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Departamento de Biología, Universidad de Puerto Rico-Río Piedras, PO Box 23360, San Juan, Puerto Rico 00931
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Mycorrhizal fungi are essential for the germination of orchid seeds. However, the specificity of orchids for their mycorrhizal fungi and the effects of the fungi on orchid growth are controversial. Mycorrhizal fungi have been studied in some temperate and tropical, epiphytic orchids, but the symbionts of tropical, terrestrial orchids are still unknown. Here we study diversity, specificity and function of mycorrhizal fungi in Vanilla, a pantropical genus that is both terrestrial and epiphytic. Mycorrhizal roots were collected from four Vanilla species in Puerto Rico, Costa Rica and Cuba. Cultured and uncultured mycorrhizal fungi were identified by sequencing the internal transcribed spacer region of nuclear rDNA (nrITS) and part of the mitochondrial ribosomal large subunit (mtLSU), and by counting number of nuclei in hyphae. Vanilla spp. were associated with a wide range of mycorrhizal fungi: Ceratobasidium, Thanatephorus and Tulasnella. Related fungi were found in different species of Vanilla, although at different relative frequencies. Ceratobasidium was more common in roots in soil and Tulasnella was more common in roots on tree bark, but several clades of fungi included strains from both substrates. Relative frequencies of genera of mycorrhizal fungi differed significantly between cultured fungi and those detected by direct amplification. Ceratobasidium and Tulasnella were tested for effects on seed germination of Vanilla and effects on growth of Vanilla and Dendrobium plants. We found significant differences among fungi in effects on seed germination and plant growth. Effects of mycorrhizal fungi on Vanilla and Dendrobium were similar: a clade of Ceratobasidium had a consistently positive effect on plant growth and seed germination. This clade has potential use in germination and propagation of orchids. Results confirmed that a single orchid species can be associated with several mycorrhizal fungi with different functional consequences for the plant.
Key words: Ceratobasidium, epiphyte, mycorrhiza, Orchidaceae, Rhizoctonia, symbiosis, Thanatephorus, Tulasnella
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Rasmussen 1995, 2002). In adult stages of photosynthetic orchids, however, the importance of mycorrhizal fungi varies and is not clearly understood (Shefferson et al 2005, Girlanda 2006).
The Orchidaceae associates with a wide range of mycorrhizal fungi, especially basidiomycetes such as Ceratobasidiales and Tulasnellales (although some individual orchid species are very specific). Some mycoheterotrophic (or nonphotosynthetic) orchids associate with different groups, usually ectomycorrhizal fungi (Currah et al 1997b, Rasmussen 2002, Taylor et al 2002). Orchid fungi can be diverse even at the level of an individual plant: more than one mycorrhizal fungus was found in a single peloton (the coils of intracellular hyphae in orchid root cells) (Kristiansen et al 2001). It is likely that these fungi have different functional consequences for the host plants but evidence of functional differences is limited (Zettler and Hofer 1998, Clements 1988).
The study of orchid mycorrhizal fungi presents several challenges. First, many orchid mycorrhizal fungi do not produce teleomorphs in pure culture (Andersen 1990, Andersen and Rasmussen 1996), so identification was difficult until the advent of molecular systematics. Second, it is not clear whether the mycorrhizal fungi in germinating seeds (a stage when all orchids need fungi) are the same as those isolated from adult orchids (when some need fungi and others are facultative) (Zelmer et al 1996, Rasmussen 2002, McCormick et al 2004). In vitro symbiotic germination experiments with fungi isolated from adult plants can help address this question (Otero et al 2004, 2005). Third, differences in ability to grow in vitro mean that some fungi might be overlooked in culture-based studies (Kristiansen et al 2001, Taylor et al 2003). Fourth, the life mechanisms of most orchid mycorrhizal fungi are unknown; they may be saprotrophs, plant pathogens, ectomycorrhizal fungi or some combination thereof (Vilgalys and Cubeta 1994, McCormick et al 2004). Fifth, the basic nature of the relationship is still mysterious. Only recently has it been demonstrated that the fungus can benefit from the interaction (Cameron et al 2006). Until now it has been assumed that the orchid was always parasitic on the fungus, in marked contrast to other types of mycorrhizal relationships.
Specificity of orchids for mycorrhizal fungi has been controversial for more than 50 y (Warcup 1981, Rasmussen 2002), as has mycorrhizal specificity in general. Recent studies have shown that terrestrial orchids are more specific for mycorrhizal fungi than previously was believed, with some exceptions (e.g. McCormick et al 2004, Shefferson et al 2005, Girlanda et al 2006). In particular some mycoheterotrophic terrestrial orchids are highly specific for groups of ectomycorrhizal fungi, which are probably more reliable sources of carbon than saprotrophs or plant pathogens (Taylor et al 2002, Leake et al 2004).
Mycorrhizal relationships of tropical, terrestrial orchids are largely unexplored (Pereira et al 2005). Although the words "tropical orchids" brings to mind epiphytes, there are many terrestrial orchids in the tropics as well; in Puerto Rico 40% of the 145 orchid species are mainly terrestrial, and others occasionally grow as terrestrials (compiled from Ackerman 1995). It is likely that the fungi available to tropical, terrestrial orchids are different from those available to temperate, terrestrial orchids: tropical soils tend to differ from temperate soils in several important respects, and many guilds of fungi differ in the tropics and temperate regions (Lodge and Cantrell 1995). However differences in mycorrhizal fungi between temperate and tropical terrestrial orchids have not been investigated. Also it is not known whether tropical, terrestrial orchids exploit the same fungi as tropical, epiphytic orchids; fungal assemblages in soil and on tree bark are presumably distinct.
The genus Vanilla is ideal to address these issues. Most Vanilla plants are hemiepiphytic, that is simultaneously terrestrial and epiphytic with roots in soil and on tree bark. Vanilla is economically important, with world production of Vanilla beans (actually capsules) about 2000 t/y (Havkin-Frenkel et al 2005). Mycorrhizal fungi are potentially useful in Vanilla cultivation for control of Fusarium root rots (Alconero 1969), so their identification has practical as well as ecological interest. But despite the economic importance and pantropical distribution of Vanilla, little is known about its mycorrhizal fungi.
Here we address these questions: (i) Do Vanilla roots in soil and roots on bark have different frequencies of mycorrhizal infection? We hypothesized that infection frequencies would be higher in roots in soil because terrestrial orchids are obligate mycorrhizal as adults whereas most epiphytic orchids are facultative (Rasmussen 1995). (ii) Are roots in soil and on bark colonized by different mycorrhizal fungi? We hypothesized that roots in soil would be colonized mainly by Tulasnella, like temperate, terrestrial orchids (Shefferson et al 2005) and the few tropical, terrestrial orchids that have been examined (e.g. Ma et al 2003, Pereira et al 2005), whereas roots on bark would be colonized mainly by Ceratobasidium, such as epiphytic orchids in Puerto Rico (Otero et al 2002). (iii) Are all mycorrhizal fungi from Vanilla equally effective at promoting seed germination and plant growth? We hypothesized that the most effective fungi at promoting seed germination in vitro and plant growth would be Ceratobasidium and that related fungi would have similar effects on orchid growth, based on studies of epiphytic orchids (Otero et al 2004, 2005). (iv) Is Vanilla specific for mycorrhizal fungi, and do different Vanilla species use the same fungi? We hypothesized that Vanilla species would vary in specificity but that the same groups of fungi would be found in different species, as in epiphytic orchids (Otero et al 2004).
To answer these questions mycorrhizal fungi from four neotropical Vanilla species were identified based on sequences of the internal transcribed spacer (nrITS) region and the mitochondrial ribosomal large subunit rDNA (mtLSU). Both pure cultures and direct DNA extractions from roots were used to sample culturable and nonculturable fungi. Representative fungi were tested for effects on Vanilla seed germination and plant growth. To determine whether growth effects were specific to Vanilla or applicable to orchids in general, we also tested the effects of fungal inoculation on a commercial cultivar of Dendrobium.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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).
Isolation of mycorrhizal fungi from Vanilla roots.— –
The velamen was removed and mycorrhizal areas were identified as brown zones along the length of the roots (FIG. 1, Porras and Bayman 2003). The rate of colonization was estimated as the number of mycorrhizal zones per centimeter of root. A total of 246 roots were processed: 60 V. planifolia, 60 V. poitaei, 15 V. phaentha and 5 V. aphylla roots from soil; 32 V. planifolia and 37 V. poitaei roots from bark; and 37 V. poitaei roots from rocks. Roots were surface sterilized with ethanol (70%) for 30 s, 50% Clorox® (= 2.6% sodium hypochlorite) for 3 min and washed twice with distilled water (Bayman et al 1997). Approximately 1500 segments of colonized roots (3–5 mm long) were plated on potatodextrose agar (PDA) with 50 µg/mL each penicillin and streptomycin. Fungi in pure culture were stained with safranin O for nuclear counts (Bandoni 1979, Sneh et al 1991).
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). The internal transcribed spacer region of nuclear rDNA (nrITS) and part of the large subunit gene of mitochondrial rDNA (mtLSU) were amplified by PCR. The nrITS was amplified with primers ITS1/ITS4 (White et al 1990) and new, specific primers for Tulasnella (Tul1/Tul4) and Ceratobasidium/Thanatephorus (CeTh1/CeTh4) (see below). The mitochondrial rDNA primers MLin3/ML6 were used only with fungal cultures because with direct amplifications from roots they amplified plant as well as fungal DNA. MLin3 primer was used instead of the more widely used ML5 because ML5 did not amplify our Tulasnella isolates (see Bruns et al 1998). The region amplified by ML7/ML8 also was amplified but was not sufficiently informative. Negative controls (without DNA template) and positive controls (fungal DNA that had been successfully amplified before) were included in all amplifications.
Design of taxon-specific ITS primers.— –
The standard nuclear ribosomal ITS primers ITS1, ITS4, ITS1F and ITS4B (White et al 1990, Gardes and Bruns 1993) successfully amplified fungal DNA from pure cultures, but in direct amplifications from Vanilla root tissue they amplified plant DNA instead of fungal DNA (presumably because plant DNA was in much higher concentration than fungal DNA). A range of annealing temperatures (52–60 C) and MgCl2 concentrations (3.5–6.5 mM) was tested to increase specificity for fungal DNA, without success. Another limitation of standard fungal ITS primers is that they do not amplify some groups of Tulasnella (Bidartondo et al 2003), so they might bias results in favor of other groups of fungi.
We therefore designed new, more specific primers based on alignments of ITS sequences from Ceratobasidium, Thanatephorus, Tulasnella and related sequences from GenBank. Orchid sequences were included to ensure that plant DNA would not be amplified. Because of sequence divergence we designed two pairs of primers: Tul1/Tul4 for Tulasnella (Tul1: ACG TTA AGG TGC TCT GGT YGA GG, Tul 4: ATG AGG TCA TGC GTT GTA GTA CC, product of 500 bp, degenerate nucleotide Y corresponds to C and T) and CeTh1/CeTh4 for Ceratobasidium and Thanatephorus (CeTh1: TTC TCT TTC ATC CAC ACA CMC C, CeTh4: ACA GGA TGC TCC ARG GAA TAC C, product of 300 bp, degenerate nucleotides M and R correspond respectively to A, C and A, G). Analysis of primer dimer and secondary structures was done with Oligo-analizer 3.0 (http://biotools.idtdna.com/gateway). PCR reactions had an annealing temperature of 56 C and 25 mM MgCl2. Both primer pairs were used on all DNA samples. All experiments included a positive control (DNA from mycelium that had successfully amplified) and two negative controls (Vanilla leaf DNA; no DNA template).
Sequencing and phylogenetic analysis.— –
PCR products were purified by digestion with alkaline phosphatase and exo-nuclease (ExoSAP-IT®, USB Corp). Sequencing was done in the Sequencing and Genotyping Facility of the University of Puerto Rico, Río Piedras using DYEnamicTM ET Terminator Cycle Sequencing Kit (Amersham Pharmacia Biotech AB) in a MegaBace sequencer. A few direct amplifications from roots appeared to contain several PCR products and were cloned before sequencing with the TOPO-TA Cloning® Kit (Invitrogen Corp.). Both strands were sequenced, aligned with Clustal W and edited by eye using Bioedit. Sequence alignments were deposited in TreeBASE (http://www.treebase.org/treebase). Alignment numbers SN2955-12168, SN2955-12169 and SN2955-12167 correspond respectively with phylogeny in FIGS. 2, 3 and 4. Phylogenetic relationships were estimated with parsimony analysis in PAUP (Version 4.0b10, Swofford 2001). A heuristic search was done with tree bisection reconnection (TBR) as the branch-swapping algorithm. All characters were equally weighted and unordered, and 100 trees were retained during analysis. Because ITS nrDNA is highly variable across fungal groups all the analyses were repeated with only the 5.8S gene; the same topology was obtained. Bootstrap values were obtained based on 1000 replicates with 50% majority rule.
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Vanilla seed germination assay.— –
Fungi representative of the diversity of mycorrhizal fungi in Vanilla and other orchids in Puerto Rico were compared for their ability to stimulate germination and plant growth in Vanilla. Thirteen treatments were used for the seed germination assay: five Ceratobasidium (AP-79a, AP-79b, NB-09d, NB-09e, and NB-09f) and two Tulasnella (AP-7a and PB-01) isolates from Vanilla, three Ceratobasidium isolates from Puerto Rican epiphytic orchids (TO-131, TO-156 and TO-160, Otero et al 2002, 2005) and three different controls. Fungi were plated on cellulose agar (Otero et al 2004) with antibiotic (50 µg/mL each penicillin and streptomycin). This medium contains cellulose as a sole carbon source, which is available to fungi but not to orchids. Controls included cellulose agar plus antibiotic (Control C+A), cellulose agar plus antibiotic and fungicide (35 µg/mL benomyl, Control C+A+F) and Knudson C modified orchid medium (a standard medium for asymbiotic germination of orchid seeds; Sigma Cell Culture) plus antibiotic and fungicide (Knudson A+F). None of the controls plates were inoculated with fungi. Lugo (1955) reported asymbiotic germination of Vanilla seeds with Knudson C medium. The antibiotics and fungicide were necessary to control growth of endophytes and contaminants in control plates without fungi. Vanilla seeds were disinfected with ethanol 70% for 1 min, sodium hypochlorite 2% for 5 min and washed with distilled water 3x. A suspension of seeds was added to each plate a week after inoculation with fungi (241 ± 116 seeds per plate, N ± SD). Fungal treatments and controls were replicated five times. The plates were incubated at 23 C in the dark. Percentage of seed germination was measured by observation at 100x 2 mo after sowing. Differences in seed germination among treatments were tested for significance with analysis of variance (ANOVA) with SPSS software (Chicago, Illinois). Percent seed germination was normalized by arcsine square root transformation (Zar 1999). Treatments with no germination were excluded from the analysis because their variance was zero.
Plant growth assays: Vanilla.— –
Fungi were compared for their ability to stimulate growth of sterile Vanilla plants. Eleven fungi were used for the plant growth assays: five Ceratobasidium (AP-79a, AP-79b, NB-09e, NB-09f, and NB-09g); three Tulasnella (AP-7b, PB-01, and AP-79c) isolates from Vanilla; three Ceratobasidium isolates from Puerto Rican epiphytic orchids (TO-131, TO-156 and TO-160, Otero et al 2002, 2005). Fungi first were inoculated on sterilized wheat grains. Mericloned V. planifolia x V. pompona plants were bought from Twyford Laboratorio de Plantas S.A. (Alajuela, Costa Rica). In vitro plants were used because potted and field-collected plants of Vanilla already were colonized by mycorrhizal and endophytic fungi, data not shown. Vanilla plants were sown in sterile peat and inoculated with 10 colonized wheat grains per plant. Plants were grown in the laboratory under natural and fluorescent light. The number of leaves and total length of each plant were measured every month. Twenty plants were used for each treatment, and 30 plants for uninoculated controls. Data analysis was the same as for seed germination. Before the analysis the number of leaves (a discrete variable) was normalized by a natural logarithm transformation; an integer was added to all data in each test to correct for zeroes and negative numbers (Zar 1999).
Plant growth assays: Dendrobium.— –
To determine whether differences among fungi in effect on plant growth were specific to Vanilla or more generalized to other distantly related orchids, plant growth assays were repeated with a commercial hybrid orchid. A subset of fungi was tested (PB-01, AP-79a, NB-09f, and TO-160), chosen on the basis of phylogeny, geographic diversity and effects on growth of Vanilla plants (above), including three representative mycorrhizal fungi from Vanilla (two Ceratobasidium and one Tulasnella) and one Ceratobasidium isolated from an epiphytic orchid in Puerto Rico by Otero et al (2002, 2004). Mericloned plants of Dendrobium hybrid ‘Jaquelyn Thomas’ were bought from Kiilani Gifts and Gardens (Hawaii). Erlenmeyer flasks with approximately 300 Dendrobium plants were inoculated with 10 colonized wheat grains per flask. Control flasks were inoculated with sterile wheat grains without fungi. Six flasks were inoculated per treatment. After 2 wk plants were taken out of the flasks, sown in sterile peat and kept in an outdoor greenhouse under natural light (719 ± 148 plants were sown for each treatment, N ± SD). Survival, the number of leaves and pseudobulbs of each plant were measured every month. Roots were sampled and checked for pelotons every month. Data analysis was the same as for seed germination. Before the analysis the number of leaves and number of pseudobulbs (discrete variables) were normalized with a natural logarithm transformation; an integer was added to data in each test to correct for zeroes and negative numbers and the percent survivorship were normalized by arcsine root transformation (Zar 1999).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Roots in soil and on rocks had much higher rates of colonization than roots on tree bark, supporting our hypothesis. No pelotons were found in roots of V. poitaei on tree bark. In V. planifolia bark roots also had fewer pelotons than soil roots, but a few roots on rough-barked trees were highly colonized (e.g. 60 mycorrhizal zones in 158 cm of root on bark of Guarea guidonea [Meliaceae]) (FIG. 1). Mycorrhizal roots on tree bark were flatter in cross-section than nonmycorrhizal roots, and mycorrhizae were found in the side in direct contact with the bark.
The rate of isolation of mycorrhizal fungi in all Vanilla species was low given the large number of mycorrhizal root segments that were sown. Pure cultures of basidiomycete fungi were recovered from only 1.3% of 1500 segments (TABLE I). This rate of isolation motivated the direct amplification of fungi from roots because it suggested that some of the mycorrhizal fungi might not be readily culturable.
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). Clade 1 included 15 fungi from V. poitaei and V. planifolia as well as two contaminants from roots of uninoculated V. planifolia plants in the greenhouse (GV-10 and GV-69, TABLE I). This clade also included isolates of T. bifrons, T. calospora and T. deliquescens (McCormick et al 2004), orchid mycorrhizal fungi from Singapore tentatively identified as Epulorhiza repens (Ma et al 2003) and members of clades L and G from orchid mycorrhizae from Michigan (McCormick et al 2004). Clade 2 included two isolates from V. poitaei and an Epulorhiza from Singapore (Ma et al 2003). Tulasnella was not found in V. phaentha from Costa Rica or V. aphylla from Cuba. However fewer samples of these species were processed so absence of Tulasnella might reflect sampling effort.
These two main clades were confirmed by a phylogenetic tree based on mtLSU sequences (FIG. 3). As in the ITS tree, Clade 1 included clades G and L from McCormick et al (2004) and T. calospora, the teleomorph of E. repens (Currah et al 1997a). Clade 2 was related to uncultured Tulasnella from the orchids Cypripedium fasciculatum and C. montanum (Shefferson et al 2005). However the mtLSU tree included fewer sequences from Vanilla than the ITS tree because only sequences from pure cultures were included; the primers used (MLIN3/ML6) amplified Vanilla DNA, so direct amplifications from roots were not possible.
Ceratobasidium/Thanatephorus.— –
Nine ITS sequences of fungi obtained from two Vanilla species grouped with Thanatephorus, forming a clade with R. solani AG-1, AG-2 and AG-4 (FIG. 4). These sequences were obtained from V. poitaei in Puerto Rico and V. aphylla in Cuba, all from roots in soil rather than bark (TABLE I). The only pure culture in this clade was multinucleate (CU-16), a defining characteristic of Thanatephorus (Sneh et al 1991). This fungus grew sparsely and slowly (at least with the methods used here). The other eight sequences in this clade came from direct amplifications from roots and therefore their nuclear number could not be determined.
The other 17 ITS sequences were obtained from three species of Vanilla grouped with Ceratobasidium, this including fungi from V. planifolia and V. poitaei from Puerto Rico and V. phaentha from Costa Rica. The most closely related known sequences were from Ceratobasidium AG-7 (= AG-S), AG-R (= CAG-5), and AG-5, pathogens on cucumber and Pittosporum in the USA (FIG. 4). The cultures examined were binucleate, a defining characteristic of Ceratobasidium (Sneh et al 1991).
However few branches in the tree had strong bootstrap support because the CeTh primer pair used amplifies a shorter product than the standard primers ITS1/4. Standard ITS and mtLSU primers could not be used because most of the samples were direct amplifications and the primers amplified plant DNA, as explained above. In addition the mtLSU product from Ceratobasidium isolates was extremely variable (with introns of 500–1500 bp) which made sequencing and analysis difficult.
Specificity.— –
Three genera of mycorrhizal fungi were found in Vanilla: Ceratobasidium, Thanatephorus and Tulasnella (FIGS. 2, 4; TABLE I). We tested for differences in relative frequencies of these genera in V. planifolia vs. V. poitaei (similar number of samples were processed for both species), in roots from soil vs. roots from bark, and in DNA from pure cultures vs. direct amplifications.
Ceratobasidium and Tulasnella were found in both V. planifolia and V. poitaei, but Thanatephorus was found only in V. poitaei. Differences in relative frequencies of fungal genera were significant (exact contingency test, P < 0.001; only data from direct amplifications were included in the test and only V. planifolia and V. poitaei were included because sample sizes were similar, but results were similar when all data were included). However several clades of Ceratobasidium and Tulasnella included fungi from both orchids. Thanatephorus was the only genus found in V. aphylla, and Ceratobasidium was the only genus found in V. phaentha, perhaps because sampling of these species was limited. However because each orchid species was sampled in a different site, these differences might reflect differences in soil type, rainfall, distribution of fungi or other factors unrelated to differences in preference among orchids.
Fungi from roots in soil vs. bark were compared for V. planifolia. (V. poitaei was not included because no mycorrhizae were found in its bark roots, FIG. 1.) Ceratobasidium was significantly more common in roots from soil whereas Tulasnella was significantly more common in roots from bark (Fisher’s exact test, P = 0.05; for consistency only data from direct amplifications were included in the test). This was the opposite of the hypothesized distribution. However most clades of Ceratobasidium and Tulasnella included closely related fungi from soil and from bark. Significantly different fungal flora were revealed by culturing vs. direct amplification (exact contingency table, P = 0.03; only fungi from Puerto Rico were included). All strains of Thanatephorus from V. poitaei grew poorly or not at all in the culture media used and would have been overlooked if culturing alone was used.
Seed germination assays: Vanilla.— –
Five of eight isolates of Ceratobasidium promoted germination of Vanilla seeds (FIG. 5). Germinated seeds had swollen embryos, broken testae and rhizoids. Ceratobasidium isolates from the epiphytic orchid Ionopsis utricularioides stimulated significantly higher germination than those from Vanilla, as hypothesized (P = 0.018). Seeds inoculated with Tulasnella, three isolates of Ceratobasidium and controls without fungi (C+A, C+A+F and K+A+F) did not germinate. The three Ceratobasidium isolates that did not stimulate germination had similar colony morphology and were in the same clade (AP-79a, AP-79b and NB-09d; FIG. 4). No isolates of Thanatephorus were included because their growth in culture was too sparse and slow to cover petri plates.
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; TABLE II). Ceratobasidium isolates AP-79a and AP-79b had a significant, negative effect on stem length and number of leaves compared to controls and other Ceratobasidium isolates (Tukey’s test, P < 0.05). Some Ceratobasidium isolates, including the three isolates from Ionopsis utricularioides, had a significant, positive effect on growth. However these isolates were not significantly higher than uninoculated controls. Isolates from the same root differed significantly in some cases (NB-09g vs. NB-09e and NB-09f) (P = 0.01, FIG. 6A).
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0.01; FIG. 6C, D; TABLE II). Plants inoculated with Ceratobasidium AP-79b had significantly shorter stems and leaves than plants inoculated with other Ceratobasidium (Tukey’s test; P < 0.01). Ceratobasidium NB-09g and TO-156 stimulated both plant length and number of leaves, but differences from controls were not significant.
Plant growth assay: Dendrobium.— –
Two isolates of Ceratobasidium (AP-79a and NB-09f) caused significant decreases in plant survivorship and number of leaves 2 wk after inoculation in flasks (P < 0.001; FIG. 7A, B). Most of the plants from these treatments were dead after a month, so they could not be included in further assays (FIG. 7C, D). Tulasnella PB-01 and Ceratobasidium TO-160 did not kill any plants, but TO-160 reduced the number of leaves compared to controls (P < 0.001, FIG. 7B); the effect was most notable on smaller plants.
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). The increase with PB-01 was significantly smaller than with TO-160 (P < 0.001). Plants inoculated with TO-160 also had significantly more pseudobulbs than controls (P = 0.012, FIG. 7D). However, although control plants were free of pelotons 3 mo after inoculation, pelotons were observed after 5 mo; at this point, the control plants were no longer mycorrhiza-free, so the experiment was ended. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Host tree species might have influenced colonization of roots on bark. Roots of V. poitaei on bark had no mycorrhizae at all. These results are consistent with those of Porras and Bayman (2003). Young Vanilla plants often start growing in the canopy and roots may not reach to the soil for months or years. In this sense Vanilla is more typical of epiphytic orchids than terrestrial orchids, although it occupies both niches. It is common to see plants hanging draped over tree branches with no roots attached to soil or bark. This suggests that adult Vanilla plants are facultative for mycorrhizae. In fact most of the pelotons found in roots on both substrates were degraded (Porras and Bayman 2003).
Tulasnella.— –
Tulasnella Clade 1 sequences from Vanilla were almost identical to sequences from orchid mycorrhizal fungi in Singapore (Ma et al 2003) and in Michigan (McCormick et al 2004; FIG. 2). This similarity is remarkable considering that ribosomal gene sequences in Tulasnella appear to evolve rapidly (Bidartondo et al 2003). The same narrow clade of Tulasnella is associated with orchids worldwide. A similar pattern of distribution was seen in Ceratobasidium strains associated with the epiphytic orchid Ionopsis utricularioides: a single clade was isolated from plants from Trinidad to Peru ( J.T. Otero pers comm). The fact that a single clade of fungi is associated with different orchids over a large area should be helpful for incorporating mycorrhizae in conservation strategies.
Uninoculated Dendrobium plants from seedling flasks were colonized within 5 mo of planting in sterilized potting mix in the greenhouse, which suggests that spores of Tulasnella are common as windborne or rainborne inoculum; however, little is known about dispersal of Tulasnella.
Tulasnella has been reported as mycorrhizal in both epiphytic orchids and terrestrial orchids in the tropics (Currah et al 1997a, Pereira et al 2005, Ma et al 2003). In V. planifolia however Tulasnella was significantly more frequent in roots on tree bark than in roots on soil. Tulasnella is generally described as saprobic (Alexopoulos et al 1996), but several Tulasnella species were shown to form ectomycorrhizae with birch and pine in Europe (Bidartondo et al 2003). 14CO2 was transferred from birch seedlings to another plant, Cryptothallus mirabilis, via Tulasnella, demonstrating a functional ectomycorrhizal relationship in which Cryptothallus uses a shared mycorrhizal partner to parasitize its neighbors (Bidartondo et al 2003). However, the forests where we collected Vanilla have few ectomycorrhizal plants, so the relationship demonstrated by Bidartondo et al is unlikely to exist here, even if the Tulasnella we found are capable of forming ectomycorrhizae. The ecological range of these fungi must be wide because they occur both in soil and on tree bark.
Clade 2 was closely related to fungi from orchids in Singapore (Ma et al 2003; FIG. 2) and more distantly to fungi from Cypripedium in USA (Shefferson et al 2005; FIG. 3). Both ITS and mtLSU trees show this clade outside described Tulasnella species, so it is possible that they belong to another genus. Relatively few sequences from identified Tulasnella species are available in GenBank despite the large number of described species, which makes identification based on DNA sequences difficult (Kristiansen et al 2001, Ma et al 2003, Bidartondo et al 2003). The ITS region is highly variable and provides greater taxonomic resolution than the coding regions of the mtLSU (Anderson and Cairney 2004). This difference in the rate of sequence divergence may explain the differences observed in the phylogenetic trees based on ITS and mtLSU.
Ceratobasidium/Thanatephorus.— –
Overall relationships (FIG. 4) agree with the ITS-based phylogeny of Gonzalez et al (2001). The relationship between Ceratobasidium and Thanatephorus (FIG. 4) also agrees with the observation of Gonzalez et al (2001) that the two genera are not adequately delimited and that Ceratobasidium is paraphyletic. Resolving these genera will require use of other genes and is beyond the scope of this study. Nuclear counts agreed with the phylogenetic tree: Isolates assigned to Thanatephorous were multinucleate and those assigned to Ceratobasidium were binucleate (Sneh et al 1991). However uni- vs. binucleate Ceratobasidium isolates from epiphytic orchids can be closely related (Otero et al 2002) suggesting that nuclear number can be misleading as a phylogenetic character.
The nine sequences assigned to Thanatephorus were from fungi associated with V. poitaei in Cambalache State Forest, Puerto Rico, and V. aphylla in Peninsula Guanahacabibes, Cuba. Both these sites have sparse, limestone-derived soils that do not retain water. The growth in culture of this clade was limited or entirely absent; eight of nine of these sequences were from direct amplifications. This limited growth was consistent on various culture media and was not improved by addition of vitamins (including thiamine) or cold-sterilized plant extracts. Thanatephorus includes important, well studied plant pathogens (Sneh et al 1991, Gonzalez et al 2001); we know of no others so recalcitrant to growth in culture.
Ceratobasidium sequences came mostly from Vanilla roots in soil, but a few Ceratobasidium strains were found in roots on tree bark (FIG. 4, TABLE I). The most related sequences in GenBank were from plant pathogens from USA (CAG-7 and CAG-R). It is not known whether the Vanilla mycorrhizal species are saprotrophs or pathogens. These results suggest that tropical orchids form mycorrhizae with a diverse assemblage of Ceratobasidium strains and that their phylogeny merits further work.
Culturing vs. direct amplification.— –
We found different groups of fungi using culturing vs. direct amplification, and the differences in frequencies were significant. In particular the use of specific primers let us detect a clade of Thanatephorus that did not grow or had limited growth in culture. On the other hand Clade 2 of Tulasnella was composed of two fungi from Vanilla and one from Singapore, all from pure cultures (FIG. 2; see Ma et al 2003). This also has been demonstrated for ericoid mycorrhizal fungi; Sebacina was the most common group identified from direct amplifications from roots, whereas Capronia sp. and Hymenoscyphus erica (Ascomycota) were the most common fungi when cultured (Allen et al 2003). Either technique reveals a subset of the fungi present and a combined approach provides a closer approximation of overall diversity. However both techniques can be biased toward specific fungal groups depending on the culture conditions, primers, DNA and PCR protocols.
Other studies have suggested that diversity of orchid mycorrhizal fungi might be higher than that reported from culture studies alone (Kristiansen et al 2001); our data support this conclusion and provide statistical support. The new primers described here should help facilitate discovery of new clades in Ceratobasidium, Thanatephorus and Tulasnella because they are more specific for these fungi and less likely to amplify plant and other fungal DNA than the ITS primers most commonly used (Bruns et al 1998). However these primers were designed based on available sequences for specific groups of fungi. There might have been other mycorrhizal fungi in Vanilla roots that these primers did not amplify. It therefore is likely that mycorrhizal fungi of Vanilla are even more diverse than we report here. The choice of primers inevitably prejudice results against certain groups; for instance some Tulasnella do not amplify with the universal fungal primer ITS1 (Bidartondo et al 2003).
Symbiotic seed germination.— –
Some Ceratobasidium isolates promoted germination of Vanilla seeds, but the Tulasnella isolates AP-7a and PB-01 did not (FIG. 1). This supports the observation of Zelmer and Currah (1997) that both Ceratorhiza goodyeraerepentis (= Ceratobasidium) and Epulorhiza repens (= Tulasnella) were isolated from adult plants of Spiranthes lacera but only Ceratorhiza goodyeraerepentis was found in young protocorms.
Ceratobasidium isolates differed significantly in their effects on seed germination of Vanilla. A clade of Ceratobasidium isolates from Ionopsis utricularioides significantly stimulated germination in Vanilla and was more effective than isolates of Ceratobasidium from Vanilla itself. This clade also promoted germination in Ionopsis and another epiphytic orchid (Otero et al 2004). This clade is widespread in Puerto Rico (Otero et al 2004) and occurs throughout the Antilles and Central America (J.T. Otero pers comm) but we did not find it associated with Vanilla roots. These results agree with previous studies, which found that orchid seeds may germinate better with fungi isolated from other species of orchids than with fungi isolated from conspecific plants (Warcup 1973, McCormick et al 2004, Otero et al 2004).
It is not clear that mycorrhizal fungi found in adult plants are the same fungi necessary for seed germination. Orchid seed germination is difficult to study in the field because of the minute size of the seeds (Rasmussen and Whigham 1993). Symbiotic germination experiments might help demonstrate that fungi isolated from adult plants (or fungi from other sources) are potentially mycorrhizal (McCormick et al 2004, Otero et al 2004, 2005). However this evidence is not definitive because orchid seeds can germinate with a wider range of fungi in vitro than they actually associate with in the field (Smreciu and Currah 1989, Masuhara and Katsuya 1994).
Propagation of Vanilla is mostly clonal, and the commercial Vanilla industry depends on a limited number of cultivars and hybrids (Soto-Arenas 2003, 2006). The recalcitrant germination of Vanilla seeds is an obstacle to the development of new clones and hybrids. The ability of certain Ceratobasidium isolates to stimulate seed germination, demonstrated here, has the potential to be useful for in vitro germination in the development of new cultivars. However, additional experiments are necessary to test for pathogenicity under different environmental and nutritional conditions in Vanilla and other plants.
Symbiotic plant growth.— –
Different isolates had negative, positive or neutral effects on plant growth and survival (FIGS. 6, 7). Related isolates often had similar effects on growth. However in a few cases closely related fungi had significantly different effects on growth of Vanilla (e.g. NB-09f and NB-09g). Differences among fungi in effects on Dendrobium plants (FIG. 7) were similar to differences among fungi on Vanilla (FIG. 6) suggesting that results of symbiotic growth tests on one orchid might be predictive of effects on other orchids as well.
Some isolates of Tulasnella and Ceratobasidium caused no mortality in flasks of mericloned plantlets of Dendrobium (FIG. 7A) despite the fact that the flasks contained nutrient agar, sterile plants and 100% humidity, conditions conducive to fungal growth and pathogenicity. Plants with these fungi (TO-160 and PB-01) had significantly more leaves and pseudobulbs than uninoculated control plants after 5 mo growth (FIG. 7C, D). The low pathogenicity and specificity suggests that these fungi could be used to reintroduce endangered orchids and to improve commercial cultivation.
Differences among mycorrhizal fungi in effects on Vanilla were less pronounced after 2 mo than after 1 mo (FIG. 6). This suggests that Vanilla plants might acquire the ability to control the parasitic behavior of some of the fungi (e.g. FIG. 6, AP-79a, AP-79b, NB-09e, NB-09f). Rhizoctonia was more pathogenic on Vanilla cuttings with one node than on bigger plants (Alconero 1969).
Specificity of Vanilla for mycorrhizal fungi.— – V. poitaei associated with a wide range of mycorrhizal fungi, Tulasnella, Ceratobasidium and Thanatephorus, in a single site. V. planifolia associated with almost as wide a range, as hypothesized. The other two Vanilla species associated with only one genus each, but this was probably due to the fact that sampling was not extensive and in only one site per species. Seeds of Vanilla germinated more readily when inoculated with fungi from other orchids than with fungi isolated from Vanilla, further evidence of a lack of specificity.
Similarly the fungi showed little specificity for the orchids. Although V. planifolia and V. poitaei differed significantly in the relative frequencies of the three genera, nearly identical ITS and mtLSU sequences often were found in fungi from different species. Closely related fungi were reported from different orchids in Europe, USA and Asia.
Terrestrial orchids in temperate areas vary widely in mycorrhizal specificity. Spiranthes lacera in Manitoba associated with Tulasnella and Ceratobasidium (Zelmer and Currah 1997), a range comparable to V. planifolia and V. poitaei. However Liparis lilifolia and Tipularia discolor, photosynthetic terrestrial orchids in USA, were far more specific (McCormick et al 2004).
Mycorrhizal fungi of Vanilla differed among sites. Thanatephorus Clade 1 (FIG. 4) included principally uncultured fungi isolated from V. poitaei from Cambalache (Puerto Rico) and V. aphylla from Guanahacabibes (Cuba). Cambalache is a subtropical moist forest with karst formations and 1479 mm annual rainfall2, and Guanahacabibes is a dry tropical forest with karst formations and 1333 mm annual rainfall3. In comparison sites where Thanatephorus was not found (El Verde and Sabana in Puerto Rico) have an annual rainfall above 3000 mm (Ramírez and Meléndez-Colon 2003). These results suggest that the unculturable Thanatephorus (Clade 1, FIG. 4) might be limited to relatively dry sites.
A single Vanilla plant can have roots in both epiphytic and terrestrial niches. This characteristic let us study specificity for substrate. Relative frequency of Ceratobasidium and Tulasnella was significantly different in V. planifolia roots from soil and roots from bark. However it is remarkable that similar fungi were found in roots on bark and in soil because they are very different environments. This suggests that distribution of the fungi studied, particularly Tulasnella, is poorly understood.
What are the advantages for Vanilla to be generalists for mycorrhizal fungi? An orchid associated with a wide range of mycorrhizal fungi increases its chance of success on the germination of its seeds (Warcup 1981, Rasmussen 2002). Considering that soil does not have continuous supply of sugars, amino acids and vitamins (Smith and Read 2002), a nonspecific symbiosis may allow an orchid to exploit more efficiently the soil resources and the variations in their availability. Vanilla is pantropical with 100 species (Ackerman 1995). It is possible that being generalists for their mycorrhizal fungi help Vanilla species to grow in a variety of conditions. Taylor et al (2003) suggest that mycorrhizae have contributed to the evolutionary diversification of the Orchidaceae. However the importance of these fungi for orchid diversification and their role in orchid distribution is still poorly understood.
Studies have detected multiple fungi in a single root, or even a single peloton (Hadley 1970, Zelmer et al 1996, Zelmer and Currah 1997, Kristiansen et al 2001, Taylor et al 2003). However, here we also show that two fungi from a single root can be functionally different (NB-09g vs. NB-09e and NB-09f, FIG. 6). Multiple colonization of an orchid by different mycorrhizal fungi increases the complexity of the interaction, but so far no experiments have tested the effects of mixed infections. It is possible that different groups of mycorrhizal fungi serve different functions for the plant.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Department of Biology, Herbarium UPR-Mayagüez. (http://www.uprm.edu/biology/profs/breckon/herbarium/cambala.html). 
3 Centro de Investigaciones y Servicios Ambientales, ECOVIDA, Cuba (http://www.ecovida.pinar.cu/Png/caractgeneral.html).
1 Corresponding author. Current address: Biology Department, University of New Mexico, MSC03 2020, Albuquerque, NM 87131. E-mail: aporras{at}unm.edu
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LITERATURE CITED |
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