Cryptostylis species (Orchidaceae) from a broad geographic and habitat range associate with a phylogenetically narrow lineage of Tulasnellaceae fungi

While many Australian terrestrial orchids have highly specialized mycorrhizal associations, we tested the hypothesis that the geographically widespread orchid genus Cryptostylis associates with a diversity of fungal species. Using fungal isolation and molecular approaches, we investigated the mycorrhizal associations of five Australian Cryptostylis species (27 sites sampled) and included limited sampling from three Asiatic Cryptostylis species (two sites). Like related orchid genera, Tulasnellaceae formed the main fungal associations of the Cryptostylis species we sampled, although some ectomycorrhizal, ericoid and saprotrophic fungi were detected infrequently. Each species of Australian Cryptostylis associated with three to seven Tulasnella Operational Taxonomic Units (OTUs), except for C. hunteriana where only one Tulasnella OTU was detected. In total, eleven Tulasnella OTUs associated with Australian Cryptostylis. The Asiatic Cryptostylis associated with four different Tulasnella OTUs belonging to the same lineage as the Australian species. While five Tulasnella OTUs (T. australiensis, T. prima, T. warcupii, T. densa, and T. punctata) were used by multiple species of Australian Cryptostylis, the most commonly used OTU differed between orchid species. The association with different Tulasnella fungi by Cryptostylis species co-occurring at the same site suggests that in any given environmental condition, Cryptostylis species may intrinsically favour different fungal OTUs.     

Continental-scale distribution and diversity of Ceratobasidium orchid mycorrhizal fungi in Australia

Background and AimsMycorrhizal fungi are a critical component of the ecological niche of most plants and can potentially constrain their geographical range. Unlike other types of mycorrhizal fungi, the distributions of orchid mycorrhizal fungi (OMF) at large spatial scales are not well understood. Here, we investigate the distribution and diversity of Ceratobasidium OMF in orchids and soils across the Australian continent.MethodsWe sampled 217 Ceratobasidium isolates from 111 orchid species across southern Australia and combined these with 311 Ceratobasidium sequences from GenBank. To estimate the taxonomic diversity of Ceratobasidium associating with orchids, phylogenetic analysis of the ITS sequence locus was undertaken. Sequence data from the continent-wide Australian Microbiome Initiative were used to determine the geographical range of operational taxonomic units (OTUs) detected in orchids, with the distribution and climatic correlates of the two most frequently detected OTUs modelled using MaxEnt.Key ResultsWe identified 23 Ceratobasidium OTUs associating with Australian orchids, primarily from the orchid genera Pterostylis, Prasophyllum, Rhizanthella and Sarcochilus. OTUs isolated from orchids were closely related to, but distinct from, known pathogenic fungi. Data from soils and orchids revealed that ten of these OTUs occur on both east and west sides of the continent, while 13 OTUs were recorded at three locations or fewer. MaxEnt models suggested that the distributions of two widespread OTUs are correlated with temperature and soil moisture of the wettest quarter and far exceeded the distributions of their host orchid species.ConclusionsCeratobasidium OMF with cross-continental distributions are common in Australian soils and frequently have geographical ranges that exceed that of their host orchid species, suggesting these fungi are not limiting the distributions of their host orchids at large spatial scales. Most OTUs were distributed within southern Australia, although several OTUs had distributions extending into central and northern parts of the continent, illustrating their tolerance of an extraordinarily wide range of environmental conditions.     

Morphological and molecular characterization of Tulasnella spp. fungi isolated from the roots of Epidendrum secundum,a widespread Brazilian orchid

Tulasnella spp. are the main fungal symbionts of Brazilian Epidendrum orchids. The taxonomy of these fungi is largely based on ITS rDNA similarity, but culture dependent techniques are still essential to establish the true biological entity of the mycobiont. The aim of this study was to characterize morphologically and molecularly 16 Tulasnella spp. fungi isolated from three different populations of E. secundum and to test the coincidences between morphological and molecular characterization. Two uninucleate rhizoctonia fungi, obtained from Oncidium barbaceniae, and two phytopathogenic isolates were included as outgroups. Qualitative and quantitative morphological characteristics were analyzed using multivariate statistics and were able to distinguish Ceratobasidium, Tulasnella and Thanatephorus genera and separate the isolates of Tulasnella spp. into two groups. Analysis of RAPD (Random Amplified Polymorphic DNA) and ITS rDNA sequences validated the morphological data. Symbionts of O. barbaceniae presented identity to ITS sequences of Ceratobasidium genus, while E. secundum isolates presented identity to two species of Tulasnella. We observed homogeneity among Tulasnella spp. obtained from a single population and from neighboring populations, but there was higher variability among isolates obtained from populations of regions that were farther apart. Morphological data associated with multivariate statistics proved to be a useful tool in the multi-level taxonomy of these orchid-associated fungi and in estimating the diversity of orchid mycorrhizal fungi.     

Strong phylogenetic congruence between Tulasnella fungi and their associated Drakaeinae orchids

The study of congruency between phylogenies of interacting species can provide a powerful approach for understanding the evolutionary history of symbiotic associations. Orchid mycorrhizal fungi can survive independently of orchids making cospeciation unlikely, leading us to predict that any congruence would arise from host-switches to closely related fungal species. The Australasian orchid subtribe Drakaeinae is an iconic group of sexually deceptive orchids that consists of approximately 66 species. In this study, we investigated the evolutionary relationships between representatives of all six Drakaeinae orchid genera (39 species) and their mycorrhizal fungi. We used an exome capture dataset to generate the first well-resolved phylogeny of the Drakaeinae genera. A total of 10 closely related Tulasnella Operational Taxonomic Units (OTUs) and previously described species were associated with the Drakaeinae orchids. Three of them were shared among orchid genera, with each genus associating with 1–6 Tulasnella lineages. Cophylogenetic analyses show Drakaeinae orchids and their Tulasnella associates exhibit significant congruence (p < 0.001) in the topology of their phylogenetic trees. An event-based method also revealed significant congruence in Drakaeinae–Tulasnella relationships, with duplications (35), losses (25), and failure to diverge (9) the most frequent events, with minimal evidence for cospeciation (1) and host-switches (2). The high number of duplications suggests that the orchids speciate independently from the fungi, and the fungal species association of the ancestral orchid species is typically maintained in the daughter species. For the Drakaeinae–Tulasnella interaction, a pattern of phylogenetic niche conservatism rather than coevolution likely explains the observed phylogenetic congruency in orchid and fungal phylogenies. Given that many orchid genera are characterized by sharing of fungal species between closely related orchid species, we predict that these findings may apply to a wide range of orchid lineages.     

Continent-wide distribution in mycorrhizal fungi: implications for the biogeography of specialized orchids

Background and Aims Although mycorrhizal associations are predominantly generalist, specialized mycorrhizal interactions have repeatedly evolved in Orchidaceae, suggesting a potential role in limiting the geographical range of orchid species. In particular, the Australian orchid flora is characterized by high mycorrhizal specialization and short-range endemism. This study investigates the mycorrhizae used by Pheladenia deformis, one of the few orchid species to occur across the Australian continent. Specifically, it examines whether P. deformis is widely distributed through using multiple fungi or a single widespread fungus, and if the fungi used by Australian orchids are widespread at the continental scale.Methods Mycorrhizal fungi were isolated from P. deformis populations in eastern and western Australia. Germination trials using seed from western Australian populations were conducted to test if these fungi supported germination, regardless of the region in which they occurred. A phylogenetic analysis was undertaken using isolates from P. deformis and other Australian orchids that use the genus Sebacina to test for the occurrence of operational taxonomic units (OTUs) in eastern and western Australia.Key Results With the exception of one isolate, all fungi used by P. deformis belonged to a single fungal OTU of Sebacina. Fungal isolates from eastern and western Australia supported germination of P. deformis. A phylogenetic analysis of Australian Sebacina revealed that all of the OTUs that had been well sampled occurred on both sides of the continent.Conclusions The use of a widespread fungal OTU in P. deformis enables a broad distribution despite high mycorrhizal specificity. The Sebacina OTUs that are used by a range of Australian orchids occur on both sides of the continent, demonstrating that the short-range endemism prevalent in the orchids is not driven by fungal species with narrow distributions. Alternatively, a combination of specific edaphic requirements and a high incidence of pollination by sexual deception may explain biogeographic patterns in southern Australian orchids.     

Isolation and identification of Rhizoctonia-like fungi from roots of three orchid genera, Paphiopedilum, Dendrobium, and Cymbidium, collected in Chiang Rai and Chiang Mai provinces of Thailand

Three orchid genera, Paphiopedilum, Cymbidium, and Dendrobium, are among the most heavily traded ornamental plants in Thailand. In this study, 27 isolates of Rhizoctonia-like fungi were isolated from root sections of mature orchids in the three orchid genera, collected from diverse horticultural settings in Chiang Mai and Chiang Rai provinces of Thailand. Fungal identification was done by the morphological characterization, the comparison of the internal transcribed spacer and 5.8S ribosomal DNA sequences, and the phylogenetic analysis. Epulorhiza repens was found to be the most common species found in the roots of various species of all three orchid genera, whereas Epulorhiza calendulina-like isolates were strictly found in the roots of Paphiopedilum species. We have also isolated and described an anamorph of Tulasnella irregularis, four new anamorphic species in the genus Tulasnella, and a new anamorphic species in the family Tulasnellaceae. Our study provides information on diversity of root-associated fungi of the orchid genera and at the sampling sites that were rarely addressed in the previous studies.     

A narrowly endemic photosynthetic orchid is non‐specific in its mycorrhizal associations

Mycorrhizal association is a common characteristic in a majority of land plants, and the survival and distribution of a species can depend on the distribution of suitable fungi in its habitat. Orchidaceae is one of the most species‐rich angiosperm families, and all orchids are fully dependent on fungi for their seed germination and some also for subsequent growth and survival. Given this obligate dependence, at least in the early growth stages, elucidating the patterns of orchid–mycorrhizal relationships is critical to orchid biology, ecology and conservation. To assess whether rarity of an orchid is determined by its specificity towards its fungal hosts, we studied the spatial and temporal variability in the host fungi associated with one of the rarest North American terrestrial orchids, Piperia yadonii. The fungal internal transcribed spacer region was amplified and sequenced by sampling roots from eight populations of P. yadonii distributed across two habitats, Pinus radiata forest and maritime chaparral, in California. Across populations and sampling years, 26 operational taxonomic units representing three fungal families, the Ceratobasidiaceae, Sebacinaceae and Tulasnellaceae, were identified. Fungi belonging to the Sebacinaceae were documented in orchid roots only at P. radiata forest sites, while those from the Ceratobasidiaceae and Tulasnellaceae occurred in both habitats. Our results indicate that orchid rarity can be unrelated to the breadth of mycorrhizal associations. Our data also show that the dominance of various fungal families in mycorrhizal plants can be influenced by habitat preferences of mycorrhizal partners.     

Members of Sebacinales subgroup B form mycorrhizae with epiphytic orchids in a neotropical mountain rain forest

Previous investigations revealed that epiphytic orchids in a mountain rain forest in southern Ecuador formed mycorrhizae with diverse members of Tulasnellales. Using specific primers, we now show that the same orchids are also associated with Sebacinales. Ultrastructural observations confirmed the Sebacinales mycobionts in situ. Mycorrhizae of flowering individuals of Stelis hallii, S. superbiens, S. concinna and Pleurothallis lilijae were sampled in different forest types of the mountain rain forest of southern Ecuador along an altitudinal gradient between 1,850 and 2,100 m a.s.l. Phylogenetic analysis of fungal nuclear rDNA sequences coding for the ribosomal large subunit (nucLSU) showed the presence of eight sequence types based on proportional differences of <1% bp. All sequence types clustered in the Sebacinales subgroup B which also contained sequences of mycobionts from ericads and terrestrial orchids. Sequences of the nuclear rDNA 5.8S subunit, including parts of the internal transcribed spacers ITS1 and ITS2 (5.8-ITS) from the mycobionts of the epiphytic orchids, were distinct from published sequences of sebacinoid mycobionts of green terrestrial orchids and ericads. Sebacinales sequences from different epiphytic orchid species differed at least by 1% bp as was previously found for Tulasnella sequences. Sebacinales occurred less frequently and with a lower number of sequence types than Tulasnellales, but distribution along the altitudinal gradient was similar.     

Effects of mycorrhizal fungi on symbiotic seed germination of Pecteilis susannae (L.) Rafin (Orchidaceae), a terrestrial orchid in Thailand

Symbiotic seed germination of Pecteilis susannae (L.) Rafin was investigated using 11 fungal isolates recovered from roots of four Thai terrestrial orchids (P. susannae, Eulophia spectabilis, Paphiopedilum bellatulum and Spathoglottis affinis). Seed germination and protocorm development were evaluated up to 133 days after sowing. Protocorm development was most advanced, up to stage 5 (elongation of the first leaf), when seeds were cultured with 4 Epulorhiza isolates obtained from roots of P. susannae (CMU-Aug 028, 4.3%, CMU-Aug 007, 4.2%, and CMU-Aug 013, 2.2%) and E. spectabilis (CMU-STE 014, 3.9%). Moreover, stage 4 protocorm development (emergence of the first leaf) occurred with fungal isolates CMU-STE 011, 5.7%, (Epulorhiza sp.) and CMU-AU 212, 4.3%, (Tulasnella sp.) obtained from roots of E. spectabilis and S. affinis respectively. When seed was incubated without fungi (control), development was limited to stage 3 of protocorm development (appearance of promeristem). This is the first report of protocorm stage 5 development in P. susannae using compatible fungal symbionts. Optimization of seed germination and seedling fitness will assist the conservation and propagation of this orchid species and other terrestrial orchids in Thailand.     

Low genetic diversity in marginal populations of Bletilla striata (Orchidaceae) in southern Korea: Insights into population history and implications for conservation

The warm-temperate vegetation of Korea, currently limited to southern coastal areas, shifted southward during the Last Glacial Maximum towards glacial refugia putatively located in southern Japan and/or southern China. We hypothesized two scenarios of post-glacial re-colonization of warm-temperate species: (i) that extant Korean populations originated from a single source or (ii) that they are derived from multiple source populations. To test which of these scenarios is more likely, we investigated the patterns of genetic diversity in 16 populations of the warm-temperate terrestrial orchid Bletilla striata, employing 20 allozyme loci. Levels of genetic variation in B. striata were substantially lower than those reported in most other terrestrial orchids. However, the degree of genetic differentiation among populations was moderate. These results suggest a pattern of post-glacial re-colonization fitting the first scenario. Both in situ and ex situ conservation strategies are suggested to preserve the genetic variation of B. striata in Korea.     

Identity and Specificity of Rhizoctonia-Like Fungi from Different Populations of Liparis japonica (Orchidaceae) in Northeast China

Mycorrhizal association is known to be important to orchid species, and a complete understanding of the fungi that form mycorrhizas is required for orchid ecology and conservation. Liparis japonica (Orchidaceae) is a widespread terrestrial photosynthetic orchid in Northeast China. Previously, we found the genetic diversity of this species has been reduced recent years due to habitat destruction and fragmentation, but little was known about the relationship between this orchid species and the mycorrhizal fungi. The Rhizoctonia-like fungi are the commonly accepted mycorrhizal fungi associated with orchids. In this study, the distribution, diversity and specificity of culturable Rhizoctonia-like fungi associated with L. japonica species were investigated from seven populations in Northeast China. Among the 201 endophytic fungal isolates obtained, 86 Rhizoctonia-like fungi were identified based on morphological characters and molecular methods, and the ITS sequences and phylogenetic analysis revealed that all these Rhizoctonia-like fungi fell in the same main clade and were closely related to those of Tulasnella calospora species group. These findings indicated the high mycorrhizal specificity existed in L. japonica species regardless of habitats at least in Northeast China. Our results also supported the wide distribution of this fungal partner, and implied that the decline of L. japonica in Northeast China did not result from high mycorrhizal specificity. Using culture-dependent technology, these mycorrhizal fungal isolates might be important sources for the further utilizing in orchids conservation.     

A diverse fungal community associated with Pseudorchis albida (Orchidaceae) roots

In addition to orchid mycorrhizal fungi (OrMF), the roots of orchids harbour plant fungal endophytes termed root-associated fungi (RAF). In the present study, the endangered photosynthetic orchid Pseudorchis albida was screened for OrMF and RAF using culture-dependent (isolations from root sections and pelotons) and culture-independent (cloning from root sections) techniques. The efficiency of the different approaches for detecting the fungi and the effect of the sampling season (summer or autumn) were evaluated. In total, 66 distinct OTUs of mycorrhizal and non-mycorrhizal fungi were found, which, to our knowledge, is the highest diversity of RAF that has yet been detected in a single orchid species. The OrMF community was dominated by Tulasnella species, which were mainly detected by isolation from pelotons or cloning from root sections. The roots and tubers showed higher mycorrhizal colonization in summer, corroborating the frequent reports of Tulasnella from pelotons in this season. In contrast, two helotialean fungi, Varicosporium elodeae and Leohumicola sp., the latter of which was repeatedly isolated from pelotons, were significantly more abundant in the autumn.     

Comparative study of nutritional mode and mycorrhizal fungi in green and albino variants of Goodyera velutina,an orchid mainly utilizing saprotrophic rhizoctonia

The majority of chlorophyllous orchids form mycorrhizal associations with so‐called rhizoctonia fungi, a phylogenetically heterogeneous assemblage of predominantly saprotrophic fungi in Ceratobasidiaceae, Tulasnellaceae, and Serendipitaceae. It is still a matter of debate whether adult orchids mainly associated with rhizoctonia species are partially mycoheterotrophic. Here, we investigated the nutritional modes of green and albino variants of Goodyera velutina, an orchid species considered to be mainly associated with Ceratobasidium spp., by measuring their ¹³C and ¹⁵N abundances, and by molecular barcoding of their mycorrhizal fungi. Molecular analysis revealed that both green and albino variants of G. velutina harbored a similar range of mycobionts, mainly saprotrophic Ceratobasidium spp., Tulasnella spp., and ectomycorrhizal Russula spp. In addition, stable isotope analysis revealed that albino variants were significantly enriched in ¹³C but not so greatly in ¹⁵N, suggesting that saprotrophic Ceratobasidium spp. and Tulasnella spp. are their main carbon source. However, in green variants, ¹³C levels were depleted and those of ¹⁵N were indistinguishable from the co‐occurring autotrophic plants. Therefore, we concluded that the albino G. velutina variants are fully mycoheterotrophic plants whose C derives mainly from saprotrophic rhizoctonia, while the green G. velutina variants are mainly autotrophic plants, at least at our study site, in spite of their additional associations with ectomycorrhizal fungi. This is the first report demonstrating that adult nonphotosynthetic albino variants can obtain their nutrition mainly from nonectomycorrhizal rhizoctonia.     

Three new species in the genus Tulasnella isolated from orchid mycorrhiza of Spiranthes sinensis var. amoena (Orchidaceae)

We isolated Tulasnella spp. from Spiranthes sinensis var. amoena, a Japanese native winter green terrestrial orchid collected in Tsukuba City, Ibaraki Pref., Japan. These isolates were classified into four morphotypes according to morphological characters, i.e., shape of monilioid cells and branch type of monilioid cell chains, while they were separated into five clades by molecular phylogenetic analysis based on the rDNA 5.8S and D1/D2 regions. The four morphotypes and five clades were correlated, and four morpho-phylogenetic groups were identified. Thus, Tulasnella deliquescens including two phylogenetic subgroups and three new species, Tulasnella dendritica sp. nov., Tulasnella ellipsoidea sp. nov., and Tulasnella cumulopuntioides sp. nov., were recognized in this study. In this study, the monilioid cell chain morphology is newly defined, and is suggested as a useful taxonomic characteristic in the asexual stage of Tulasnella spp.     

Untangling above‐ and belowground mycorrhizal fungal networks in tropical orchids

Orchids typically depend on fungi for establishment from seeds, forming mycorrhizal associations with basidiomycete fungal partners in the polyphyletic group rhizoctonia from early stages of germination, sometimes with very high specificity. This has raised important questions about the roles of plant and fungal phylogenetics, and their habitat preferences, in controlling which fungi associate with which plants. In this issue of Molecular Ecology, Martos et al. (2012) report the largest network analysis to date for orchids and their mycorrhizal fungi, sampling a total of over 450 plants from nearly half the 150 tropical orchid species on Reunion Island, encompassing its main terrestrial and epiphytic orchid genera. The authors found a total of 95 operational taxonomic units of mycorrhizal fungi and investigated the architecture and nestedness of their bipartite networks with 73 orchid species. The most striking finding was a major ecological barrier between above‐ and belowground mycorrhizal fungal networks, despite both epiphytic and terrestrial orchids often associating with closely related taxa across all three major lineages of rhizoctonia fungi. The fungal partnerships of the epiphytes and terrestrial species involved a diversity of fungal taxa in a modular network architecture, with only about one in ten mycorrhizal fungi partnering orchids in both groups. In contrast, plant and fungal phylogenetics had weak or no effects on the network. This highlights the power of recently developed ecological network analyses to give new insights into controls on plant–fungal symbioses and raises exciting new hypotheses about the differences in properties and functioning of mycorrhiza in epiphytic and terrestrial orchids.     

Terrestrial orchid conservation in the age of extinction

Background

Conservation through reserves alone is now considered unlikely to achieve protection of plant species necessary to mitigate direct losses of habitat and the pervasive impact of global climate change. Assisted translocation/migration represent new challenges in the face of climate change; species, particularly orchids, will need artificial assistance to migrate from hostile environments, across ecological barriers (alienated lands such as farmlands and built infrastructure) to new climatically buffered sites. The technology and science to underpin assisted migration concepts are in their infancy for plants in general, and orchids, with their high degree of rarity, represent a particularly challenging group for which these principles need to be developed. It is likely that orchids, more than any other plant family, will be in the front-line of species to suffer large-scale extinction events as a result of climate change.

Scope

The South West Australian Floristic Region (SWAFR) is the only global biodiversity hotspot in Australia and represents an ideal test-bed for development of orchid conservation principles. Orchids comprise 6 % of all threatened vascular plants in the SWAFR, with 76 out of the 407 species known for the region having a high level of conservation risk. The situation in the SWAFR is a portent of the global crisis in terrestrial orchid conservation, and it is a region where innovative conservation solutions will be required if the impending wave of extinction is to be averted. Major threatening processes are varied, and include land clearance, salinity, burning, weed encroachment, disease and pests. This is compounded by highly specialized pollinators (locally endemic native invertebrates) and, in the most threatened groups such as hammer orchids (Drakaea) and spider orchids (Caladenia), high levels of mycorrhizal specialization. Management and development of effective conservation strategies for SWAFR orchids require a wide range of integrated scientific approaches to mitigate impacts that directly influence ecological traits critical for survival.

Conclusions

In response to threats to orchid species, integrated conservation approaches have been adopted (including ex situ and translocation principles) in the SWAFR with the result that a significant, multidisciplinary approach is under development to facilitate conservation of some of the most threatened taxa and build expertise to carry out assisted migration to new sites. Here the past two decades of orchid conservation research in the SWAFR and the role of research-based approaches for managing effective orchid conservation in a global biodiversity hotspot are reviewed.Key words: Orchids, pollination, mycorrhiza, integrated conservation, terrestrial, threats, ex situ conservation, in situ conservation     

Essential mycorrhizal partners of the endemic Chilean orchids Chloraea collicensis and C. gavilu

Orchids are obligate mycoheterotrophic plants, relying on fungal nutrient resources to grow for their entire life or until they develop into photosynthetic seedlings. In Chile, orchids are represented by 7 genera and 63 species, 27 of which are endemic. Some Chilean species are considered endangered or rare, but many are insufficiently known. This study aims to isolate, culture, and identify fungal species found in symbiosis with the endemic Chilean orchids Chloraea collicensis Kraenzl. and Chloraea gavilu Lindl. for their potential to be used in future conservation programs. Roots of both species of orchids were collected in the field and those presenting pelotons were firstly cultured in agar-water and thereafter sub-cultured in potato dextrose agar media. Fungal colony growth was measured under the dissecting microscope. Fungal isolates from C. gavilu showed a higher growth rate than isolates from C. collicensis and could be used as inoculum for seed germination in further studies. Isolated colonies showed morphological characteristics of the form genus Rhizoctonia and presented two nuclei per cell. The ITS-nrDNA sequences confirmed their morphological identification as species of Tulasnella.     

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

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.     

The colonization patterns of different fungi on roots of Cymbidium hybridum plantlets and their respective inoculation effects on growth and nutrient uptake of orchid plantlets

Cymbidium hybridum is one of the most popular pot orchids and cut flowers worldwide. However, the long vegetative growth period and the discordant blooming retarded its mass production. The mixotrophic nutritional mode of some chlorophyllous Cymbidium suggested the essential role of mycorrhizal fungi in the growth of adult green orchids. Here 34 root-associated endophytes were obtained from wild and cultivated Cymbidium and eight strains exhibited obvious growth-promoting effects on the C. hybridum plantlets with increasing root number, root diameter or new bud initiation. Among these, three isolates CL01, ZH3A-3 and CY5-1 with distinct cultural traits and colonization patterns showed better growth-promoting effects. Internal transcribed spacer sequence analyses and morphological observation revealed isolate CL01 belonged to Tulasnella-like Rhizoctonia, ZH3A-3, Umbelopsis nana and CY5-1, Scytalidium lignicola. Microscopic study showed isolate CL01 formed typical orchid mycorrhiza and isolate CY5-1 formed pseudo-mycorrhiza with orchid, whereas hyphae of isolate ZH3A-3 aggregated in the host velamen cells at regular intervals and caused the hypertrophied nucleus and aggregated cytoplasm of neighboring host cell. These three isolates significantly enhanced the increased percentage of total fresh weight of plantlets compared with un-inoculated control (83, 99 and 75 %, respectively). In addition, isolate CL01 increased the N, P, Zn, Cu, Fe contents and ZH3A-3 significantly improved K, Ca, Cu, Mn contents of the symbiotic plantlets compared with control. These results suggested that the mass production of C. hybridum and related orchids could be improved by different beneficial fungi from its parents.     

Studies of Mycorrhizal Fungi of Chinese Orchids and Their Role in Orchid Conservation in China—A Review

China has over 1,200 species of native orchids in nearly 173 genera. About one fourth of native species are of horticultural merit. Some species are of Chinese medicinal value. In fact, the demand on orchid species with high Chinese medicinal values such as Gastrodia elata, Dendrobium offcinale, along with demands on species of cultural importance, such as those in the genus of Cymbidium, is a major factor causing wild populations to diminish and in some cases, drive wild populations to the brink of extinction. These market demands have also driven studies on the role of mycorrhizal fungi in orchid seed germination, seedling and adult growth, and reproduction. Most of these mycorrhizal studies of Chinese orchids, however, are published in Chinese, some in medical journals, and thus overlooked by the mainstream orchid mycorrhizal publications. Yet some of these studies contained interesting discoveries on the nature of the mycorrhizal relationships between orchids and fungi. We present a review of some of these neglected publications. The most important discovery comes from the mycorrhizal studies on G. elata, in which the researchers concluded that those fungi species required to stimulate seed germination are different from those that facilitate the growth of G. elata beyond seedling stages. In addition, presence of the mycorrhizal fungi associated with vegetative growth of post-seedling G. elata hindered the germination of seeds. These phenomena were unreported prior to these studies. Furthermore, orchid mycorrhizal studies in China differ from the mainstream orchid studies in that many epiphytic species (in the genus of Dendrobium, as medicinal herbs) were investigated as well as terrestrial orchids (mostly in the genus Cymbidium, as traditional horticultural species). The different responses between epiphytic and terrestrial orchid seeds to fungi derived from roots suggest that epiphytic orchids may have a more general mycorrhizal relationship with fungi than do terrestrial orchid species during the seed germination stage. To date, orchid mycorrhizal research in China has had a strongly commercial purpose. We suggest that this continuing research on orchid mycorrhizal relationships are a solid foundation for further research that includes more rare and endangered taxa, and more in-situ studies to assist conservation and restoration of the endangered orchids. Knowledge on the identities and roles of mycorrhizal fungi of orchids holds one of the keys to successful restoration and sustainable use of Chinese orchids.