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Tropical Biology |
Department of Biology, University of Puerto Rico, Río Piedras, P.O. Box 23360, San Juan, Puerto Rico 00931-3360 USA
Received for publication February 5, 2002. Accepted for publication May 16, 2002.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: Ceratobasidium • orchid mycorrhizae • Puerto Rico • Rhizoctonia • specificity
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Understanding the mycorrhizal symbiosis is of great importance, as availability of the fungal symbionts may play a key role in determining orchid distribution and diversity. Furthermore, most studies of orchid mycorrhizal interactions have concentrated on terrestrial orchids from temperate regions, whereas the majority of orchid species are epiphytes in tropical regions.
Studies to date regarding the specificity of orchid mycorrhizal relationships have drawn conflicting conclusions. Two distinct approaches have been taken in the investigation of specificity of orchid mycorrhizal relationships: (1) in vitro seed germination experiments and (2) taxonomic comparisons of fungi present in orchid roots of mature plants. One germination study of epiphytic orchids suggesting specificity (Clements, 1987
) is balanced by another that suggests more generalist interactions (Zettler, Burkhead, and Marshall, 1999
). Answering the question of specificity depends on a thorough sampling and detailed taxonomy of orchid mycorrhizal fungi.
The systematics of orchid mycorrhizal fungi has been studied using both morphological (Warcup and Talbot, 1966
, 1971
; Currah, Hambleton, and Smreciu, 1988
; Rasmussen, 1995
), and molecular characters (Taylor and Bruns, 1997
, 1999
; Kristiansen et al., 2001
). Rhizoctonia-like fungi include the anamorphic (asexual) genera Ceratorhiza, Epulorhiza, Moniliopsis, 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 (Sneh, Burpee, and Ogoshi, 1991
). Rhizoctonia are characterized 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 inflated hyphae, known as monilioid cells (Sneh, Burpee, and Ogoshi, 1991
). Sexual stages are rarely encountered in the field or laboratory. Consequently, the broad vegetative criteria for identification have resulted in paraphyletic taxonomy, with various unrelated fungi being grouped together.
One morphological feature that has helped in the classification 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 has 13 AGs. The binucleate Rhizoctonia spp. include 21 AGs (Sneh, Burpee, and Ogoshi, 1991
), and the uninucleate Rhizoctonia spp. include 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-6 and AG-12 (Carling, 1996
; Carling et al., 1999
; but see Masuhara, Katsuya, and Yamaguchi, 1993
). Nevertheless, the most common group of orchid mycorrhizal fungi is binucleate Rhizoctonia (Currah et al., 1997
). Uninucleate Rhizoctonia have not been previously reported from orchid roots.
Molecular systematics has substantially advanced the taxonomy of Rhizoctonia spp. (Vilgalys and González, 1990
; Cubeta and Vilgalys, 1997
; Kuninaga et al., 1997
; Salazar et al., 2000
; González et al., 2001
) and orchid mycorrhizae (Taylor and Bruns, 1997
, 1999
; Pope and Carter, 2001
; Kristiansen et al., 2001
), mostly based on nuclear ribosomal internal transcribed spacer (ITS) sequences. Pope and Carter (2001)
studied ITS sequences of Rhizoctonia solani AGs including orchid mycorrhizal fungi from Australia. Kristiansen et al. (2001)
amplified 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 find a variety of fungi related to Ceratobasidium, Thanatephorus, and Tulasnella. (2) What is the level of specificity of epiphytic orchids for their Rhizoctonia-like fungi? If there is specificity, then we expect that the fungi isolated from a single orchid species will belong to a single fungal clade. And finally, (3) what is the phylogenetic relationship between uni- and binucleate Rhizoctonia-like fungi associated with orchids? We expected consistent differences in morphology and phylogeny between uni- and binucleate Rhizoctonia-like fungi from Puerto Rico.
To answer these questions we sampled 44 plants from nine tropical orchid species belonging to seven genera and five subtribes. The orchids were collected from a number of ecologically diverse sites on Puerto Rico and Mona islands. We used ITS sequence data together with morphological data to estimate relationships among fungi.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). 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 50 ppm 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 identified using the concept of Sneh, Burpee, and Ogoshi (1991)
. A total of 108 Rhizoctonia-like isolates were found. No perfect stages were observed on PDA or water agar (Sneh, Burpee, and Ogoshi, 1991
). Of these isolates, we selected 26 for sequencing representing all orchids species sampled. All isolates from orchids with less than three total isolates were included. Among isolates from T. variegata and I. utriculariodes (the most heavily sampled species) isolates were chosen based on morphological diversity on PDA.
DNA isolation and ITS amplification
The DNA of 26 of 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-1 and ITS-4 (White et al., 1990
). The PCR products were cleaned using Qiaquick columns (Qiagen, Valencia, California, USA), according to the manufacturer's instructions, and sequenced in both directions. Each 5-µL sequencing reaction consisted of 1 µL ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Mix (Applied Biosystems, Foster City, California, USA), 1 µL 5x dilution buffer (40 mmol/L Tris HCl, pH 9, 1 mmol/L MgCl2), 1 µmol/L of primer, and 2.5 µmol/L template DNA. The sequencing cycle consisted of 95°C for 10 s, 50°C for 5 s, and 60°C for 4 min, for 40 cycles. The sequencing product was precipitated in 60% isopropanol and resuspended in 1.5 µL of loading buffer (5:1 de-ionized formamide: 25 mmol/L EDTA (pH 8.0), 0.05% mass/volume blue dextran Amresco, Solon, Ohio, USA). Reactions were denatured at 90°C for 3 min, loaded onto 6% thermopage acrylamide gels, and run on the ABI PRISM 377 Sequencer (Applied Biosystems, Foster City, California, USA) for 4 h.
We edited both strands of the sequences using the Sequencher 3.0 program (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), sequence alignment was performed using SOAP v1.05a (Löytynoja and Milinkovitch, 2001
). Twenty-four Clustal W (Thomson, Higgins, and Gibbons, 1994
) alignments were produced, varying the gap opening penalty between 15 and 25 in steps of 2, and the gap extension penalty between 8 and 14 also 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 mycorrhizal fungi, and (3) the sequences were so divergent that they precluded unambiguous alignment. The remaining sequences were subjected to a Modeltest 3.06 to 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 (http://ajbsupp.botany.org/v89/) using a BLAST search with a minimum score. The optimal distance model for our data was an HKW85 distance. A neighbor joining (NJ) and a heuristic search with maximum likelihood (ML) method were done with PAUP (Swofford, 1998
) and MacClade (Maddison and Maddison, 1992
) using multinucleate Rhizoctonia fungi as the outgroup to construct a phylogram. Because the data generated the same topology using both methods, we present the ML tree with bootstrap values of 1000 replications from the NJ tree. Finally, a homology distance using a corrected p was constructed to produce a similarity matrix.
Morphological measurements
Fungal growth rate was measured from the average of two perpendicular diameters of the colony taken every 24 h. Hyphal length and width and monilioid cell length and width were measured in 20 cells for each culture using light microscopy at 400x after staining with toluidine blue (Goh, Sim, and Lim, 1992
). The number of nuclei per cell was determined in 20 young cells by fluorescence microscopy at 500x after staining with 1% Hoescht 33342 solution for 10 min. Each set of measurements was repeated in three different subcultures.
Statistical analysis
Student's t tests were used to test significance of differences in growth and morphological data. This was done to compare uni- and binucleate fungi and to compare uninucleate fungi isolated from T. variegata with those from I. utricularioides. All analyses were performed using the Minitab 11 statistical 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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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 anastomosis groups of Ceratobasidium spp.
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) (Fig. 1). The D clade grouped with Ceratobasidium AG-H (AF354089, isolated from soil in Japan; González et al., 2001
). The two remaining isolates were jto048 (from Psychilis monensis of Mona Island), and jto109 (from the terrestrial orchid E. plantaginea). The sequence from isolate jto109 grouped with Ceratobasidium sp. AG-A, AG-Bo and Rhizoctonia AG-A. The sequence from jto048 had no close matches.
Four groups of isolates had identical ITS sequences when the informative alignable bases were considered (jto075 and jto091 from I. satyroides; jto024 and jto032, isolated from I. utricularioides; jto043 and jto047 from I. utricularioides; and jto071, jto072, jto115, jto118, and jto124, isolated from I. satyroides, C. fasciola, and C. filiforme; Fig. 1). In all cases the fungi with identical sequences came from the same study site; in one case (jto071 and 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 than 92.7% (Table 3). The homology among uninucleate Rhizoctonia from Finland and C. bicorne was 99.3% for ITS-1 and 99.1% for ITS-2. On the other hand, the homology among the R. solani samples was lower, 69.7% for ITS-1 and 87.7% for ITS-2.
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Uni- vs. binucleate Rhizoctonia
Of 108 Rhizoctonia-like fungi, 66 (62.2%) were uninucleate (Table 4). One isolate had both uni- and binucleate cells. There were no consistent genetic 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 composed entirely of binucleate fungi was clade D, which had only two samples. Tropical, mycorrhizal Rhizoctonia that were uninucleate belonged to a distinct clade from the pathogenic uninucleate fungi from Finland and Norway (Hietala, Vahala, and Hantula, 2001
).
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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 site 97.1% of fungi were uninucleate. Other well-represented sites were San Cristóbal, Tortuguero, and Cambalache with 50.0, 44.4, and 55.5%, respectively. All fungi from Sabana 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 significant difference among sites in the proportion of uninucleate Rhizoctonia-like fungi (2 = 18.5; df = 2; P < 0.001).
Morphological data were significantly different between uni- and binucleate fungi for two of five 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 µm; binucleate: mean = 10.9 µm; t = 2.10, df = 82, P = 0.04), but not in hyphal length (uninucleate: mean = 45.0 µm; binucleate: mean = 47.4 µm; t = 1.22, df = 73, P = 0.23), hyphal width (uninucleate: mean = 5.8 µm; binucleate: mean = 5.8 µm; t = 0.03, df = 65, P = 0.98) and monilioid cell length (uninucleate: mean = 21.6 µm; binucleate: mean = 21.4 µm; t = 0.23, df = 62, P = 0.82).
Morphological differences among fungi from different orchid hosts were significant for growth rate (F2,291 = 10.66, P << 0.001), but not for hyphal length (F2,291 = 0.38, P = 0.68) or hyphal width (F2,291 = 0.47, P = 0.63).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), 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 conditions, 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 phylogenetic 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 Currah, 1995
; Currah et al., 1997
), but has only once been reported from Neotropical orchids (Richardson, Currah, and Hambleton, 1993
).
There have been few attempts to isolate and identify Rhizoctonia-like fungi from neotropical orchids. Ceratorhiza goodyerae-repentis was reported in Campylocentrum maculatum (Lindl.) Rolfe and Rodriguezia compacta Schltr. from Costa Rica (Richardson, Currah, and Hambleton, 1993
; Richardson and Currah, 1995
). In Puerto Rico multinucleate Rhizoctonia-like fungi were isolated from Lepanthes spp. (Bayman 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 Rhizoctonia-like and non-Rhizoctonia-like fungi (Richardson, Currah, and Hambleton, 1993
; Richardson and Currah, 1995
; Bayman et al., 1997
) yet little is known of their role in orchid biology. To choose only Rhizoctonia-like endophytes for mycorrhizal studies may miss fungi critical for orchid establishment. An alternative is to isolate pelotons for culturing (Hadley, 1970
; Rasmussen, 1995
) or DNA amplification (Kristiansen et al., 2001
). Unfortunately, many epiphytic tropical orchids do not have the massive mycorrhizal infections of terrestrial orchids, making it difficult 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. Additionally, symbiotic germination experiments in vitro with seeds from I. utricularioides and T. variegata resulted in significantly 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-1 and ITS-2 sequence 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-3 and 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. However, these comparisons are approximate because the algorithm used by Kuninaga et al. (1997)
employed different weightings than the one used here. ITS-1 was more variable than ITS-2 among Puerto Rican clades and also in R. solani (Kuninaga et al., 1997
).
Uninucleate Rhizoctonia-like endophytes
The uninucleate condition has been reported in Rhizoctonia quercus (Burpee, Sanders, and Cole, 1980
) and in a Rhizoctonia pathogenic on wheat (Hall, 1986
). The only well-known species of uninucleate 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 produced under laboratory conditions were typical of Ceratobasidium and fit the morphological concept of Ceratobasidium bicorne Erikss. & Ryv. (Hietala, 1997
). Molecular data confirmed this liaison (Hietala, Vahala, and Hantula, 2001
; Fig. 1). From molecular markers, uninucleate Rhizoctonia was considered a genetically homogenous group, distinct from binucleate Rhizoctonia spp. (Lilja, Hietala, and Karjalainen, 1996
; Hietala, Vahala, and Hantula, 2001
).
The hyphal widths reported from the Finnish and Norwegian fungi (Hietala, Sen, and Lilja, 1994
) were similar to those obtained in this study. Additionally, monilioid cell widths and lengths from Puerto Rico fit within the range reported by Hietala, Sen, and Lilja (1994)
, but they found much more variation than we did. Nevertheless, the analysis of ITS sequences shows that they are in different lineages within Ceratobasidium (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 binucleate 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
). Morphological 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 processes.
In general, the uninucleate condition is rare (Hietala, 1997
), but our data suggest that it may be more common in epiphytic orchids. Nonetheless, the proportion of uni- and binucleate Rhizoctonia-like fungi varied among species (Table 4). Binucleate fungi were more broadly distributed among orchid species than uninucleate ones, but T. variegata and I. utricularioides had significantly more uninucleate isolates than would be expected.
Specificity in orchid mycorrhizae
Since the discovery that fungi are involved in orchid seed germination (Bernard, 1909
), specificity in orchid mycorrhizae has been controversial (Harley and Smith, 1983
). Previous studies found either that orchids are specific (Clements, 1987
; Taylor and Bruns, 1997
) or generalist (Hadley, 1970
; Smreciu and Currah, 1989
; Masuhara and Katsuya, 1989
, 1991
; Masuhara, Katsuya, and Yamaguchi, 1993
; Rasmussen, 1995
) in their mycorrhizal symbioses. However, data from an extensive study of European orchids suggests that the degree of specificity is variable among species (Muir, 1989
).
Specificity is best assessed in a comparative context. For that reason, the difference in specificity between Tolumnia variegata and Ionopsis utricularioides—two species similar in distribution, ecology, and taxonomic affinity—is striking. Endophytes 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-Pérez, 1991
) and isozymes (Ackerman and Ward, 2000), but most components of variation were found to be within populations. Furthermore, Puerto Rican populations form a single taxon (Ackerman, 1995
). These data suggest that I. utricularoides may be more specific than T. variegata in its association with Rhizoctonia-like fungi. Variation in specificity was also evident within a single genus, as I. satyrioides had fungi in three subclades (A, B, and C) whereas those from I. utricularioides were in only one (subclade B).
Non-photosynthetic orchids are considered to have specialist 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 ectomycorrhizae (Taylor and Bruns, 1997
) is comparable to that of Rhizoctonia-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 uninucleate; (3) the uninucleate condition is not a good phylogenetic character in mycorrhizal fungi from Puerto Rico; and (4) specificity of the orchid mycorrhizal association varies dramatically 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 specificity in their mycorrhizal interactions more than previously thought.
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FOOTNOTES |
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2 Author for reprint requests (is975785{at}rrpac.upr.clu.edu
)
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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