Mode of carbon gain and fungal associations of Neuwiedia malipoensis within the evolutionarily early-diverging orchid subfamily Apostasioideae.

BACKGROUND AND AIMS: The earliest-diverging orchid lineage, Apostasioideae, consists only of two genera: Apostasia and Neuwiedia. Previous reports of Apostasia nipponica indicated a symbiotic association with an ectomycorrhiza-forming Ceratobasidiaceae clade and partial utilization of fungal carbon during the adult stage. However, the trophic strategy of Neuwiedia throughout its development remains unidentified. To further improve our understanding of mycoheterotrophy in the Apostasioideae, this study focused on Neuwiedia malipoensis examining both the mycorrhizal association and the physiological ecology of this orchid species across various development stages. METHODS: We identified the major mycorrhizal fungi of N. malipoensis protocorm, leafy seedling and adult stages using molecular barcoding. To reveal nutritional resources utilized by N. malipoensis, we compared stable isotope natural abundances (δ13C, δ15N, δ2H, δ18O) of different developmental stages with those of autotrophic reference plants. KEY RESULTS: Protocorms exhibited an association with saprotrophic Ceratobasidiaceae rather than ectomycorrhiza-forming Ceratobasidiaceae and the 13C signature was characteristic of their fully mycoheterotrophic nutrition. Seedlings and adults were predominantly associated with saprotrophic fungi belonging to the Tulasnellaceae. While 13C and 2H stable isotope data revealed partial mycoheterotrophy of seedlings, it is unclear to what extent the fungal carbon supply is reduced in adult N. malipoensis. However, the 15N enrichment of mature N. malipoensis suggests partially mycoheterotrophic nutrition. Our data indicated a transition in mycorrhizal partners during ontogenetic development with decreasing dependency of N. malipoensis on fungal nitrogen and carbon. CONCLUSIONS: The divergence in mycorrhizal partners between N. malipoensis and A. nipponica indicates different resource acquisition strategies and allows various habitat options in the earliest-diverging orchid lineage, Apostasioideae. While A. nipponica relies on the heterotrophic carbon gain from its ectomycorrhizal fungal partner and thus on forest habitats, N. malipoensis rather relies on own photosynthetic carbon gain as an adult, allowing it to establish in habitats as widely distributed as those where Rhizoctonia fungi occur.

Temporal turnover of Ceratobasidiaceae orchid mycorrhizal fungal communities with ontogenetic and phenological development in Prasophyllum (Orchidaceae).

BACKGROUND AND AIMS: Plant-fungus symbioses may experience temporal turnover during the host's ontogenetic or phenological development, which can influence the host plant's ecological requirements. This study investigates temporal turnover of Ceratobasidiaceae orchid mycorrhizal fungal (OMF) communities in Prasophyllum (Orchidaceae), asking if OMF communities are subject to temporal change due to orchid phenology or ontogeny. METHODS: Roots of adult Prasophyllum frenchii, P. lindleyanum and P. sp. aff. validum from Australia were sampled between autumn and spring. Seed was sown in situ as 'baits' to explore the mycorrhizal associations of germinating protocorms, which were compared to OMF in roots of co-occurring adult plants. Culture dependent and independent sequencing methods were used to amplify the internal transcribed spacer and mitochondrial large subunit loci, with sequences assigned to Operational Taxonomic Units (OTUs) in phylogenetic analyses. Germination trials were used to determine if fungal OTUs were mycorrhizal. KEY RESULTS: A persistent core of OMF associated with Prasophyllum, with Ceratobasidiaceae OMF dominant in all three species. Phenological turnover occurred in P. lindleyanum and P. sp. aff. validum, but not in P. frenchii, which displayed specificity to a single OTU. Ontogenetic turnover occurred in all species. However, phenological and ontogenetic turnover was typically driven by the presence or absence of infrequently detected OTUs in populations that otherwise displayed specificity to one or two dominant OTUs. Ex situ germination trials showed 13 of 14 tested OTUs supported seed germination in their host orchid, including eight OTUs that were not found in protocorms in situ. CONCLUSIONS: An understanding of OMF turnover can have practical importance for the conservation of threatened orchids and their mycorrhizal partners. However, frameworks for classifying OMF turnover should focus on OTUs important to the life cycle of the host plant, which we suggest are likely to be those that are frequently detected or functionally significant.

Co-occurring orchid species associated with different low-abundance mycorrhizal fungi from the soil in a high-diversity conservation area in Denmark.

Plant-fungal interactions are ubiquitous across ecosystems and contribute significantly to plant ecology and evolution. All orchids form obligate symbiotic relationships with specific fungi for germination and early growth, and the distribution of terrestrial orchid species has been linked to occurrence and abundance of specific orchid mycorrhizal fungi (OMF) in the soil. The availability of OMF can therefore be a habitat requirement that is relevant to consider when establishing management and conservation strategies for threatened orchid species, but knowledge on the spatial distribution of OMF in soil is limited. We here studied the mycorrhizal associations of three terrestrial orchid species (Anacamptis pyramidalis, Orchis purpurea and Platanthera chlorantha) found in a local orchid diversity hotspot in eastern Denmark, and investigated the abundance of the identified mycorrhizal fungi in the surrounding soil. We applied ITS metabarcoding to samples of orchid roots, rhizosphere soil and bulk soil collected at three localities, supplemented with standard barcoding of root samples with OMF specific primers, and detected 22 Operational Taxonomic Units (OTUs) putatively identified as OMF. The three orchid species displayed different patterns of OMF associations, supporting the theory that association with specific fungi constitutes part of an orchid's ecological niche allowing co-occurrence of many species in orchid-rich habitats. The identified mycorrhizal partners in the basidiomycete families Tulasnellaceae and Ceratobasidiaceae (Cantharallales) were detected in low abundance in rhizosphere soil, and appeared almost absent from bulk soil at the localities. This finding highlights our limited knowledge of the ecology and trophic mode of OMF outside orchid tissues, as well as challenges in the detection of specific OMF with standard methods. Potential implications for management and conservation strategies are discussed.

Diversity of Mycorrhizal Fungi in Temperate Orchid Species: Comparison of Culture-Dependent and Culture-Independent Methods.

Many orchid species are endangered due to anthropogenic pressures such as habitat destruction and overharvesting, meanwhile, all orchids rely on orchid mycorrhizal fungi (OMF) for seed germination and seedling growth. Therefore, a better understanding of this intimate association is crucial for orchid conservation. Isolation and identification of OMF remain challenging as many fungi are unculturable. In our study, we tested the efficiency of both culture-dependent and culture-independent methods to describe OMF diversity in multiple temperate orchids and assessed any phylogenetic patterns in cultivability. The culture-dependent method involved the cultivation and identification of single pelotons (intracellular hyphal coils), while the culture-independent method used next-generation sequencing (NGS) to identify root-associated fungal communities. We found that most orchid species were associated with multiple fungi, and the orchid host had a greater impact than locality on the variability in fungal communities. The culture-independent method revealed greater fungal diversity than the culture-dependent one, but despite the lower detection, the isolated fungal strains were the most abundant OMF in adult roots. Additionally, the abundance of NGS reads of cultured OTUs was correlated with the extent of mycorrhizal root colonization in orchid plants. Finally, this limited-scale study tentatively suggests that the cultivability character of OMF may be randomly distributed along the phylogenetic trees of the rhizoctonian families.

Variability in Nutrient Use by Orchid Mycorrhizal Fungi in Two Medium Types.

Orchid mycorrhizal fungi (OMF) from the rhizoctonia aggregate are generally considered to be soil saprotrophs, but their ability to utilize various nutrient sources has been studied in a limited number of isolates cultivated predominantly in liquid media, although rhizoctonia typically grow on the surface of solid substrates. Nine isolates representing the key OMF families (Ceratobasidiaceae, Tulasnellaceae and Serendipitaceae), sampled in Southern France and the Czech Republic, were tested for their ability to utilize carbon (C), nitrogen (N) and phosphorus (P) sources in vitro in both liquid and solid media. The isolates showed significant inter- and intra-familiar variability in nutrient utilization, most notably in N sources. Isolates produced generally larger amounts of dry biomass on solid medium than in liquid one, but some isolates showed no or limited biomass production on solid medium with particular nutrient sources. The largest amount of biomass was produced by isolates from the family Ceratobasidiaceae on most sources in both medium types. The biomass production of Tulasnellaceae isolates was affected by their phylogenetic relatedness on all sources and medium types. The ability of isolates to utilize particular nutrients in a liquid medium but not a solid one should be considered when optimizing solid media for symbiotic orchid seed germination and in understanding of OMF functional traits under in situ conditions.

Molecular evidence supports simultaneous association of the achlorophyllous orchid Chamaegastrodia inverta with ectomycorrhizal Ceratobasidiaceae and Russulaceae.

BACKGROUND: Achlorophyllous orchids are mycoheterotrophic plants, which lack photosynthetic ability and associate with fungi to acquire carbon from different environmental sources. In tropical latitudes, achlorophyllous forest orchids show a preference to establish mycorrhizal relationships with saprotrophic fungi. However, a few of them have been recently found to associate with ectomycorrhizal fungi and there is still much to be learned about the identity of fungi associated with tropical orchids. The present study focused on mycorrhizal diversity in the achlorophyllous orchid C. inverta, an endangered species, which is endemic to southern China. The aim of this work was to identify the main mycorrhizal partners of C. inverta in different plant life stages, by means of morphological and molecular methods. RESULTS: Microscopy showed that the roots of analysed C. inverta samples were extensively colonized by fungal hyphae forming pelotons in root cortical cells. Fungal ITS regions were amplified by polymerase chain reaction, from DNA extracted from fungal mycelia isolated from orchid root samples, as well as from total root DNA. Molecular sequencing and phylogenetic analyses showed that the investigated orchid primarily associated with ectomycorrhizal fungi belonging to a narrow clade within the family Ceratobasidiaceae, which was previously detected in a few fully mycoheterotrophic orchids and was also found to show ectomycorrhizal capability on trees and shrubs. Russulaceae fungal symbionts, showing high similarity with members of the ectomycorrhizal genus Russula, were also identified from the roots of C. inverta, at young seedling stage. Ascomycetous fungi including Chaetomium, Diaporthe, Leptodontidium, and Phomopsis genera, and zygomycetes in the genus Mortierella were obtained from orchid root isolated strains with unclear functional role. CONCLUSIONS: This study represents the first assessment of root fungal diversity in the rare, cryptic and narrowly distributed Chinese orchid C. inverta. Our results provide new insights on the spectrum of orchid-fungus symbiosis suggesting an unprecedented mixed association between the studied achlorophyllous forest orchid and ectomycorrhizal fungi belonging to Ceratobasidiaceae and Russulaceae. Ceratobasidioid fungi as dominant associates in the roots of C. inverta represent a new record of the rare association between the identified fungal group and fully mycoheterotrophic orchids in nature.

Draft genome sequence of fastidious pathogen Ceratobasidium theobromae, which causes vascular-streak dieback in Theobroma cacao.

BACKGROUND: Ceratobasidium theobromae, a member of the Ceratobasidiaceae family, is the causal agent of vascular-streak dieback (VSD) of cacao, a major threat to the chocolate industry in the South-East Asia. The fastidious pathogen is very hard to isolate and maintain in pure culture, which is a major bottleneck in the study of its genetic diversity and genome. RESULT: This study describes for the first time, a 33.90 Mbp de novo assembled genome of a putative C. theobromae isolate from cacao. Ab initio gene prediction identified 9264 protein-coding genes, of which 800 are unique to C. theobromae when compared to Rhizoctonia spp., a closely related group. Transcriptome analysis using RNA isolated from 4 independent VSD symptomatic cacao stems identified 3550 transcriptionally active genes when compared to the assembled C. theobromae genome while transcripts for only 4 C. theobromae genes were detected in 2 asymptomatic stems. De novo assembly of the non-cacao associated reads from the VSD symptomatic stems uniformly produced genes with high identity to predicted genes in the C. theobromae genome as compared to Rhizoctonia spp. or genes found in Genbank. Further analysis of the predicted C. theobromae transcriptome was carried out identifying CAZy gene classes, KEGG-pathway associated genes, and 138 putative effector proteins. CONCLUSION: These findings put forth, for the first time, a predicted genome for the fastidious basidiomycete C. theobromae causing VSD on cacao providing a model for testing and comparison in the future. The C. theobromae genome predicts a pathogenesis model involving secreted effector proteins to suppress plant defense mechanisms and plant cell wall degrading enzymes.

A leafless epiphytic orchid, Taeniophyllum glandulosum Blume (Orchidaceae), is specifically associated with the Ceratobasidiaceae family of basidiomycetous fungi.

Leafless epiphytes in the Orchidaceae undergo a morphological metamorphosis in which the root has chloroplast-containing cortical cells and is the sole photosynthetic organ for carbon gain. All orchids are entirely dependent on mycorrhizal fungi for their carbon supply during seed germination, and this mycorrhizal association generally persists in adult plants. However, our knowledge of the mycorrhizal association of leafless epiphytic orchids remains limited, and the contribution of the mycorrhizal association to nutrient acquisition in these orchid species is largely unknown. In this study, the mycorrhizal fungi of a leafless epiphytic orchid, Taeniophyllum glandulosum, were identified molecularly using 68 mature plants and 17 seedlings. In total, 187 fungal internal transcribed spacer sequences were obtained, of which 99% were identified as Ceratobasidiaceae. These sequences were classified into five operational taxonomic units (OTUs) based on 97% sequence similarity. The most frequent sequence was OTU1, which accounted for 91% of all Ceratobasidiaceae sequences, although other phylogenetically distinct Ceratobasidiaceae fungi were detected. These results show that T. glandulosum is specifically associated with a particular group of Ceratobasidiaceae. All mycorrhizal fungi found in T. glandulosum seedlings belonged to OTU1, which was also found in adult plants on the same host tree. The mycorrhizal fungi from 13 host tree species were compared, and T. glandulosum was preferentially associated with OTU1 on 11 tree species. In conclusion, T. glandulosum is specifically associated with Ceratobasidiaceae fungi and this specific association remains throughout the orchid life cycle and is found on divergent host tree species.

Fine-scale spatial distribution of orchid mycorrhizal fungi in the soil of host-rich grasslands.

Mycorrhizal fungi are essential for the survival of orchid seedlings under natural conditions. The distribution of these fungi in soil can constrain the establishment and resulting spatial arrangement of orchids at the local scale, but the actual extent of occurrence and spatial patterns of orchid mycorrhizal (OrM) fungi in soil remain largely unknown. We addressed the fine-scale spatial distribution of OrM fungi in two orchid-rich Mediterranean grasslands by means of high-throughput sequencing of fungal ITS2 amplicons, obtained from soil samples collected either directly beneath or at a distance from adult Anacamptis morio and Ophrys sphegodes plants. Like ectomycorrhizal and arbuscular mycobionts, OrM fungi (tulasnelloid, ceratobasidioid, sebacinoid and pezizoid fungi) exhibited significant horizontal spatial autocorrelation in soil. However, OrM fungal read numbers did not correlate with distance from adult orchid plants, and several of these fungi were extremely sporadic or undetected even in the soil samples containing the orchid roots. Orchid mycorrhizal 'rhizoctonias' are commonly regarded as unspecialized saprotrophs. The sporadic occurrence of mycobionts of grassland orchids in host-rich stands questions the view of these mycorrhizal fungi as capable of sustained growth in soil.

Temporal patterns of orchid mycorrhizal fungi in meadows and forests as revealed by 454 pyrosequencing.

Orchid mycorrhizal (OrM) symbionts play a key role in the growth of orchids, but the temporal variation and habitat partitioning of these fungi in roots and soil remain unclear. Temporal changes in root and rhizosphere fungal communities of Cypripedium calceolus, Neottia ovata and Orchis militaris were studied in meadow and forest habitats over the vegetation period by using 454 pyrosequencing of the full internal transcribed spacer (ITS) region. The community of typical OrM symbionts differed by plant species and habitats. The root fungal community of N. ovata changed significantly in time, but this was not observed in C. calceolus and O. militaris. The rhizosphere community included a low proportion of OrM symbionts that exhibited a slight temporal turnover in meadow habitats but not in forests. Habitat differences in OrM and all fungal associates are largely attributable to the greater proportion of ectomycorrhizal fungi in forests. Temporal changes in OrM fungal communities in roots of certain species indicate selection of suitable fungal species by plants. It remains to be elucidated whether these shifts depend on functional differences inside roots, seasonality, climate or succession.