicorne Erikss.&Ryv.(Hietala,1997).Molecular data con-?rmed this liaison(Hietala,Vahala,and Hantula,2001;Fig.

1).From molecular markers,uninucleate Rhizoctonia was con-sidered a genetically homogenous group,distinct from binu-cleate Rhizoctonia spp.(Lilja,Hietala,and Karjalainen,1996; Hietala,Vahala,and Hantula,2001).

The hyphal widths reported from the Finnish and Norwe-gian fungi(Hietala,Sen,and Lilja,1994)were similar to those obtained in this study.Additionally,monilioid cell widths and lengths from Puerto Rico?t within the range reported by Hie-tala,Sen,and Lilja(1994),but they found much more varia-tion than we did.Nevertheless,the analysis of ITS sequences shows that they are in different lineages within Ceratobasi-dium(Fig.1).This observation suggests that the uninucleate condition has appeared independently in two clades,the ABC clade from Puerto Rico and the uninucleate clade from Finland and Norway.We found an isolate with both uni-and binucle-ate cells,suggesting that the uninucleate condition may be plastic even in a single mycelium.The co-occurrence of uni-and binucleate cells in the same hyphae was also observed in Rhizoctonia from Finland(Hietala,Sen,and Lilja,1994).Mor-phological data show that the main difference between uni-and binucleate Rhizoctonia of orchid roots is the mycelial growth rate.Thus,changes between uni-and binucleate states may be associated with physiological or developmental pro-cesses.

In general,the uninucleate condition is rare(Hietala,1997), but our data suggest that it may be more common in epiphytic

November2002]1857

O TERO ET AL.—T ROPICAL ORCHID MYCORRHIZAE

orchids.Nonetheless,the proportion of uni-and binucleate Rhizoctonia-like fungi varied among species(Table4).Binu-cleate fungi were more broadly distributed among orchid spe-cies than uninucleate ones,but T.variegata and I.utricular-ioides had signi?cantly more uninucleate isolates than would be expected.

Speci?city in orchid mycorrhizae—Since the discovery that fungi are involved in orchid seed germination(Bernard,1909), speci?city in orchid mycorrhizae has been controversial(Har-ley and Smith,1983).Previous studies found either that or-chids are speci?c(Clements,1987;Taylor and Bruns,1997) or generalist(Hadley,1970;Smreciu and Currah,1989;Ma-suhara and Katsuya,1989,1991;Masuhara,Katsuya,and Ya-maguchi,1993;Rasmussen,1995)in their mycorrhizal sym-bioses.However,data from an extensive study of European orchids suggests that the degree of speci?city is variable among species(Muir,1989).

Speci?city is best assessed in a comparative context.For that reason,the difference in speci?city between Tolumnia variegata and Ionopsis utricularioides—two species similar in distribution,ecology,and taxonomic af?nity—is striking.En-dophytes from T.variegata appeared in all four subclades,but those from I.utricularoides appeared only in the B subclade (Fig.1).Tolumnia variegata has also been shown to be highly variable in morphology(Ackerman and Galarza-Pe′rez,1991) and isozymes(Ackerman and Ward,2000),but most compo-nents of variation were found to be within populations.Fur-thermore,Puerto Rican populations form a single taxon(Ack-erman,1995).These data suggest that I.utricularoides may be more speci?c than T.variegata in its association with Rhi-zoctonia-like fungi.Variation in speci?city was also evident within a single genus,as I.satyrioides had fungi in three sub-clades(A,B,and C)whereas those from I.utricularioides were in only one(subclade B).

Non-photosynthetic orchids are considered to have special-ist mycorrhizal associations in the sense that they can involve only ectomycorrhizal fungi rather than Rhizoctonia-like fungi (Taylor and Bruns,1997;but see McKendrick et al.,2002). However,the amount of variation shown by these ectomycor-rhizae(Taylor and Bruns,1997)is comparable to that of Rhi-zoctonia-like fungi of T.variegata,an orchid that we argue is a relative generalist in its mycorrhizal associations.

In conclusion,our data show(1)Puerto Rican epiphytic orchids are associated with Ceratobasidium spp.that may be undescribed but closely related to temperate isolates;(2)many mycorrhizal fungi from Puerto Rican orchids were uninucle-ate;(3)the uninucleate condition is not a good phylogenetic character in mycorrhizal fungi from Puerto Rico;and(4)spec-i?city of the orchid mycorrhizal association varies dramatical-ly even among closely related species.Although some species, such as non-photosynthetic orchids from temperate regions, have particular mycorrhizal fungi(Taylor and Bruns,1997), orchids appear to vary in degree of speci?city in their mycor-rhizal interactions more than previously thought.

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