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. 2012 Dec 28;111(3):409–418. doi: 10.1093/aob/mcs294

Mycorrhizal preference promotes habitat invasion by a native Australian orchid: Microtis media

Jonathan R De Long 1,2, Nigel D Swarts 3,*, Kingsley W Dixon 2, Louise M Egerton-Warburton 1,4
PMCID: PMC3579446  PMID: 23275632

Abstract

Background and Aims

Mycorrhizal specialization has been shown to limit recruitment capacity in orchids, but an increasing number of orchids are being documented as invasive or weed-like. The reasons for this proliferation were examined by investigating mycorrhizal fungi and edaphic correlates of Microtis media, an Australian terrestrial orchid that is an aggressive ecosystem and horticultural weed.

Methods

Molecular identification of fungi cultivated from M. media pelotons, symbiotic in vitro M. media seed germination assays, ex situ fungal baiting of M. media and co-occurring orchid taxa (Caladenia arenicola, Pterostylis sanguinea and Diuris magnifica) and soil physical and chemical analyses were undertaken.

Key Results

It was found that: (1) M. media associates with a broad taxonomic spectrum of mycobionts including Piriformospora indica, Sebacina vermifera, Tulasnella calospora and Ceratobasidium sp.; (2) germination efficacy of mycorrhizal isolates was greater for fungi isolated from plants in disturbed than in natural habitats; (3) a higher percentage of M. media seeds germinate than D. magnifica, P. sanguinea or C. arenicola seeds when incubated with soil from M. media roots; and (4) M. media–mycorrhizal fungal associations show an unusual breadth of habitat tolerance, especially for soil phosphorus (P) fertility.

Conclusions

The findings in M. media support the idea that invasive terrestrial orchids may associate with a diversity of fungi that are widespread and common, enhance seed germination in the host plant but not co-occurring orchid species and tolerate a range of habitats. These traits may provide the weedy orchid with a competitive advantage over co-occurring orchid species. If so, invasive orchids are likely to become more broadly distributed and increasingly colonize novel habitats.

Keywords: Terrestrial orchid, mycorrhizal fungi, disturbed habitats, south-western Australia, invasive species, Microtis media

INTRODUCTION

Orchidaceae are well known for their rarity and specialization to certain biomes and habitats (Linder, 1995; Shefferson et al., 2008), pollinators (e.g. Cozzolino et al., 2004) and mycorrhizal fungal associates (e.g. Taylor and Bruns, 1997; McCormick et al., 2004; Shefferson et al., 2007). However, an increasing number of orchids are exhibiting weed-like attributes, e.g. they are disturbance-tolerant and readily proliferate in novel environments. Over 90 orchid species are currently listed as weed-like or naturalized plants (Ackerman, 2007), but orchids are rarely the focus of invasive species studies. We examined the roles of mycorrhizal fungi and edaphic factors in the weediness of a terrestrial orchid, Microtis media.

Terrestrial orchids are notable for their obligate, often specific, relationship with mycorrhizal fungi, making them a useful test for the hypothesis that mycorrhizal fungi may be involved in their invasive capacity. All orchids investigated to date demonstrate an obligate relationship with mycorrhizal fungi for seed germination and progression through an achlorophyllous protocorm stage to the seedling stage (Rasmussen, 1995, 2002). After this time, most orchid species establish photosynthesis but may remain reliant on mycorrhizal fungi for carbon and nutrients to varying degrees.

Seed germination and protocorm development often require specific mycorrhizal fungal species (Warcup, 1971, 1973; Taylor et al., 2002). Specificity is defined here as the phylogenetic breadth of the mycorrhizal associations of a particular orchid. DNA sequence data have shown that orchid mycorrhizas are formed with a relatively narrow phylogenetic breadth of fungi in comparison with the mycorrhizas of photosynthetic plants. Most of the fungi are members of Basidiomycota (Rasmussen, 1995; Taylor et al., 2002) belonging to the anamorphic form genus Rhizoctonia (Rasmussen, 2002), with teleomorphs corresponding to families Tulasnellaceae, Sebacinaceae and Ceratobasidiaceae (Taylor et al., 2002). Mycorrhizas may also be formed with Ascomycota (e.g. Tuber; Selosse et al., 2004; Shefferson et al., 2005) and typically ectomycorrhizal taxa such as Russulaceae (Taylor and Bruns, 1997, 1999; McKendrick et al., 2000; Taylor et al., 2002). However, not all fungi are equally effective at supporting germination and progression beyond the seedling stage (Otero et al., 2005) and some orchids will utilize different mycorrhizal fungi at different life-history stages (Bidartondo and Read, 2008).

Patterns of symbiotic germination success are also affected indirectly by edaphic factors that contribute to the local availability and abundance of mycorrhizal fungi. Although less well documented, factors such as soil moisture levels (McCormick et al., 2006), soil type (Ogura-Tsujita and Yukawa, 2008; Phillips et al., 2011), organic matter content (Bowles et al., 2005; McCormick et al., 2012), and pH and nutrients (Batty et al., 2001; Bowles et al., 2005) can influence the abundance and diversity of mycorrhizal fungi capable of supporting seed germination and development. For example, increasing levels of soil moisture and organic content have been correlated with greater germination success (Diez, 2007).

Less attention has been paid to the inevitable corollaries, i.e. do weed-like orchids form novel mycorrhizal associations to avoid the loss of mycobionts, utilize a diverse range of fungi that are widespread and/or utilize fungi with broad edaphic amplitudes? Although evidence to support these possibilities is still limited, recent analyses have shown that weed-like orchids form mycorrhizas with widespread fungi (e.g. Epipactis helleborine and Pezizales such as Wilcoxina and Tuber; Ogura-Tsujita and Yukawa, 2008) or exploit a spectrum of fungi (Disa bracteata; Bonnardeaux et al., 2007). In addition, weed-like orchids may show an unusual breadth of habitat tolerance (e.g. Oeceoclades; Cohen and Ackerman, 2009).

Here, the overarching question we ask is: what role(s) might mycorrhizal specificity and edaphic factors play in the distribution of the weed-like orchid Microtis media? This species has been described as weed-like due to its ability to rapidly colonize and proliferate in a wide range of habitats. Over the last 30 years, M. media has been appearing opportunistically in terrestrial orchid collections, horticultural beds, disturbed roadsides and bushlands in south-western Australia and is now established in New Zealand, south-east Asia, Japan and China.

In this study, we used molecular identification of individual pelotons isolated from M. media roots, symbiotic M. media seed germination assays, ex situ fungal baiting and soil analyses to address three hypotheses: (1) M. media is a generalist and forms mycorrhizal associations with a diversity of fungi across its geographical and habitat range; (2) these generalist mycorrhizal fungi significantly enhance the germination of M. media seed but not those of co-occurring orchid species; and (3) the distribution of M. media mycorrhizal fungi can be correlated with environmental variables.

METHODS

Species

Microtis media is a member of the onion orchid alliance, comprising three genera (Hydrorchis, Microtidium and Microtis). It is a terrestrial herbaceous perennial, clonal and morphologically variable species with leaves 250–650 mm long and 5–8 mm wide and inflorescences 200–560 mm long (Hoffman and Brown, 1998). Microtis media produces a spike with 10–100 self-pollinated flowers resulting in near 100 % flower–fruit conversion and the development of many thousands of wind-dispersed seeds. The seed germinates prolifically under horticultural conditions (Fig. 1A) producing a flowering plant in 6–8 months, thereby representing one of the most quickly maturing terrestrial orchids.

Fig. 1.

Fig. 1.

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(A) Site colonization by M. media; (B) Australia continental view of M. media sample sites; (C) symbiotic germination of M. media with fungus from horticultural beds; (D) ex situ baiting.

Study sites and specimen collection

Microtis media is found in a range of habitats throughout the south-west biodiversity hotspot in Western Australia. This region is recognized for its remarkable diversity and endemism, and the principal determinant of vegetation structure is the nature and nutrient status of the substrate and absolute amount of precipitation. Refugial species persist in higher rainfall areas, whereas fragmented relictual species and suites of newly derived taxa occur in the drier areas (Hopper and Gioia, 2004). Microtis media plants were sampled from sites within the geographical limits of the species as currently known: north to Chapman (–28·82, 114·67); south to Walpole (–35·0, 116·66); and from the west coast to Newman Rock in the east (–32·16, 123·33; Fig. 1B). Five plants were collected from each of 30 sites within the distribution (Supplementary Data Table S1). Microtis media plants were also sampled from four habitats in Kings Park and Botanic Garden (KPBG): high-quality bushland, disturbed bushland, anthropogenically altered (i.e. horticultural beds, wood chip/organic litter covered walkways) and vagrant plants found in glasshouse potted plants. Whole plant samples of M. media were excavated from the soil using a hand trowel, with care taken to remove as much of the root and tuber system as possible. Upon collection, samples were gently rinsed under tap water and above- and below-ground structures were separated and weighed. Below-ground structures were wrapped in damp paper towels and stored at 5 °C (<5 d) until peloton isolation. Above-ground structures were placed in paper bags, dried at ambient temperature (23 °C) and weighed.

Peloton isolation

To obtain mycorrhizal cultures for seed germination assays and DNA sequencing, the single peloton isolation method following Rasmussen (1995) was performed under sterile conditions. Three plants per site were selected at random for the isolation of pelotons. Root structures were washed with tap water, rinsed three times with sterile deionized water to remove surface contaminants and, under sterile conditions in a lamina flow cabinet, abraded with a scalpel to release the fungal pelotons into a pool of sterile deionized water. Pelotons were rinsed in three 450-μL pools of sterile deionized water and subsequently plated onto soil solution equivalent plates (SSE; Brundrett et al., 2003). Plates were sealed with Parafilm®, stored in airtight plastic bags and incubated at 21 °C with 24-h fluorescent lighting (ambient conditions in the laboratory) for 4–8 d. Hyphal tips were subcultured onto fresh SSE plates for an additional 4–8 d, after which successful cultures were sub-cultured (hyphal tips) onto potato dextrose agar (PDA) plates.

DNA extraction, PCR amplification and sequencing

A representative selection of cultures isolated from habitat types and wild sites were used for DNA sequencing. Approximately 2–5 mg of hyphae was scraped from the surface of single PDA plates under sterile conditions, and DNA extracted using the protocols of Gardes et al. (1991) and Halász et al. (2005). Following extraction, the nuclear large subunit (nLSU) of the ribosomal DNA (approx. 900 bp) was amplified for each DNA sample by PCR using primer LROR (5′-ACCCGCTGAACTTAAGC-3′) with either LR5 (5′-TCCTGAGGGAAACTTCG-3′) or LR7 (5′-TACTACCACCAAGATCT-3′). PCR reactions were performed using the following programme: 95 °C for 3 min; 40 cycles of 95 °C for 35 s, 47 °C for 1 min and 72 °C for 2 min; and 72 °C for 7 min. Successfully amplified PCR products were purified using an Agentcourt AMPure PCR clean-up kit or QIAquick® PCR Purification Kit prior following the manufacturers' directions. Purified samples were prepared for sequencing on a Beckman-Coulter CEQ 880 Genetic Analysis System or Applied Biosystems (ABI) 377XL Prism DNA Sequencer. Sequencing reactions were performed using primer LROR with either LR5 or LR7 for the nLSU. BLAST searches (www.ncbi.nlm.nih.gov/BLAST) were conducted on all the sequences to determine the closest known relative. Sequences were edited using Aligner v.2·0.4 ©2002–2007 (Codon Code Inc., Dedham, MA, USA). Samples with excess background noise or unclear signal readout were re-sequenced. Sequences were then auto-aligned using Mult-Alin (http://bioinfo.genotoul.fr/multalin/) to obtain an initial alignment. The sequence alignment was then manually edited using Se-Al Version 2·0a11. The sequence matrix was analysed with PAUP version 4·0 (Swofford, 2002). A midpoint rooting was utilized as opposed to an outgroup because the objective of the study was to compare mycorrhizal fungal composition between habitat types rather than determine phylogenetic relationships among fungal species. A bootstrap analysis was conducted with 100 replicates of 100 heuristic searches in order to test the statistical strength of the phylogenetic trees created.

Ex situ baiting and scoring

We used ex situ seed baiting to investigate mycorrhizal distribution/germination in soils collected from a variety of habitats. The methods are described in detail in Brundrett et al. (2003). Briefly, sterile Sarstedt microcosms (each 100 × 100 × 20 mm) were packed with soil (sieved to 2 mm) collected from each habitat (Fig. 1D). The soil was moistened with sterile deionized water and covered with a Millipore vinyl membrane before adding seed that had been dried upon collection at 15 °C and 15 % relative humidity for 2 weeks and in storage at –20 °C for one year. Species used were M. media and three co-occurring orchid species with different mycorrhizal colonization patterns, Caladenia arenicola (colonization within the stem collar), Diuris magnifica (within the root) and Pterostylis sanguinea (throughout the underground stem) (Ramsay et al., 1986), which were attached to 3 × 3 mm squares of Millipore™ filter (0·45 µm). Sixteen squares were placed in each microcosm, comprising seven with M. media seeds, and three each with P. sanguinea, C. arenicola and D. magnifica seeds. A four by four matrix was laid out within each microcosm on the surface of the Millipore™ vinyl with samples positioned according to numbers generated randomly in the program R®. Each microcosm was covered with its own plastic top, and stored in near total darkness at a constant temperature of 23 °C for 7 weeks. Microcosms were monitored weekly for desiccation, with deionized water being added as appropriate. The microcosms were then incubated under fluorescent light for 1 week, after which seed development was scored and assigned a rank on the following scale: 0 = ungerminated; 1 = splits in testa; 2 = initial protocorm development; 3 = trichomes present and swollen embryo; 4 = leaf primordium present; 5 = leaf present (Ramsay et al., 1986). Seeds reaching Stage 3 and beyond were considered to have successfully germinated.

Symbiotic germination trials

Symbiotic germination trials were conducted using M. media seed collected and stored at KPBG as above. Seeds were encased in a small pouch consisting of Millipore™ mesh (0·45 µm) and surface sterilized in 4 % (w/v) bleach solution (calcium hypochlorite) followed by three rinses in rinsed sterile water. Seeds were then removed from pouches and aseptically transferred to Petri dishes containing oatmeal agar (OMA: 5 g crushed oats, 2 L reverse osmosis water and 16 g sugar; pH adjusted to 5·5 before autoclaving for 20 min at 121 °C at 1·05 kg cm−2/15–20 p.s.i) inoculated with a single mycorrhizal fungal isolate. Control OMA plates (without fungi) and asymbiotic nutrient media plates were established to control for the influence of fungi and nutrients, respectively. Plates were wrapped in aluminium foil and incubated at a constant temperature of 21 °C for 3 weeks. Plates were checked on a weekly basis to monitor contamination. Plates were then unwrapped and transferred to a fluorescently lit growing room with 24 h of light for a further 5 weeks and then scored for seed germination using the same categories as ex situ baiting and scoring (Fig. 1C).

Root clearing and staining

Root samples to be scored for peloton colonization were stored in 50 % ethanol at 4 °C until processed (Grace and Stribley, 1991). Root clearing and staining followed a modified version of the protocol outlined by Koske and Gemma (1989), and samples were stored in acidic glycerol until scoring. Stained samples were placed on microscope slides in random horizontal/vertical patterns and peloton colonization was determined in 30 random microscope fields using the magnified line intersect method (McGonigle et al., 1990).

Soil analysis

Soil samples were analysed by CSBP Limited Fertilizers (Western Australia) for texture, gravel content, plant-available ammonium, nitrate, phosphorus, potassium, sulphur, reactive iron, total nitrogen, total carbon, electrical conductivity, pH, CaCl2 and H2O content, and organic carbon using standard chemical soil analyses.

Statistical analyses

Statistical analyses were conducted in JMP Version 5·1.2. All data sets were checked for normal distribution and homoscedastic tendencies before analyses were undertaken. All percentages from ex situ baiting and symbiotic germination trials were arcsin transformed before analysis; re-transformed data are presented in the Results. Differences in the percentage of root colonization, root and shoot biomass, seed germination and each soil nutrient were analysed between sites using one-way ANOVA, followed by post-hoc Tukey's honestly significant difference (HSD) test for significant variables (P < 0·05).

A Mantel test was used to test whether mycorrhizal composition among locations (species dissimilarity) could be explained by abiotic factors (soil pH, nutrients) or spatially structured (geographical) residual variability (Smouse et al., 1986; Legendre and Fortin, 1989; Fortin and Gurevitch, 1993). Three distance matrices were constructed. The genetic matrix was constructed for each pair-wise combination of sites using the dissimilarity distance based on the phylogenetic data. The abiotic matrix was constructed for each pair-wise combination of sites using the raw soil data and Euclidean distance and the geographical distance matrix was calculated as the linear distance (km) from one site to another.

RESULTS

Diversity of endophytic fungi

Molecular typing of orchid endophytic fungi recovered potentially 11 taxa in M. media root systems based on nLSU sequences, all of which could be identified to genus and/or species level based on publicly available and closely related sequences (Table 1). BLAST searches indicated that two genera represented the diversity of endophytic fungi in M. media (Table 1, Fig. 2). Most sequences from Microtis media endophytic fungi were most closely related to Sebacina vermifera (sensu Warcup and Talbot, 1967; GenBank accession numbers EU626000·1, EU625994·1) and Piriformospora indica (GenBank accession number AY505557·1), both of which belong to Sebacinaceae. These searches also supported the identification of Tulasnella calospora (Tulasnellaceae) and a single genotype of Ceratobasidium sp. (Ceratobasidiaceae). Further, we identified genetic diversity within each endophytic fungal genus. Four genotypes were recognized in Sebacina vermifera, and three in Tulasnella calospora, and sequence variation (pair-wise distances) in both species was >3 %. Even though the number of endophytic isolates examined was limited, these results suggest that a considerable taxonomic breadth of the mycobiont may occur within a single genus.

Table 1.

Identification of M. media mycorrhizal fungi based on BLAST search results

Habitat Sample Two closest sequences found in GenBank by BLAST analysis Accession number Percent identity Alignment length (bp)
Bushland EE-4 Piriformospora indica AY505557·1 95 1908
Chaetospermum camelliae EF589738·1 94 1829
GG6-2B Tulasnella calospora DQ388045·1 91 1037
Uncultured Tulasnella AY298949·1 91 1033
Granite outcrop AA3-1D Sebacina vermifera EU625994·1 99 1472
Sebacina vermifera EU625993·1 99 1472
AA3-1E Sebacina vermifera EU625994·1 97 1369
Sebacina vermifera EU625993·1 97 1369
AA3-2 Sebacina vermifera EU625994·1 98 1530
Sebacina vermifera EU625993·1 98 1530
Seepage FF-1 Piriformospora indica AY505557·1 95 1831
Sebacina vermifera EU625994·1 99 1698
JJ2-D Piriformospora indica AY505557·1 97 904
Piriformospora indica AY293202·1 97 904
JJ2-C Piriformospora indica AY505557·1 97 857
Piriformospora indica AY293202·1 97 857
JJ2-D Piriformospora indica AY505557·1 98 2420
Tremellodendron sp. AY745701·1 92 1910
JJ2-E Piriformospora indica AY505557·1 98 2370
Chaetospermum artocarpi EF589735·1 92 1884
Horticultural beds E4-RE Sebacina vermifera DQ983814·1 97 782
Mycorrhizal basidiomycete EU526285·1 97 780
F2-C Sebacina vermifera EU626001·1 98 1563
Sebacina vermifera EU626000·1 98 1563
O2-B Ceratobasidium sp. AY293171·1 85 774
Ceratobasidium sp. DQ520098·1 85 763
O5-B Sebacina vermifera EU626001·1 99 1663
Sebacina vermifera EU626000·1 99 1663
P1 Piriformospora indica AY505557·1 94 1873
Sebacina vermifera EU626001·1 99 1812
Glasshouse W2 Piriformospora indica AY505557·1 93 1917
Chaetospermum artocarpi EF589735·1 92 1834
KK1 Piriformospora indica AY505557·1 94 2041
Chaetospermum artocarpi EF589735·1 92 1908
KK2-E Piriformospora indica AY505557·1 94 1962
Chaetospermum artocarpi EF589735·1 92 1838
Disturbed bushland C1 Tulasnella calospora DQ388045·1 99 1328
Tulasnella calospora DQ388042·1 98 1314
G4 Piriformospora indica AY505557·1 94 1749
Tremellodendron sp. AY745701·1 92 1635
L2-B Tulasnella calospora DQ388045·1 95 1256
Tulasnella calospora DQ388042·1 95 1243
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Fig. 2.

Fig. 2.

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Phylogenetic relationships among mycorrhizal fungal associates of M. media derived from nLSU sequences (bootstrap 50 % majority-rule consensus tree), and M. media percentage seed germination (Stages: 1, splits in testa; 3, initial trichomes present; 5, leaf present) in symbiotic oat plate trials. Vertical bars indicate the standard error of the mean; columns with the same letter do not differ significantly at P < 0·05 (Tukey's HSD, d.f. = 5). For Stage 3 germinants, habitats denoted with an asterisk have significantly greater seed germination than those without (P < 0·05).

The results of the phylogenetic analysis using nLSU produced an alignment containing 21 sequences and 911 characters, 214 of which were potentially parsimoniously informative (Fig. 2). A heuristic parsimony search was conducted with 1000 replicates. A total of 54 most-parsimonious trees were created with 351 steps. The consistency index was 0·78 and the retention index was 0·87. These analyses demonstrated that M. media associated primarily with members of Sebacinaceae. In disturbed bushlands, M. media associated with Tulasnella calospora, whereas Ceratobasidium sp. was recovered only from M. media plants in horticultural beds. These analyses also showed that four of the five endophytic Piriformospora indica genotypes isolated from M. media at seepage sites were clustered together with strong bootstrap support (99 %), three of the four Sebacina vermifera genotypes isolated from horticultural bed plants (100 % bootstrap support) and the three Sebacina vermifera genotypes from granite outcrop plants (100 % bootstrap support) formed groups.

Symbiotic germination trials

Symbiotic germination trials demonstrated that the origin of mycorrhizal fungi significantly influenced the development of M. media seed to Stage 1 (F = 5·01, P = 0·0006) and/or Stage 3 (F = 11·99, P < 0·0001; Fig. 2); Stages 2 (P = 0·928), 4 (P = 0·831) and 5 (P = 0·305) were not significantly influenced by mycorrhizal origin. Fungi isolated from disturbed bushland or horticultural beds resulted in the highest levels of seeds reaching Stage 1. However, these levels of development did not differ significantly from those in control (85·7 ± 3·1 %) or asymbiotic treatments (83·1 ± 2·8 %), suggesting that the presence or absence of fungi may not constitute a bottleneck to the initiation of seed germination. In contrast, the presence and source of fungi influenced seeds reaching Stage 3, deemed successful germination. Mycorrhizal fungal isolates from seeps, undisturbed bushland and glasshouses resulted in the highest levels of seed germination (36–53 %; Fig. 2). However, mycorrhizal fungi from horticultural beds, granite outcrops and disturbed bushland produced significantly lower levels of seed germination (4–8 %); these levels did not differ significantly from those in the control (6·6 ± 3·1 %) or asymbiotic (5·9 ± 2·2 %) treatments.

Ex situ baiting and scoring

Significant differences in seed germination were detected among the four orchid taxa in response to habitat type (Fig. 3). Irrespective of soil source, M. media seeds reached significantly higher levels of germination (Stage 3) than seed of D. magnifica, P. sanguinea or C. arenicola (Species, F = 98·61, P < 0·0001). Notably, M. media seed germinated in soil from disturbed habitats whereas seed of D. magnifica, P. sanguinea and C. arenicola did not (Habitat × Species, F = 4·53, P < 0·0001).

Fig. 3.

Fig. 3.

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Ex situ baiting trials percentage germination (Stage 3) of M. media and sympatric orchid taxa Caladenia, Diuris and Pterostylis on soil collected from a variety of sites. Vertical bars indicate the standard error of the mean; columns with the same letter do not differ significantly at P < 0·05 (Tukey's HSD, d.f. = 7).

Plant biomass and root colonization

Shoot biomass (F = 3·61, P = 0·018) and consequently total plant biomass (F = 6·41, P = 0·001) were significantly higher in plants collected from horticultural beds and seepage sites than other locations (data not shown). There was no significant difference in root biomass among habitats (F = 1·74, P = 0·175). Cortical cells of all M. media roots were heavily colonized by fungal hyphae. However, there was no significant difference in mycorrhizal root colonization between habitat types (71–99 % roots colonized; F = 1·38, P = 0·264) and no significant correlation with any soil factor (P > 0·05).

Soil resources

ANOVA indicated that only pH (F = 15·56, P = 0·001) and plant-available P differed significantly among habitats (F = 3·34, P = 0·019; Table 2). Soil pH and P levels were highest in horticultural beds and lowest in native bushland and glasshouse substrates.

Table 2.

Soil pH and P levels in each of the six habitat types sampled

Habitat Site Soil pH Soil P [μg (g soil)−1]
Native Bushland 5·78 (0·1)b 5 (1)b
Granite outcrop 5·37 (0·3)ab 13 (1)ab
Seepage 6·00 (0·3)ab 6 (3)ab
Disturbed Horticultural beds 7·00 (0·2)a 33 (13)a
Glasshouse 5·52 (0·1)b 4 (1)b
Bushland 6·65 (0·2)ab 7 (2)ab
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Means within each column with the same letter do not differ significantly at P < 0·05 by Tukey's HSD test.

Correlations between mycorrhizal fungi composition, habitat and geography

Simple Mantel tests indicated that spatial structure (autocorrelation) was not a factor influencing the mycorrhizal fungal community in M. media (Table 3). However, the significant correlation between genetic and habitat distances suggests that habitat variables were significant correlates of mycorrhizal fungi community composition. When the effect of autocorrelation (geographical distance) was controlled, this correlation was further strengthened.

Table 3.

Simple and partial Mantel correlations between fungal species compositional dissimilarity, habitat dissimilarity and geographical distance

Mycorrhizal fungal composition Habitat Geographical distance
Mycorrhizal fungal composition – –0·219 (0·03) –0·125 (0·16)
Habitat –0·235 (0·03) – –0·106 (0·68)
Geographic distance –0·153 (0·08) 0·006 (0·70) –
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Elements in the upper triangle of the matrix are simple correlations, whereas elements in the lower triangle are partial correlations. P values are given in parentheses; significant P and r values are denoted in bold text.

DISCUSSION

In this study, we considered some of the factors that might contribute to weediness in a terrestrial orchid. By combining molecular and experimental studies, we showed that: adult M. media plants formed mycorrhizal associations with a broad phylogenetic range of fungi; a high proportion of these fungi were capable of initiating germination in seed of M. media but not sympatric terrestrial orchid species; mycorrhizal fungi isolated from M. media from natural habitats had higher germination efficacy than fungi from M. media in disturbed environments; and M. media–mycorrhizal associations display an unusual breadth of habitat tolerance.

Orchid mycorrhizal characterization and specificity

We hypothesized that broad associations between M. media and mycorrhizal fungal species might contribute to weediness in M. media. Indeed, molecular analysis showed that M. media formed symbiotic associations with a broad taxonomic range of mycorrhizal fungi. This apparent lack of specificity is in agreement with studies investigating another invasive orchid, Disa bracteata, (Bonnardeaux et al., 2007), and supports the concept that invasive or weedy plant species reliant upon mutualisms will probably be generalists and associate with an array of fungi (Richardson et al., 2000). In contrast, other terrestrial orchids that occupy the same geographical range as M. media have been shown to associate with a phylogenetically narrow suite of mycobionts, but not always (e.g. Pyrorchis nigricans; Bonnardeaux et al., 2007). For instance, certain Caladenia spp. exhibit a high specificity toward one or two closely related Sebacina vermifera (Swarts et al., 2010), whereas Diuris (Smith et al., 2010), Chiloglottis (Roche et al., 2010) and Drakaea spp. all associate with narrow monophyletic groups of Tulasnella mycorrhizal symbionts (Phillips et al., 2011).

It might also be expected that a disturbance-tolerant orchid may form associations with disturbance-tolerant fungi. Most fungi identified from M. media roots were sebacinoid species, suggesting that this group of fungi may be more likely to occur in disturbed habitats. However, many terrestrial orchids, including Australian taxa that primarily occur in undisturbed habitats, also associate with sebacinoid species. For example, the most commonly identified symbiont was Piriformospora indica. This species belongs to a well-supported group of closely related endophytic species with acknowledged benefits to plant growth (Weiß et al., 2011) including some orchids (Schafer and Kogel, 2009). Six fungal accessions comprised Sebacina vermifera sensu Warcup & Talbot (1967) and were closely allied to mycorrhizal fungi in Caladenia (Swarts et al., 2010); an additional three isolates were identified as Tulasnella calospora (bushland) and were related to mycorrhizal fungi previously identified in Prasophyllum giganteum and Diuris magnifica (Bonnardeaux et al., 2007). Only one sequence yielded Ceratobasidium sp. (horticultural beds), which is known to form mycorrhizas with Pterostylis and Prasophyllum (Warcup, 1973; Irwin et al., 2007).

Populations of M. media also appeared to share the same or closely related fungi, resulting in limited symbiont diversity. For example, P. indica AY505557·1 was identified in all sites apart from the granite outcrop. However, two lines of evidence suggest otherwise. First, maximum sequence divergence was >3 %, which would lead P. indica isolates to be classified as six different species based on the criterion of <3 % changes in base pairs (Tedersoo et al., 2008). In addition, some phylogeographical structure was evident among isolates. Second, the fungal isolates differed functionally. Few seeds reached successful germination (Stage 3 or 5) when inoculated with mycorrhizal fungal isolates from disturbed bushlands, horticultural beds and granite outcrops. Although low seed germination rates are not unusual in orchids (Huynh et al., 2009), these differences may reflect a variation in germination effectiveness among the orchid mycorrhizal isolates. Similar variations in germination have been observed in response to different Ceratobasidium isolates in Tolumnia (Otero et al., 2005) and among fungal isolates on Caladenia (Huynh et al., 2009). Alternatively, habitats that facilitate germination may not harbour the fungi required by the orchid to reach adulthood and ultimately reproduce. For example, certain mycobionts may be effective in stimulating seed germination (e.g. bushland, glasshouse), whereas others may be more effective mycobionts for adult plants (e.g. seeps, granite outcrop; see McCormick et al., 2006).

A number of factors may have contributed to the limited diversity of mycorrhizal fungi. First, our results are based on a single collection of root tissue. However, the abundance and identity of orchid mycorrhizal fungi can vary seasonally or in response to edaphic conditions (Huynh et al., 2009; McCormick et al., 2006). Second, some fungi may have been non-responsive to primers that target the nLSU gene region. Finally, peloton species that grew well in culture, such as Sebacinales, may have been over-represented in our analyses, whereas those that did not grow or grew only slowly in culture media may have been overlooked. Further sampling and analysis may reveal a greater diversity of mycorrhizal fungi. In addition, direct amplification of fungi from orchid tissue may reveal a more diverse array of fungal species, including various ascomycetes (Otero and Flanagan, 2006; Dearnaley, 2007), but not always (see Roche et al., 2010).

Symbiotic germination

We also hypothesized that mycorrhizal fungi from disturbed habitats might selectively enhance the germination of M. media seed. This was true when seed germination was compared between M. media and co-occurring taxa of Caladenia, Pterostylis and Diuris. Ex situ baiting clearly showed that soils from the disturbed bushland induced the highest percentage of M. media seeds to germinate successfully (Stage 3) in comparison with other habitats. Furthermore, germination was significantly higher in M. media seed than in Caladenia, Pterostylis and Diuris, irrespective of habitat. These results support the following concepts: (1) the broad capacity of M. media to use mycorrhizal fungi from different habitat types; (2) disturbed sites harbour mycorrhizal fungi more conducive to germination in M. media than other sites; and (3) the capacity for prolific germination in M. media in comparison with other orchid taxa. The ability of M. media to germinate prolifically may provide a competitive advantage similar to that recorded in other weedy terrestrial orchids such a Zeuxine oblonga, Phaius tankervilleae and Spathoglottis plicata (Dixon et al., 2003). Indeed, M. media is well known to produce large numbers of seedlings in a variety of horticultural situations and is often well ahead of seedling development found in other terrestrial orchids.

Plant and edaphic linkages

Environmental conditions were expected to influence M. media through effects on fungal diversity or plant growth and colonization. Tulasnellaceae were recovered only from bushland sites and Ceratobasidium sp. from horticultural beds, indicating that environment may play a role in determining the distribution of mycorrhizal fungal associates in M. media. However, only soil P fertility and pH differed significantly among habitat types, and we were unable to detect strong links between soil pH and P levels and mycorrhizal fungi or seed germination. In contrast, previous studies show soil moisture, organic content, pH and nutrient levels to be correlated with orchid mycorrhizal distributions (Batty et al., 2001; Diez, 2007; McCormick et al., 2012). We found that M. media grows in soils with 5 to 33 mg P (g soil)−1, and levels of endophytic root colonization did not differ significantly across this range. This result probably reflects an unusual breadth of habitat tolerance in M. media plants and/or M. media–mycorrhizal fungal associations. This does not, however, exclude the possibility that other extrinsic factors such as soil moisture fluxes or plant community composition may influence mycorrhizal associations. Likewise, the presence of endophytic colonization alone is not indicative of functional significance (Huynh et al., 2004), and it is still possible that differences in functional capacity exist within and among fungi and colonized roots (Huynh et al., 2009).

Implications

Our findings from M. media support the hypothesis that invasive species are possibly governed by more general rather than idiosyncratic patterns (Arim et al., 2006). For potentially invasive orchids, these general patterns include high phylogenetic breadth of associating mycorrhizal fungi, capacity for germination with a broad range of fungi and broad habitat tolerance, as seen here in M. media. Indeed, orchids such as M. media that associate with a broad taxonomic spectrum of mycobionts are likely to be broadly distributed and colonize new habitats because the probability of encountering a compatible fungus should be high (Swarts and Dixon, 2008). This advantage may be further enhanced by higher germination potential and the capacity to develop mycorrhizal associations in soils of varying levels of fertility (P).

With its clear potential to act as an invasive species (rapid maturation of seedlings; clonal habit; self pollination; mycorrhizal generalist), M. media may impact populations of rare or threatened orchids through a resource competition process whereby it will eventually replace existing populations given its remarkable ability to utilize mycorrhizal and nutrient resources. Alternatively, M. media may exploit previously unutilized resources such as ecologically underutilized or unused mycobionts through niche differentiation (Vila and Weiner, 2004) at a much faster rate than the existing native species. Future research should test the competitive advantage of invasive orchid species and their role as a threat to other rare or ecologically constrained orchids.

SUPPLEMENTARY DATA

Supplementary data are available online at www.aob.oxfordjournals.org and consist of Table S1: latitude and longitude of M. media specimen collection sites, and habitat type.

Supplementary Data
supp_111_3_409__index.html (942B, html)

ACKNOWLEDGEMENTS

This study was made possible by generous funding provided by Potash-Corp., The Froehlich Foundation, The University of Western Australia, Kings Park and Botanic Garden, the Plant Biology and Conservation Research Grant from Northwestern University and the Chicago Botanic Garden. We thank the volunteers at Kings Park and Botanic Garden and the Chicago Botanic Garden and Nyree Zerega, Janet Anthony, Matthew Barrett, Jeremie Fant, Michalie Foley, Annette Johnson, Siegy Krauss, Ryan Phillips and Ann Smithson for field and laboratory assistance.

LITERATURE CITED

  1. Ackerman JD. Invasive orchids: weeds we hate to love? Lankesteriana. 2007;7:19–21. [Google Scholar]
  2. Arim M, Abades SR, Neill PE, Lima M, Marquet PA. Spread dynamics of invasive species. Proceedings of the National Academy of Sciences of the USA. 2006;103:374–378. doi: 10.1073/pnas.0504272102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Batty AL, Dixon KW, Brundrett M, Sivasithamparam K. Constraints to symbiotic germination of terrestrial orchid seed in a mediterranean bushland. New Phytologist. 2001;152:511–520. doi: 10.1046/j.0028-646X.2001.00277.x. [DOI] [PubMed] [Google Scholar]
  4. Bidartondo MI, Read DJ. Fungal specificity bottlenecks during orchid germination and development. Molecular Ecology. 2008;17:3707–3716. doi: 10.1111/j.1365-294X.2008.03848.x. [DOI] [PubMed] [Google Scholar]
  5. Bonnardeaux Y, Brundrett M, Batty A, Dixon K, Koch J, Sivasithamparam K. Diversity of mycorrhizal fungi of terrestrial orchids: compatibility webs, brief encounters, lasting relationships and alien invasions. Mycological Research. 2007;111:51–61. doi: 10.1016/j.mycres.2006.11.006. [DOI] [PubMed] [Google Scholar]
  6. Bowles M, Zettler L, Bell T, Kelsey P. Relationships between soil characteristics, distribution and restoration potential of the federal threatened eastern prairie fringed orchid, Platanthera leucophaea (Nutt.) Lindl. The American Midland Naturalist. 2005;154:273–285. [Google Scholar]
  7. Brundrett MC, Scade A, Batty AL, Dixon KW, Sivasithamparam K. Development of in situ and ex situ seed baiting techniques to detect mycorrhizal fungi from terrestrial orchid habitats. Mycological Research. 2003;107:1210–1220. doi: 10.1017/s0953756203008463. [DOI] [PubMed] [Google Scholar]
  8. Cohen IM, Ackerman JD. Oeceoclades maculata, an alien tropical orchid in a Caribbean rainforest. Annals of Botany. 2009;104:557–563. doi: 10.1093/aob/mcn191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cozzolino S, D'Emerico S, Widmer A. Evidence for reproductive isolate selection in Mediterranean orchids: karyotype differences compensate for the lack of pollinator specificity. Proceedings of the Royal Society of London Series B – Biological Sciences. 2004;271:S259–S262. doi: 10.1098/rsbl.2004.0166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dearnaley JDW. Further advances in orchid mycorrhizal research. Mycorrhiza. 2007;17:475–486. doi: 10.1007/s00572-007-0138-1. [DOI] [PubMed] [Google Scholar]
  11. Diez JM. Hierarchical patterns of symbiotic orchid germination linked to adult proximity and environmental gradients. Journal of Ecology. 2007;95:159–170. [Google Scholar]
  12. Dixon KW, Kell SP, Barrett RL, Cribb PJ, editors. Orchid conservation. Borneo: Natural History Publications; 2003. [Google Scholar]
  13. Fortin M, Gurevitch J. Mantel tests: spatial structure in field experiments. In: Scheiner SM, Gurevitch J, editors. Design and analysis of ecological experiments. New York: Chapman and Hall; 1993. pp. 308–326. [Google Scholar]
  14. Gardes M, White TJ, Fortin JA, Bruns TD, Taylor JW. Identification of indigenous and introduced symbiotic fungi in ectomycorrhizae by amplification of nuclear and mitochondrial ribosomal DNA. Canadian Journal of Botany. 1991;69:180–190. [Google Scholar]
  15. Grace C, Stribley DP. A safer procedure for routine staining of vesicular–arbuscular mycorrhizal fungi. Mycological Research. 1991;95:1160–1162. [Google Scholar]
  16. Halász K, Bratek Z, Szegó D, et al. Tests of species concepts of the small, white, European group of Tuber spp. based on morphology and rDNA ITS sequences with special reference to Tuber rapaedorum. Mycological Progress. 2005;4:281–290. [Google Scholar]
  17. Hoffman N, Brown A. Orchids of South-West Australia. 2nd edn. Nedlands, WA: University of Western Australia Press; 1998. [Google Scholar]
  18. Hopper SD, Gioia P. The Southwest Australia Floristic Region: evolution and conservation of a global hot spot of biodiversity. Annual Review of Ecology, Evolution, and Systematics. 2004;35:623–650. [Google Scholar]
  19. Huynh TT, McLean CB, Coates F, Lawrie AC. Effects of developmental stage and peloton morphology on success in isolation of mycorrhizal fungi in Caladenia formosa (Orchidaceae) Australian Journal of Botany. 2004;52:231–241. [Google Scholar]
  20. Huynh TT, Thomson R, McLean CB, Lawrie AC. Functional and genetic diversity of mycorrhizal fungi from single plants of Caladenia formosa (Orchidaceae) Annals of Botany. 2009;104:757–765. doi: 10.1093/aob/mcp153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Irwin MJ, Bougoure JJ, Dearnaley JDW. Pterostylis nutans (Orchidaceae) has a specific association with two Ceratobasidium root-associated fungi across its range in eastern Australia. Mycoscience. 2007;48:231–239. [Google Scholar]
  22. Legendre P, Fortin M. Spatial pattern and ecological analysis. Vegetatio. 1989;80:107–138. [Google Scholar]
  23. Linder HP. Setting conservation priorities—The importance of endemism and phylogeny in the southern African orchid genus Herschelia. Conservation Biology. 1995;9:585–595. [Google Scholar]
  24. Koske R, Gemma J. A modified procedure for staining roots to detect VA mycorrhizas. Mycological Research. 1989;92:486–505. [Google Scholar]
  25. McCormick MK, Whigham DF, O'Neill J. Mycorrhizal diversity in photosynthetic orchids. New Phytologist. 2004;163:425–438. doi: 10.1111/j.1469-8137.2004.01114.x. [DOI] [PubMed] [Google Scholar]
  26. McCormick MK, Whigham DF, Sloan D, O'Malley K, Hodkinson B. Orchid fungus fidelity: a marriage meant to last? Ecology. 2006;87:903–91. doi: 10.1890/0012-9658(2006)87[903:ofammt]2.0.co;2. [DOI] [PubMed] [Google Scholar]
  27. McCormick M, Taylor DL, Juhszova K, Burnett RK, Whigham D, O'Neill JP. Limitations on orchid recruitment: not a simple picture. Molecular Ecology. 2012;21:1511–1523. doi: 10.1111/j.1365-294X.2012.05468.x. [DOI] [PubMed] [Google Scholar]
  28. McGonigle TP, Miller MH, Evans DG, Fairchild GL, Swan JA. A new method which gives an objective measure of colonization of roots by vesicular–arbuscular mycorrhizal fungi. New Phytologist. 1990;115:495–501. doi: 10.1111/j.1469-8137.1990.tb00476.x. [DOI] [PubMed] [Google Scholar]
  29. McKendrick SL, Leake JR, Taylor DL, Read DJ. Symbiotic germination and development of myco-heterotrophic plants in nature: ontogeny of Corallorhiza trifida and characterization of its mycorrhizal fungi. New Phytologist. 2000;145:523–537. doi: 10.1046/j.1469-8137.2000.00603.x. [DOI] [PubMed] [Google Scholar]
  30. Ogura-Tsujita Y, Yukawa T. Epipactis helleborine shows strong mycorrhizal preference towards ectomycorrhizal fungi with contrasting geographic distributions in Japan. Mycorrhiza. 2008;18:331–338. doi: 10.1007/s00572-008-0187-0. [DOI] [PubMed] [Google Scholar]
  31. Otero JT, Flanagan NS. Orchid diversity: beyond deception. Trends in Ecology and Evolution. 2006;21:64–65. doi: 10.1016/j.tree.2005.11.016. [DOI] [PubMed] [Google Scholar]
  32. Otero JT, Bayman P, Ackerman JD. Variation in mycorrhizal performance in the epiphytic orchid Tolumnia variegata in vitro: the potential for natural selection. Evolutionary Ecology. 2005;19:29–43. [Google Scholar]
  33. Phillips RD, Barrett MD, Dixon KW, Hopper SD. Do mycorrhizal symbioses cause rarity in orchids? Journal of Ecology. 2011;99:858–869. [Google Scholar]
  34. Ramsay RR, Dixon KW, Sivasithamparam K. Patterns of infection and endophytes associated with Western Australian orchids. Lindleyana. 1986;1:203–214. [Google Scholar]
  35. Rasmussen HN. Terrestrial orchids from seed to mycotrophic plant. Cambridge: Cambridge University Press; 1995. [Google Scholar]
  36. Rasmussen HN. Recent developments in the study of orchid mycorrhiza. Plant and Soil. 2002;244:149–163. [Google Scholar]
  37. Richardson DM, Pyšek P, Rejmánek M, Barbour MG, Panetta FD, West CJ. Naturalization and invasion of alien plants: concepts and definitions. Diversity and Distributions. 2000;6:93–107. [Google Scholar]
  38. Roche SA, Carter RJ, Peakall R, Smith LM, Whitehead MR, Linde C. A narrow group of monophyletic Tulasnella (Tulasnellaceae) symbionts lineages are associated with multiple species of Chiloglottis (Orchidaceae): implications for orchid diversity. American Journal of Botany. 2010;97:1313–1327. doi: 10.3732/ajb.1000049. [DOI] [PubMed] [Google Scholar]
  39. Schafer P, Kogel KH. The sebacinoid fungus Piriformospora indica: an orchid mycorrhiza which may increase host plant reproduction and fitness. In: Deising H, editor. The Mycota, volume 5. Plant Relationships. Berlin/Heidelberg: Springer-Verlag; 2009. pp. 99–112. [Google Scholar]
  40. Selosse M-A, Faccio A, Scappaticci G, Bonfante P. Chlorophyllous and achlorophyllous specimens of Epipactis microphylla (Neottieae, Orchidaceae) are associated with ectomycorrhizal Septomycetes, including truffles. Microbial Ecology. 2004;47:416–426. doi: 10.1007/s00248-003-2034-3. [DOI] [PubMed] [Google Scholar]
  41. Shefferson RP, Weiß M, Kull T, Taylor DL. High specificity generally characterizes mycorrhizal association in rare lady's slipper orchids, genus Cypripedium. Molecular Ecology. 2005;14:613–626. doi: 10.1111/j.1365-294X.2005.02424.x. [DOI] [PubMed] [Google Scholar]
  42. Shefferson RP, Taylor DL, Weiß M, et al. The evolutionary history of mycorrhizal specificity in rare lady's slipper orchids, genus Cypripedium. Molecular Ecology. 2007;14:613–626. doi: 10.1111/j.1365-294X.2005.02424.x. [DOI] [PubMed] [Google Scholar]
  43. Shefferson RP, Kull T, Tali K. Mycorrhizal interactions of orchids colonizing Estonian mine tailings hills. American Journal of Botany. 2008;95:156–164. doi: 10.3732/ajb.95.2.156. [DOI] [PubMed] [Google Scholar]
  44. Smith ZF, James EA, McLean CB. Mycorrhizal specificity of Diuris fragrantissima (Orchidaceae) and persistence in a reintroduced population. Australian Journal of Botany. 2010;58:97–106. [Google Scholar]
  45. Smouse PE, Long JC, Sokal RR. Multiple regression and correlation extensions of the Mantel test of matrix correspondence. Systematic Zoology. 1986;35:627–632. [Google Scholar]
  46. Swarts N, Dixon KW. Terrestrial orchid conservation in the age of extinction. Annals of Botany. 2008;104:543–556. doi: 10.1093/aob/mcp025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Swarts N, Sinclair EA, Francis A, Dixon KW. Ecological specialization in mycorrhizal symbiosis leads to rarity in an endangered orchid. Molecular Ecology. 2010;19:3226–3242. doi: 10.1111/j.1365-294X.2010.04736.x. [DOI] [PubMed] [Google Scholar]
  48. Swofford DL. PAUP* Phylogenetic analysis using parsimony and other methods, Version 4·0b10. Sunderland, MA: Sinauer Associates; 2002. [Google Scholar]
  49. Taylor DL, Bruns TD. Independent, specialized invasions of ectomycorrhizal mutualism by two nonphotosynthetic orchids. Proceedings of the National Academy of Sciences, USA. 1997;94:4510–4515. doi: 10.1073/pnas.94.9.4510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Taylor DL, Bruns TD. Population, habitat and genetic correlates of mycorrhizal specialization in the ‘cheating’ orchids Corallorhiza maculata and C. mertensiana. Molecular Ecology. 1999;8:1719–1732. doi: 10.1046/j.1365-294x.1999.00760.x. [DOI] [PubMed] [Google Scholar]
  51. Taylor DL, Bruns TD, Leake JR, Read DJ. Mycorrhizal specificity and function in myco-heterotrophic plants. In: Sanders I, van der Hijden M, editors. Mycorrhizal Ecology. Vol. 157. Berlin: Springer; 2002. pp. 375–413. [Google Scholar]
  52. Tedersoo L, Jairus T, Horton BM, et al. Strong host preference of ectomycorrhizal fungi in a Tasmanian wet sclerophyll forest as revealed by DNA barcoding and taxon-specific primers. New Phytologist. 2008;180:479–490. doi: 10.1111/j.1469-8137.2008.02561.x. [DOI] [PubMed] [Google Scholar]
  53. Vila M, Weiner J. Are invasive plant species better competitors than native plant species? Evidence from pair-wise experiments. Oikos. 2004;105:229–238. [Google Scholar]
  54. Warcup JH. Specificity of mycorrhizal association in some Australian terrestrial orchids. New Phytologist. 1971;70:41–46. [Google Scholar]
  55. Warcup JH. Symbiotic germination of some Australian terrestrial orchids. New Phytologist. 1973;72:387–392. [Google Scholar]
  56. Warcup JH, Talbot PHB. Perfect states of Rhizoctonia associated with orchids. New Phytologist. 1967;66:631–641. [Google Scholar]
  57. Weiß M, Sýkorová Z, Garnica S, et al. Sebacinales everywhere: previously overlooked ubiquitous fungal endophytes. PLoS ONE. 2011;6 doi: 10.1371/journal.pone.0016793. e16793. http://dx.doi.org/10.1371/journal.pone.0016793 . [DOI] [PMC free article] [PubMed] [Google Scholar]

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