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Background and Aims Plant–fungus symbioses may experience temporal turnover during the ontogenetic or phenological development of the host, which can influence the ecological requirements of the host plant. In this study, we investigate temporal turnover of Ceratobasidiaceae orchid mycorrhizal fungal (OMF) communities in Prasophyllum (Orchidaceae), asking whether OMF communities are subject to temporal change attributable to orchid phenology or ontogeny. Methods Roots of adult Prasophyllum frenchii , Prasophyllum lindleyanum and Prasophyllum 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 with 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 whether fungal OTUs were mycorrhizal. Key Results A persistent core of OMF was 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 that 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. [ABSTRACT FROM AUTHOR]

Fungal biodiversity significantly influences ecosystem dynamics through various interactions with plants, ranging from pathogenic to mutually beneficial associations. This study explores the fungal diversity associated with an ornamental orchid genus Epidendrum that is widely propagated globally but native to northern South America. Root samples were collected from Epidendrum in diverse geographic locations: Brazil, Colombia, Germany, Spain and six South African provinces. Fungal biodiversity was catalogued from the genomic DNA extracted from these roots using fungal-specific primers and Illumina MiSeq sequencing. Bioinformatic and statistical analyses revealed significant fungal diversity in the roots, with distinct dominant orders in each geographic region. Among the South African samples, significant differences were found in alpha diversity indices and species richness. Even though samples originating from different provinces overlapped in the PCoA plot, PERMANOVA indicated a significant difference in the fungal biodiversity, which was further supported by PERMDISP. In the global dataset, alpha diversity indices were insignificant, but species richness was. In the PCoA plot, data points clustered by sampling sites, indicating substantial differences in fungal biodiversity between the samples. This was validated by PERMANOVA and PERMDISP analyses. Outcomes from the core fungal analyses showed Epidendrum retained a conserved set of fungal orders from its native habitat when it transitioned to exotic regions, while it also formed new associations with local fungal communities in these introduced regions. These findings highlight the role of both core and region-specific fungal communities in the ecological adaptability and success of this widely planted orchid genus. [ABSTRACT FROM AUTHOR]

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 (δ 13C, δ 15N, δ 2H, δ 18O) 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. [ABSTRACT FROM AUTHOR]

Societal Impact Statement Vanilla is one of the most valuable spices in the world. In Madagascar and La Réunion, the world's leading producers, vanilla is of great economic and cultural importance. Like all orchids, vanilla plants associate with mycorrhizal fungi in their roots forming mutualistic associations that allow them to grow and thrive. Understanding the diversity of mycorrhizal fungi adapted to vanilla cultivation, particularly in the Indian Ocean islands where they have never previously been studied, is becoming a necessity for maintaining vanilla crops in these regions in the face of climate change and the emergence of new pathogens. Summary The vanilla orchid (Vanilla spp.) is one of the most valuable cultivated plants worldwide. As with all orchids, vanillas form mycorrhizal associations with fungi in their roots, but their fungal partners have not been investigated outside their native geographic range in Central America. We investigated the whole fungal and mycorrhizal associations in cultivated vanilla (Vanilla planifolia) by sequencing the fungal ITS‐2 marker in the terrestrial and aerial roots using a metabarcoding approach. We selected plants cultivated in three conditions (i.e., cultivation under shade house, in openfield, or in the understory) in one locality of La Réunion island (Indian Ocean) and tested for a possible effect of cultivation practices on fungal communities. Cultivated vanillas in La Réunion mainly associate with Tulasnellaceae (75 OTUs) and Ceratobasidiaceae (8 OTUs). Among the seven most abundant Tulasnellaceae, six are similar to fungi detected in the roots of cultivated vanillas in Central America or in the roots of native orchids in La Réunion. Cultivation practices impacted both total fungal and mycorrhizal community compositions with no clear effect on fungal richness. Notably, Tulasnellaceae and Ceratobasidiaceae were scarce in aerial roots, except in the traditional cultivation in the forest understory. These results shed light on the geographical origins of mycorrhizal fungi of cultivated vanillas in La Réunion and show that they form a pool of both locally and globally distributed fungal partners. These mycorrhizal communities vary according to cultivation practices, and their potential roles in plant nutrition and resistance against pathogens await further attention.

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Epiphytic orchids comprise 68% of vascular epiphytes globally; nevertheless, many are endangered. One such epiphytic orchid is Phalaenopsis japonica, which is widely used in the floricultural industry. This study aimed to identify the mycorrhizal fungi of adult P. japonica and their roles in seed germination and seedling development. Root samples were collected from 32 adults across 4 sites in southern Japan, and mycorrhizal fungi were identified using Sanger and high-throughput sequencing (HTS). The results show phylogenetically diverse mycobionts, mainly Ceratobasidiaceae (CE) and Tulasnellaceae (TU), with dominant OTUs designated CE6 and CE22. Sanger sequencing found 9 OTUs, 4 CE, and 5 TU; HTS detected 22 OTUs, 4 CE, 16 TU, and 2 Serendipitaceae. Seeds inoculated with CE6 improved germination and protocorm development compared with other strains. In addition, asymbiotic seedlings inoculated with CE6, CE22, and TU18 displayed varying effects in growth, with CE6 being the most notable. While TU18 did not promote seed germination, it effectively promoted leaf development in seedlings. Overall, Ceratobasidiaceae was predominantly associated with seed germination, seedling growth, and the adult stages, with CE6 and CE22 becoming the primary partners throughout the life history of P. japonica. Our findings illuminate mycorrhizal symbiosis in epiphytic habitats, offering conservation and commercial production insights. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Social Impact Statement Vanilla planifolia is the source of the spice vanilla. This study is part of an international initiative to study the biology, including mycorrhizal fungi and cultivation practices of vanilla to improve its production in Mexico. The study focused on documenting mycorrhizal fungal diversity in vanilla. It also provided preliminary data on differences in mycorrhizal fungal communities associated with cultivation practices. A richer mycorrhizal community was observed in vanilla growing in a wild natural farm compared with those from a highly managed farm. Our research provides insights for sustainable vanilla production that can benefit Mexican farming communities. Summary Relatively little is known regarding differences in root symbionts (i.e., mycorrhizae) between epiphytic and terrestrial orchids. We characterized the mycorrhizal fungal communities of aerial and terrestrial roots of the orchid, Vanilla planifolia, from four Mexican farms representing different management systems. Amplicon sequencing identified 40 putative mycorrhizal fungi based on ITS sequence data, these included traditional orchid mycorrhizal fungi such as Ceratobasidium, and Thanatephorus in the order Cantharellales as well as Serendipitaceae in the order Sebacinales, and species of several genera traditionally considered as ectomycorrhizal fungi. Mycorrhizal fungal communities were similar in aerial and terrestrial roots, but differed in read abundances. Plants growing in wild‐natural conditions hosted a richer, but not statistically different, community of mycorrhizal fungi in comparison with plants in the highly managed farm. Soil characteristics including texture, organic matter, N, P, and K do not explain differences between fungal communities at these farms. This is one of the first reports of a diverse community of fungi traditionally considered to form ectomycorrhizas in association with aerial orchid roots. Further research is needed to understand the functional role of these putative mycorrhizal fungi in the ecology of V. planifolia, and if ectomycorrhizal fungi commonly occur in other hemiepiphytic and epiphytic orchids.

Partially mycoheterotrophic plant species obtain organic carbon, via both photosynthesis and mycorrhizal symbiosis. In this study, we investigated the mycorrhizal fungi association and nutritional mode of Cheirostylis liukiuensis, which is suspected to be a partial mycoheterotrophic plant, due to its characteristic reduced underground organs, low-light growth environment, and some fully mycoheterotrophic species in the phylogenetically related genera. Molecular analysis of the dominant mycobiont and stable isotope analysis suggested that C. liukiuensis is a partial mycoheterotrophic plant predominantly associate with non-ectomycorrhizal Ceratobasidiaceae fungi. As examples of partial mycoheterotrophic orchids exploiting non-ectomycorrhizal rhizoctonia are still limited, this study provides valuable information on the nutritional modes of green orchids. [ABSTRACT FROM AUTHOR]

Abstract 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.

Social Impact Statement: Vanilla planifolia is the source of the spice vanilla. This study is part of an international initiative to study the biology, including mycorrhizal fungi and cultivation practices of vanilla to improve its production in Mexico. The study focused on documenting mycorrhizal fungal diversity in vanilla. It also provided preliminary data on differences in mycorrhizal fungal communities associated with cultivation practices. A richer mycorrhizal community was observed in vanilla growing in a wild natural farm compared with those from a highly managed farm. Our research provides insights for sustainable vanilla production that can benefit Mexican farming communities. Summary: Relatively little is known regarding differences in root symbionts (i.e., mycorrhizae) between epiphytic and terrestrial orchids. We characterized the mycorrhizal fungal communities of aerial and terrestrial roots of the orchid, Vanilla planifolia, from four Mexican farms representing different management systems.Amplicon sequencing identified 40 putative mycorrhizal fungi based on ITS sequence data, these included traditional orchid mycorrhizal fungi such as Ceratobasidium, and Thanatephorus in the order Cantharellales as well as Serendipitaceae in the order Sebacinales, and species of several genera traditionally considered as ectomycorrhizal fungi. Mycorrhizal fungal communities were similar in aerial and terrestrial roots, but differed in read abundances.Plants growing in wild‐natural conditions hosted a richer, but not statistically different, community of mycorrhizal fungi in comparison with plants in the highly managed farm. Soil characteristics including texture, organic matter, N, P, and K do not explain differences between fungal communities at these farms.This is one of the first reports of a diverse community of fungi traditionally considered to form ectomycorrhizas in association with aerial orchid roots. Further research is needed to understand the functional role of these putative mycorrhizal fungi in the ecology of V. planifolia, and if ectomycorrhizal fungi commonly occur in other hemiepiphytic and epiphytic orchids. [ABSTRACT FROM AUTHOR]

Abstract 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.

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. [ABSTRACT FROM AUTHOR]

Aim: Biodiversity is known to be unevenly distributed along latitudinal gradients. While this pattern has been observed for many different organisms, it is unclear whether the distributions of ecologically important belowground mutualists, such as orchid mycorrhizal fungi (OMF), also vary according to latitude. Location: Europe. Taxon: Spiranthes spiralis (L. Chevall) Orchidaceae. Methods: We sampled 37 populations over a >3,000 km latitudinal gradient of the European orchid Spiranthes spiralis to test whether the diversity and community composition of OMF are influenced by latitude. For this, we sequenced the broad spectrum ITS3/4OF region to identify OMF and quantified environmental variables that may influence their occurrence. To assess whether seed germination was affected by suitable OMF in different regions, we performed a reciprocal translocation germination experiment at the northern, central and southern part of its range. Results: In total, 75 OMF taxa were associated with S. spiralis, the majority belonging to the Ceratobasidiaceae and Thelephoraceae. Both taxonomic and phylogenetic OMF diversity decreased with increasing latitude. Three Ceratobasidiaceae taxa were widespread, but the number of sequences of two of these taxa decreased with increasing latitude. Reciprocal translocation germination experiments revealed that S. spiralis seeds germinated in each region regardless of seed provenance, but germination rates were highest in the southern part of its range where mycorrhizal diversity was highest. Main conclusions: OMF diversity decreased with increasing latitude, indicating that communities of OMF may be shaped by latitudinal gradients. These results highlight the need to understand the factors that determine the distribution of belowground mutualists, which are essential to predict how plant distributions will shift under future environmental change. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Knowledge of fungal root-associates is essential for effective conservation of tropical epiphytic orchids. We investigated the diversity of root-associated fungi of Cyrtochilum myanthum , Scaphyglottis punctulata and Stelis superbiens from a tropical mountain rainforest in southern Ecuador, using a culture dependent approach. We identified 115 fungal isolates, corresponding to 49 fungal OTUs, based on sequences of the nrDNA ITS and partial 28S region. Members of Ascomycota were unambiguously dominant (37 OTUs), including Trichoderma sp. as the most frequent taxon. Members of Basidiomycota (Agaricales and Polyporales) and Mucoromycota (Umbelopsidales and Mortierellales) were also identified. Four potential mycorrhizal OTUs of Tulasnellaceae and Ceratobasidiaceae were isolated from C. myanthum and S. superbiens . Fungal community composition was examined using Sørensen and Jaccard indices of similarity. Alfa diversity was significantly different between C. myanthum and S. superbiens . No difference in beta diversity of the fungal communities between the 3 orchid species and the collecting sites was detected. The study revealed a high diversity of fungi associated with orchid roots. Our results contribute to a better understanding of specific relationships between epiphytic orchids and their root-associated fungi. [ABSTRACT FROM AUTHOR]

• 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Host breadth is often assumed to have no evolutionary significance in broad interactions because of the lack of cophylogenetic patterns between interacting species. Nonetheless, the breadth and suite of hosts utilized by one species may have adaptive value, particularly if it underlies a common ecological niche among hosts. Here, we present a preliminary assessment of the evolution of mycorrhizal specificity in 12 closely related orchid species (genera Goodyera and Hetaeria) using DNA-based methods. We mapped specificity onto a plant phylogeny that we estimated to infer the evolutionary history of the mycorrhiza from the plant perspective, and hypothesized that phylogeny would explain a significant portion of the variance in specificity of plants on their host fungi. Sampled plants overwhelmingly associated with genus Ceratobasidium, but also occasionally with some ascomycetes. Ancestral mycorrhizal specificity was narrow in the orchids, and broadened rarely as Goodyera speciated. Statistical tests of phylogenetic inertia suggested some support for specificity varying with increasing phylogenetic distance, though only when the phylogenetic distance between suites of fungi interacting with each plant taxon were taken into account. These patterns suggest a role for phylogenetic conservatism in maintaining suits of fungal hosts among plants. We stress the evolutionary importance of host breadth in these organisms, and suggest that even generalists are likely to be constrained evolutionarily to maintaining associations with their symbionts. [ABSTRACT FROM AUTHOR]

We demonstrated that "orchid mycorrhiza," a specialized mycorrhizal type, appeared in the common ancestor of the largest plant family Orchidaceae and that the fungal partner shifted from Glomeromycota to a particular dade of Basidiomycota in association with this character evolution. Several unique mycorrhizal characteristics may have contributed to the diversification of the family. However, the origin of orchid mycorrhiza and the diversity of mycobionts across orchid lineages still remain obscure. In this study, we investigated the mycorrhizae of five Apostasia taxa, members of the earliest-diverging dade of Orchidaceae. The results of molecular identification using nrDNA ITS and LSU regions showed that Apostasia mycorrhizal fungi belong to families Botryobasidiaceae and Ceratobasidiaceae, which fall within the order Cantharellales of Basidiomycota. Most major clades in Orchidaceae also form mycorrhizae with members of Cantharellales, while the sister group and other closely related groups to Orchidaceae (i.e., Asparagales except for orchids and the "commelinid" families) ubiquitously form symbioses with Glomeromycota to form arbuscular mycorrhizae. This pattern of symbiosis indicates that a major shift in fungal partner occurred in the common ancestor of the Orchidaceae. [ABSTRACT FROM AUTHOR]

Lady's slipper orchids (Cypripediumspp.) are rare terrestrial plants that grow throughout the temperate Northern Hemisphere. Like all orchids, they require mycorrhizal fungi for germination and seedling nutrition. The nutritional relationships of adultCypripediummycorrhizae are unclear; however,Cypripediumdistribution may be limited by mycorrhizal specificity, whether this specificity occurs only during the seedling stage or carries on into adulthood. We attempted to identify the primary mycorrhizal symbionts for 100Cypripediumplants, and successfully did so with twoCypripedium calceolus, 10Cypripedium californicum, sixCypripedium candidum, 16Cypripedium fasciculatum, twoCypripedium guttatum, 12Cypripedium montanum, and 11Cypripedium parviflorumplants from a total of 44 populations in Europe and North America, yielding fungal nuclear large subunit and mitochondrial large subunit sequence and RFLP (restriction fragment length polymorphism) data for 59 plants. Because orchid mycorrhizal fungi are typically observed without fruiting structures, we assessed fungal identity through direct PCR (polymerase chain reaction) amplification of fungal genes from mycorrhizally colonized root tissue. Phylogenetic analysis revealed that the great majority ofCypripediummycorrhizal fungi are members of narrow clades within the fungal family Tulasnellaceae. Rarely occurring root endophytes include members of the Sebacinaceae, Ceratobasidiaceae, and the ascomycetous genus,Phialophora.C. californicumwas the only orchid species with apparently low specificity, as it associated with tulasnelloid, ceratobasidioid, and sebacinoid fungi in roughly equal proportion. Our results add support to the growing literature showing that high specificity is not limited to nonphotosynthetic plants, but also occurs in photosynthetic ones. [ABSTRACT FROM AUTHOR]

The environmental distribution of non-obligate orchid mycorrhizal (OM) symbionts belonging to the 'rhizoctonia' complex remains elusive. Some of these fungi, indeed, are undetectable in soil outside the host rhizosphere. A manipulation experiment was performed to assess the importance of neighbouring non-orchid plants and soil as possible reservoirs of OM fungi for Spiranthes spiralis , a widespread photosynthetic European terrestrial orchid species. Fungi of S. spiralis roots were identified by DNA metabarcoding before and 4 months after the removal of the surrounding vegetation and soil. Although such a treatment significantly affected fungal colonization of newly-formed orchid roots, most OM fungi were consistently associated with the host roots. Frequency patterns in differently aged roots suggest that these fungi colonize new orchid roots from either older roots or other parts of the same plant, which may thus represent an environmental source for the subsequent establishment of the OM symbiosis. • We addressed the importance of vegetation and soil as reservoirs of orchid mycobionts. • Removal of other plants and soil impacted Spiranthes spiralis root fungal community. • Most orchid mycorrhizal fungi were consistently associated with the host roots. • Some fungi may colonize mature orchid roots from other parts of the same plant. • Our findings have implications for the interpretation of orchid distribution. [ABSTRACT FROM AUTHOR]