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Species from Ceratobasidium are known as orchid mycorrhizal, plant pathogens or saprophytic. In mycorrhizal association with orchids, Ceratobasidium can establish a highly specific interaction. Gomesa recurva is an epiphytic orchid occurring in the Brazilian Atlantic Forest. In the present study mycorrhizal fungi were isolated from roots of G. recurva and its phylogenetic positions was investigated. A total of nine isolates were obtained directly from pelotons and submitted to phylogenetic analysis based on alignments of sequences of the internal transcribed spacer (ITS) of the nuclear ribosomal DNA. Phylogenetic analyzes, along with morphological data, revealed that isolates are distinct from all known Ceratobasidium species and a new species, namely Ceratobasidium gomesae sp. nov., is proposed. Given the dependence on mycorrhizae for germination and protocorm development, knowledge about orchid symbionts is an important factor in managing the conservation of these plants, and future studies may investigate the ability of C. gomesae to promote seed germination.

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.

Mixotrophy (MX, also called partial mycoheterotrophy) in plants is characterized by isotopic abundances that differ from those of autotrophs. Previous studies have evaluated mycoheterotrophy in MX plants associated with fungi of similar ecological characteristics, but little is known about the differences in the relative abundances of 13C and 15N in an orchid species that associates with several different mycobionts species. Since the chlorophyllous orchid Cremastra variabilis Nakai associates with various fungi with different ecologies, we hypothesized that it may change its relative abundances of 13C and 15N depending on the associated mycobionts. We investigated mycobiont diversity in the chlorophyllous orchid C. variabilis together with the relative abundance of 13C and 15N and morphological underground differentiation (presence or absence of a mycorhizome with fungal colonization). Rhizoctonias (Tulasnellaceae, Ceratobasidiaceae, Sebacinales) were detected as the main mycobionts. High differences in δ13C values (– 34.7 to – 27.4 ‰) among individuals were found, in which the individuals associated with specific Psathyrellaceae showed significantly high relative abundance of 13C. In addition, Psathyrellaceae fungi were always detected on individuals with mycorhizomes. In the present study, MX orchid association with non-rhizoctonia saprobic fungi was confirmed, and the influence of mycobionts on morphological development and on relative abundance of 13C and 15N was discovered. Cremastra variabilis may increase opportunities to gain nutrients from diverse partners, in a bet-hedging plasticity that allows colonization of various environmental conditions.

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.

PREMISE Orchids depend primarily on mycorrhizal fungi to obtain nutrients throughout their life cycle. Epiphytic orchids account for 69% of orchid diversity. The unstable availability of water and nutrients in their arboreal habitats often results in severe water and nutrient stresses. Consequently, mycorrhizal associations may be important for the survival of epiphytic orchids, but our understanding thereof remains limited. Here, we investigated the mycorrhizal community in a single epiphytic orchid species, using more samples than in any previous study. METHODS We assessed the mycorrhizal communities of Thrixspermum japonicum, one of the most common epiphytic orchids in the temperate region of Japan. In total, 144 individuals were collected from 28 host tree species at 20 sites across 1300 km. The mycorrhizal fungi were identified based on nuclear ribosomal DNA internal transcribed spacer sequences and assigned operational taxonomic units (OTUs) based on 97% sequence similarity. RESULTS We obtained 24 OTUs; 9 belonged to the Ceratobasidiaceae and 15 to the Tulasnellaceae. These OTUs are widely distributed throughout the phylogenetic trees of the two fungal families. However, a single Ceratobasidiaceae OTU accounted for 49.7% of all fungal sequences and was predominant in samples from 15 host tree species and 12 sites. CONCLUSIONS Our results imply that despite having a broad range of mycorrhizal partners, T. japonicum was predominantly associated with a single fungal taxon at most of the sites among the host-tree species investigated. These findings contribute to elucidating mycorrhizal symbiosis in epiphytic habitats.

Interaction with orchid mycorrhizal fungi (OMF) is essential to all members of the Orchidaceae, yet we know little about whether or how OMF abundances in substrates shape orchid populations. While root-associated OMF diversity is catalogued frequently, technological constraints have impeded the assessments of OMF communities in substrates until recently, thereby limiting the ability to link OMF communities in a habitat to population responses. Furthermore, there is some evidence that edaphic and microclimatic conditions impact OMF in soil, yet we lack an understanding of the coupled influences of abiotic environment and OMF structure on orchid population dynamics. To discover the linkages between abiotic environment, OMF community structure, and population size, we characterized the microclimatic conditions, soil physicochemistry, and OMF communities hosted by roots and soil across large and small populations of a terrestrial orchid endemic to California Floristic Province in North America. By using high-throughput sequencing of the ITS2 region of nrDNA amplified from root and soil DNAs, we determined that both roots and soil of larger populations, which were high in phosphorus but low in zinc, organic matter, and silt, were dominated by Tulasnellaceae OTUs. In comparison, roots and soil from smaller populations of the orchid hosted higher relative abundances of the Ceratobasidiaceae. In this multiyear, range-wide study that simultaneously measured habitat environmental conditions, and soil and root OMF communities, our results suggest that soil chemistry is clearly linked to soil and root OMF communities, which then likely alter and shape orchid populations.

Orchids produce small seeds with no energy reserves, relying entirely on orchid mycorrhizal fungi (OMF) for germination. This process, known as symbiotic germination, can lead to different outcomes depending on abiotic factors, such as nutrient availability. Previous studies have shown that nutrient addition has a negative effect on the outcome of symbiotic germination. However, if this effect varies across OMF species, and if it is related to a fungal response to nutrients, remains unknown. This paper examines the effect of fungal identity and nutrient addition (nitrogen and phosphorus) on the germination of the orchid Bipinnula fimbriata using seven OTUs of mycorrhizal fungi from the families Tulasnellaceae and Ceratobasidiaceae. We also evaluated the effect of nutrient addition on mycorrhizal fungi growth rates. Results showed that the nutrient effect on symbiotic germination varied depending on fungal identity. While there was a strong negative effect on symbiotic germination with all Tulasnella OTUs and two Ceratobasidium OTUs, less or no effect was observed on the other two Ceratobasidium OTUs. Further studies are needed to understand the mechanism underlying this variation and how variable is the effect of nutrient addition on symbiotic germination in Orchidaceae and OMF species.

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.

Interspecific interactions play an important role in community assembly. A basic ecological question is whether interactions are specialized (one to one) or generalized (many to many). Specialization of interactions should ideally be assessed across several populations because species could be specialists at a particular site but generalists when several sites are considered. Mycorrhizal interactions are fundamental for orchid life and distribution, but their level of specialization is still under debate. To understand the extent to which epiphytic orchids are specialists in their mycorrhizal interactions, we studied the richness and phylogenetic structure of mycobionts across different sites, and the similarity in the mycobiont composition between coexisting orchid species. We sequenced the nrDNA ITS2 region and explored the mycobiont communities associated with two epiphytic orchids, Epidendrum marsupiale and Cyrtochilum pardinum, at two elevations within two sites in Ecuador. We found 108 OTUs belonging to Serendipitaceae (66), Ceratobasidiaceae (22), Atractiellales (11) and Tulasnellaceae (9). Orchids at the highest elevations hosted the highest OTU richness. The two orchid species shared a high percentage of mycobionts between all sites. No phylogenetic structure within orchid mycorrhizal communities was found at any sites or elevations. Our results indicate that the studied orchids are generalists and share a broad group of mycobionts (16 OTUs) with no apparent niche segregation within or between sites.

Urbanisation has contributed to significant biodiversity loss, yet, urban areas can facilitate biodiversity conservation. For instance, there is evidence of urban trees supporting natural establishments of orchids, the most species-rich plant family on Earth. However, the germination niches-which include both suitable biophysical conditions and orchid mycorrhizal fungus/fungi (OMF)-are not sufficiently known for most species, especially tropical epiphytic orchids. The fate of their dispersed seeds is poorly understood as well. We conducted fungal baiting and seed sowing experiments, next-generation sequencing, generalised linear models, and seed viability tests to detect and identify potential OMF, investigate biophysical factors that influenced OMF availability and orchid germination, and assess seed longevity. Ceratobasidiaceae- and Serendipitaceae-associated OMF were successfully detected in three of four orchid species. In general, orchid species and humus presence had significant effects on OMF availability. Orchid species and temperature were predictive of germination. Post-experiment viability tests revealed that one orchid species, Grammatophyllum speciosum Blume, may produce long-lived seeds. The results suggest that urban trees can support OMF and orchid germination, but both processes are limited by biophysical factors. This study also indicates the possibility of seed persistence among epiphytic species. As orchid germination niches are complex and tend to be unique to individual species, we do not encourage generalisations. In contrast, species-specific information can help formulate useful recommendations towards conservation.

Root-associated endophytic fungi (RAF) are found asymptomatically in almost all plant groups. However, little is known about the compositions and potential functions of RAF communities associated with most Orchidaceae species. In this study, the diversity of RAF was examined in four wild epiphytic orchids, Acampe rigida, Doritis pulcherrima, Renanthera coccinea, and Robiquetia succisa, that occur in southern China. A culture-independent method involving Illumina amplicon sequencing, and an in vitro culture method, were used to identify culturable fungi. The RAF community diversity differed among the orchid roots, and some fungal taxa were clearly concentrated in a certain orchid species, with more OTUs being detected. By investigating mycorrhizal associations, the results showed that 28 (about 0.8%) of the 3527 operational taxonomic units (OTUs) could be assigned as OMF, while the OTUs of non-mycorrhizal fungal were about 99.2%. Among the OMFs, Ceratobasidiaceae OTUs were the most abundant with different richness, followed by Thelephoraceae. In addition, five Ceratobasidium sp. strains were isolated from D. pulcherrima, R. succisa, and R. coccinea roots with high separation rates. These culturable Ceratobasidium strains will provide materials for host orchid conservation and for studying the mechanisms underlying mycorrhizal symbiosis.

Some epiphytic orchids in the tribe Vandeae are characterized by extremely vestigial leaves (even leafless). Thus, their leaves provide only a small proportion of carbon required for their growth and development, while a large portion of carbon may need to be supplied by their roots and mycorrhizal fungi (MF). The MF richness and composition of leafless epiphytic orchids, which belong to numerous genera with diverse ecophysiologies and wide geographical ranges, remain poorly understood. In this study, we identified the MF communities of seven leafless epiphytic species from three orchid genera from up to 17 sites in China using high-throughput sequencing. Our analyses revealed that the leafless epiphytic orchids have a highly specialized association with Ceratobasidiaceae. Several fungal OTUs were found in three different orchid genera and have promoted germinations of Chiloschista and Phalaenopsis, which may have been caused by convergent evolution of leafless epiphytic orchids. Furthermore, the MF composition of Taeniophyllum glandulosum was significantly affected by collection site and host tree. Our study provides new insights into mycorrhizal associations of epiphytic orchids.

Partial mycoheterotrophy, the ability of plants to obtain carbon from fungi throughout their life cycle in combination with photosynthesis, appears to be more common within the Plant Kingdom than previously anticipated. Recent studies using stable isotope analyses have indicated that isotope signatures in partially mycoheterotrophic plants vary widely among species, but the relative contributions of family- or species-specific characteristics and the identity of the fungal symbionts to the observed differences remain unclear. Here, we investigated in detail mycorrhizal communities and isotopic signatures in four co-occurring terrestrial orchids (Platanthera chlorantha, Epipactis helleborine, E. neglecta and the mycoheterotrophic Neottia nidus-avis). All investigated species were mycorrhizal generalists (i.e., associated with a large number of fungi simultaneously), but mycorrhizal communities differed significantly between species. Mycorrhizal communities associating with the two Epipactis species consisted of a wide range of fungi belonging to different families, whereas P. chlorantha and N. nidus-avis associated mainly with Ceratobasidiaceae and Sebacinaceae species, respectively. Isotopic signatures differed significantly between both Epipactis species, with E. helleborine showing near autotrophic behavior and E. neglecta showing significant enrichment in both carbon and nitrogen. No significant differences in photosynthesis and stomatal conductance were observed between the two partially mycoheterotrophic orchids, despite significant differences in isotopic signatures. Our results demonstrate that partially mycoheterotrophic orchids of the genus Epipactis formed mycorrhizas with a wide diversity of fungi from different fungal families, but variation in mycorrhizal community composition was not related to isotope signatures and thus transfer of C and N to the plant. We conclude that the observed differences in isotope signatures between E. helleborine and E. neglecta cannot solely be explained by differences in mycorrhizal communities, but most likely reflect a combination of inherent physiological differences and differences in mycorrhizal communities.

Most green orchids form mycorrhizal associations with rhizoctonia fungi, a polyphyletic group including Serendipitaceae, Ceratobasidiaceae, and Tulasnellaceae. Although accumulating evidence indicated that partial mycoheterotrophy occurs in such so-called rhizoctonia-associated orchids, it remains unclear how much nutrition rhizoctonia-associated orchids obtain via mycoheterotrophic relationships. We investigated the physiological ecology of green and albino individuals of a rhizoctonia-associated orchid Cypripedium debile, by using molecular barcoding of the mycobionts and stable isotope (13C and 15 N) analysis. Molecular barcoding of the mycobionts indicated that the green and albino individuals harbored Tulasnella spp., which formed a clade with the previously reported C. debile mycobionts. In addition, stable isotope analysis showed that both phenotypes were significantly enriched in 13C but not in 15 N. Therefore, green and albino individuals were recognized as partial and full mycoheterotrophs, respectively. The green variants were estimated to obtain 42.5 ± 8.2% of their C from fungal sources, using the 13C enrichment factor of albino individuals as a mycoheterotrophic endpoint. The proportion of fungal-derived C in green C. debile was higher than that reported in other rhizoctonia-associated orchids. The high fungal dependence may facilitate the emergence of albino mutants. Our study provides the first evidence of partial mycoheterotrophy in the subfamily Cypripedioideae. Partial mycoheterotrophy may be more general than previously recognized in the family Orchidaceae.

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.

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.

Fungal endophytes, both mycorrhizal and non-mycorrhizal, are involved in the development of the life cycle of orchids, providing potential beneficial relationships. Here, we assess the succession of changes in the diversity of fungal symbionts associated with a terrestrial temperate orchid species, Anacamptis morio subsp. champagneuxii, over three phenological stages: developed leaves but no stem elongation, flowering, and fruiting. Fungi endophyte associated with roots were obtained by culture in sterile conditions. A total of 18 morphotypes-one Mortierellomycota, two Basidiomycota and 15 Ascomycota-were differentiated, and were also characterized using PCR and DNA sequencing techniques. Only three of the 18 OTUs are shared among the three phenological stages examined: Westerdykella sp., a member of Ceratobasidiaceae, and Fusarium oxysporum, representing a relative abundance of between 28% (fruiting) to 41% (flowering). Our research confirmed that fungal symbionts varied among the different phenological stages examined, the peak of endophyte diversity appearing in the flowering stage. The availability of a diverse mycobiota seems to be important for the survival of orchid plants because it may cover particular physiological needs, and knowledge concerning this mycobiota is of special relevance in the establishment of reliable conservation programmes.

Mycorrhizal fungi play an important role in the germination and growth of orchids essentially influencing their survival, abundance, and spatial distribution. In this study, we investigated the composition of the mycorrhizal fungal community in seven terrestrial orchid species inhabiting Song Mountain, Beijing, China, using Illumina MiSeq high-throughput sequencing. The mycorrhizal communities in the seven orchids were mainly composed of members of the Ceratobasidiaceae, Sebacinales, and Tulasnellaceae, while a number of ectomycorrhizal fungi belonging to the Russulaceae, Tricholomataceae, Thelephoraceae, and Cortinariaceae were occasionally observed. However, the dominant fungal associates and mycorrhizal community differed significantly among the orchid species as well as subhabitats. These findings confirm the previous observation that sympatric orchid species show different preferences for mycorrhizal fungi, which may drive niche partitioning and contribute to their co-occurrence.

Fungal mutualisms are essential for the evolution and diversification of Orchidaceae, yet the fungal symbionts of Pleione orchids are poorly understood because molecular data are unavailable for this genus. Based on ITS-rDNA sequencing for mycobionts of 15 Pleione species (both wild and cultivated plants were included), we conducted phylogenetic analyses for the most dominant mycobionts, and compared the operational taxonomic units (OTUs) of mycorrhizal fungi among species within Pleione. Tulasnellaceae, Ceratobasidiaceae, Serendipitaceae (Sebacinales), Atractiellales, and Auriculariales were reported as putative mycobionts of Pleione. In particular, the mycorrhizal associations between subtropical orchids and Atractiellales have not been observed before. For the dominant mycobionts in the roots of Pleione and its related genera, Bletilla and Coelogyne, we detected no fungal OTU that was shared. Within Pleione, species with a sympatric distribution showed preferences for different fungi. Epiphytic and lithophytic individuals of Pleione albiflora shared OTUs of Tulasnellaceae but harbored different OTUs of Sebacinales, indicating some degree of fungal specificity toward certain habitats. These findings provide new insights into the ecological adaptation and evolution of orchids, and will contribute to the conservation and utilization of species resources.

The presence of appropriate fungal mycobiont for seedling recruitment and plant nutritional support is essential for the long-term survival of orchids in managed or restored habitats. Vanda thwaitesii Hook. f., an epiphytic orchid endemic to the Western Ghats in India and Sri Lanka, endangered due to habitat destruction, necessitates a restoration programme in order to conserve them in the native habitats. Understanding of mycorrhizal association is important for restoration and therefore a symbiotic fungus from natural seedlings of V. thwaitesii was isolated and symbiotic activity evaluated. The isolated fungus having symbiotic activity designated as ‘VT3’ is a slow growing species with cottony growth, binucleate and barrel shaped monilioid cells; typical characters of a Ceratorrhiza sp., anamorph of Ceratobasidium sp. Molecular identification through sequencing the internal transcribed spacer (ITS) regions of the ribosomal RNA gene revealed that it possesses 92% similarity with an uncultured Ceratobasidiaceae clone. V. thwaitesii seeds showed higher germination rate when inoculated with VT3. Majority of the control seeds failed to develop further after the initiation of germination. After the entry of fungus, the seed showed germination and 81% of the germinated seeds was transformed into protocorms in 30 days. Promeristem formation, first leaf development or second leaf initiation occurred in 95% of the protocorms in 60 days. The protocorms in symbiotic culture developed their second leaf (55%) and roots (19%) and only 24% remained at one leaf stage by 90 days. In 150 days, 43% of the seedlings obtained were with 2–3 leaves and 2 roots and 50% of the seedlings with 3–4 leaves and 2–3 roots. Asymbiotic seedlings co-cultured with the symbiotic fungus showed vigorous growth due to active colonization in their root cortex. The results suggest that the symbiotic fungus Ceratobasidiaceae clone VT3 isolated from V. thwaitesii seedlings is promising enough to continue efforts on restoration procedures for this endangered orchid species endemic to Southern Western Ghats region in India and Sri Lanka.

Orchids are critically dependent on fungi for seedling establishment and growth, so the distribution and diversity of orchids might depend on the associated fungal communities. We characterised the communities associated with eight orchid species in three Mediterranean protected areas, using a combination of above-ground analyses of sporophores and below-ground molecular analyses of orchid root samples. In three years of sporophore collection in 25 plots around flowering orchid plants, 268 macrofungal species belonging to 84 genera were observed. Statistical analyses indicated a correlation between macrofungal diversity and orchid community variation, regardless of the effect of environmental and spatial factors characterizing the investigated orchid sites. Fungal ITS-DNA PCR amplification, cloning, and sequencing revealed Rhizoctonia-like fungi belonging to Ceratobasidiaceae (26 %), Tulasnellaceae (22.5 %), and Sebacinaceae (3.5 %), as well as other basidiomycetes and ascomycetes, in the roots of 99 orchid plants. Mycorrhizal specificity was low but co-occurring orchid species showed preferences for different partners. The diverse macrofungal communities found in the sites may contribute to orchid community variation without colonizing the orchid roots. Molecular analyses revealed a segregation of associated fungi, which may contribute to Mediterranean orchid coexistence in nature.

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 Sorensen 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.

Urban expansion threatens biodiversity worldwide, therefore urban spaces need to be amenable to biodiversity conservation. On trees in urban environments, natural colonisation and successful translocation of epiphytic orchids are necessary to enhance urban biodiversity, and depend on the availability of compatible orchid mycorrhizal fungi (OMF). However, the extent of OMF presence and distribution, as well as niche requirements for the OMF, remain poorly studied. To identify and quantify OMF on urban trees as well as assess their suitability for native epiphytic orchids, we conducted high-throughput sequencing on tree bark and orchid root samples. OMF were detected at 60% of the study sites on 16% of 270 bark samples (from stem, fork, and branch microsites within each tree). OMF presence and richness on bark samples were related to multiple biophysical factors; in general, humus presence and precipitation levels were positively predictive of OMF presence and richness. We found Ceratobasidiaceae- and Serendipitaceae-associated OMF both on bark and within roots. Orchid species also showed differing mycorrhizal specificity. Sites associated with fungal genera Ceratobasidium, Rhizoctonia, and Serendipita were considered suitable habitats for seven orchid species. The results suggest that urban trees support OMF and are therefore suitable for native orchid species; however, OMF availability are largely constrained by biophysical factors. To maximise the likelihood of translocation success and consequent natural establishment, we propose that (micro)sites are screened for compatible OMF prior to any intervention.

Species-rich seminatural grasslands in Central Europe have suffered a dramatic loss of biodiversity due to conversion to arable land, but vast areas are being restored. Population recovery of orchids, which depend on mycorrhizal fungi for germination, is however limited. We hypothesised that ploughing and fertilisation caused shifts in orchid mycorrhizal communities in soil and restricted orchid germination. We examined edaphic conditions in 60 restored and seminatural grasslands, and germination success in 10 restored grasslands. Using a newly designed primer, we screened the composition of rhizoctonias in soil, seedlings and roots of seven orchid species. Seminatural and restored grasslands differed significantly in nutrient amounts and rhizoctonia assemblages in soil. While Serendipitaceae prevailed in seminatural grasslands with a higher organic matter content, Ceratobasidiaceae were more frequent in phosphorus-rich restored grasslands with increased abundance on younger restored sites. Tulasnellaceae displayed no preference. Germination success in restored grasslands differed significantly between orchid species; two mycorrhizal generalist species germinated with a broad range of rhizoctonias at most restored grasslands, while germination success of specialists was low. Past agricultural practices have a long-lasting effect on soil conditions and orchid mycorrhizal communities. Altered mycorrhizal availability may be the main reason for low germination success of specialist orchid species.

Interactions with mycorrhizal fungi have been increasingly recognized as one of the most important ecological factors determining the distribution and local abundance of orchids. While some orchid species may interact with a variety of fungal associates, others are more specific in their choice of mycorrhizal partners. Moreover, orchids that co-occur at a given site, often associate with different partners, possibly to avoid competition and to allow stable coexistence. However, whether differences in mycorrhizal partners directly affect seed germination and subsequent protocorm formation remains largely unknown. In this research, we used in vitro germination experiments to investigate to what extent seed germination and protocorm formation of Gymnadenia conopsea was affected by the origin and identity of fungal associates. Fungi were isolated from G. conopsea and three other co-occurring orchid species (Dactylorhiza viridis (Coeloglossum viride), Herminium monorchis, and Platanthera chlorantha). In total, eight fungal associates, belonging to Tulasnellaceae, Ceratobasidiaceae, and Serendipitaceae, were successfully isolated and cultured. While all eight fungal strains were able to promote early germination of G. conopsea seeds, only fungal strain GS2, a member of the Ceratobasidiaceae isolated from G. conopsea itself, was able to promote protocorm formation and subsequent growth to a seedling. Two other fungal strains isolated from G. conopsea only supported seed germination until the protocorm formation stage. The other five fungal strains isolated from the co-occurring orchid species did not support seed germination beyond the protocorm stage. We conclude that, although G. conopsea is considered a mycorrhizal generalist that associates with a wide range of fungi during its adult life, it requires specific fungi to promote protocorm formation and growth to a seedling. ispartof: MYCORRHIZA vol:30 issue:2-3 pages:221-228 ispartof: location:Germany status: published

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.

Orchid mycorrhizal (OrM) fungi play a crucial role in the ontogeny of orchids, yet little is known about how the structure of OrM fungal communities varies with space and environmental factors. Previous studies suggest that within orchid patches, the distance to adult orchids may affect the abundance of OrM fungi. Many orchid species grow in species-rich temperate semi-natural grasslands, the persistence of which depends on moderate physical disturbances, such as grazing and mowing. The aim of this study was to test whether the diversity, structure and composition of OrM fungal community are influenced by the orchid patches and management intensity in semi-natural grasslands. We detected putative OrM fungi from 0 to 32 m away from the patches of host orchid species (Orchis militaris and Platanthera chlorantha) in 21 semi-natural calcareous grasslands using pyrosequencing. In addition, we assessed different ecological conditions in semi-natural grasslands but primarily focused on the effect of grazing intensity on OrM fungal communities in soil. We found that investigated orchid species were mostly associated with Ceratobasidiaceae and Tulasnellaceae and, to a lesser extent, with Sebacinales. Of all the examined factors, the intensity of grazing explained the largest proportion of variation in OrM fungal as well as total fungal community composition in soil. Spatial analyses showed limited evidence for spatial clustering of OrM fungi and their dependence on host orchids. Our results indicate that habitat management can shape OrM fungal communities, and the spatial distribution of these fungi appears to be weakly structured outside the orchid patches.

BACKGROUND AND AIMS Mycorrhizal associations are influenced by abiotic and biotic factors, including climate, soil conditions and the identity of host plants. However, the effect of environmental conditions on orchid mycorrhizal associations remains poorly understood. The present study examined how differences in soil nutrient availability are related to the diversity and composition of mycorrhizal fungi associated with two terrestrial orchid species from central Chile. METHODS For 12 populations of Bipinnula fimbriata and B. plumosa, OTU (operational taxonomic unit) richness, phylogenetic diversity and community composition of mycorrhizal fungi in root samples were estimated using internal transcribed spacer (ITS) sequences. Then, these mycorrhizal diversity variables were related to soil nutrients and host species using generalized linear models and non-metric multidimensional scaling. KEY RESULTS Variation in OTU composition of mycorrhizal fungi among sites was explained mainly by orchid host species. Fungi in Tulasnellaceae and Ceratobasidiaceae were isolated from both orchid species, but the former were more frequent in B. fimbriata and the latter in B. plumosa. Soil nutrients and orchid host species had significant effects on OTU richness and phylogenetic diversity. Mycorrhizal diversity decreased in habitats with higher N in both species and increased with P availability only in B. fimbriata CONCLUSIONS The results suggest that soil nutrient availability modulates orchid mycorrhizal associations and provide support for the hypothesis that specialization is favoured by higher soil nutrient availability. Inter-specific differences in mycorrhizal composition can arise due to a geographical pattern of distribution of orchid mycorrhizal fungi, host preferences for fungal partners or differential performance of mycorrhizal fungi under different nutrient availabilities. Further experiments are needed to evaluate these hypotheses.

20 I 21 II 21 III 23 IV 29 V 33 VI 35 36 36 References 36 SUMMARY: Root endophytism and mycorrhizal associations are complex derived traits in fungi that shape plant physiology. Sebacinales (Agaricomycetes, Basidiomycota) display highly diverse interactions with plants. Although early-diverging Sebacinales lineages are root endophytes and/or have saprotrophic abilities, several more derived clades harbour obligate biotrophs forming mycorrhizal associations. Sebacinales thus display transitions from saprotrophy to endophytism and to mycorrhizal nutrition within one fungal order. This review discusses the genomic traits possibly associated with these transitions. We also show how molecular ecology revealed the hyperdiversity of Sebacinales and their evolutionary diversification into two sister families: Sebacinaceae encompasses mainly ectomycorrhizal and early-diverging saprotrophic species; the second family includes endophytes and lineages that repeatedly evolved ericoid, orchid and ectomycorrhizal abilities. We propose the name Serendipitaceae for this family and, within it, we transfer to the genus Serendipita the endophytic cultivable species Piriformospora indica and P. williamsii. Such cultivable Serendipitaceae species provide excellent models for root endophytism, especially because of available genomes, genetic tractability, and broad host plant range including important crop plants and the model plant Arabidopsis thaliana. We review insights gained with endophytic Serendipitaceae species into the molecular mechanisms of endophytism and of beneficial effects on host plants, including enhanced resistance to abiotic and pathogen stress.

Lectins are widespread proteins found in plants, fungi, bacteria, and vertebrates, and they play the critical roles in many physiological functions. Two lectin molecules (namely, RSAI and RSAII) were extracted from Rhizoctonia solani Kuhn and their effects on Pieris brassicae L. larvae were determined by larval survival rate, body mass, nutritional indices, digestive enzyme activities, and caspase-3 gene expression. The highest mortality caused by RSA treatment was recorded up to 80%, the larval weight decreased to 0.05 g and Similarly, RSAs significantly decreased nutritional indices including conversion efficiency of ingested food (ECI), conversion efficiency of digested food (ECD), approximate digestibility (AD), relative consumption rate (RCR), and relative growth rate (RGR) in a dose-dependent manner. Activities of α-amylase and α- and β-glucosidases significantly decreased in the larvae fed with RSA-treated diets. Also, activities of TAG-lipase and proteases significantly reduced after feeding with different concentrations of RSAs. Gene expression analysis of caspase-3 in control and treated larvae revealed significant increment of its expression in the larvae fed with RSAI and RSAII, respectively, 9.52- and 1.47-fold compared to control. These results clearly demonstrated insecticidal effects of R. solani lectins on P. brassicae via several physiological pathways, thus rendering RSA as a good target for furthering our knowledge and suggesting new strategies to overcome pesticide side effects.

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 13 C and 15 N 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 13 C but not so greatly in 15 N, suggesting that saprotrophic Ceratobasidium spp. and Tulasnella spp. are their main carbon source. However, in green variants, 13 C levels were depleted and those of 15 N 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.

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 com-position 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 germina-tion 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.

Orchids are known for their vast diversity and dependency on mycorrhizal fungi. Under in situ conditions, the biotic and abiotic factors determining the composition and distribution of orchid mycorrhizal fungi (OMF) communities remain largely unexplored. Therefore in situ experiments are needed to better understand the interactions between orchids and fungi. A seedling-trap experiment was conducted in the Reserva Biológica San Francisco, a well-known biodiversity hotspot located in the Andes of southern Ecuador. The objective was to investigate the effect of orchid species, site, elevation or temporal variation on the assembly and structure of OMF associated with Cyrtochilum retusum and Epidendrum macrum. The OMF community composition was determined using the Illumina MiSeq sequencing of the internal transcribed spacer 2 (ITS2) region. The results exhibited 83 OMF operational taxonomic units belonging to Tulasnellaceae, Ceratobasidiaceae, Serendipitaceae and Atractiellales. It was observed that the composition of the OMF communities was different among orchid species and temporal variation but was not different among sites. The results further support that orchids have a core of keystone OMF that are ubiquitously distributed and stable across temporal change, whereas the majority of these fungi are randomly associated with the plants.

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.

Plant dependence on fungal carbon (mycoheterotrophy) evolved repeatedly. In orchids, it is connected with a mycorrhizal shift from rhizoctonia to ectomycorrhizal fungi and a high natural (13)C and (15)N abundance. Some green relatives of mycoheterotrophic species show identical trends, but most of these remain unstudied, blurring our understanding of evolution to mycoheterotrophy. We analysed mycorrhizal associations and (13)C and (15)N biomass content in two green species, Neottia ovata and N. cordata (tribe Neottieae), from a genus comprising green and nongreen (mycoheterotrophic) species. Our study covered 41 European sites, including different meadow and forest habitats and orchid developmental stages. Fungal ITS barcoding and electron microscopy showed that both Neottia species associated mainly with nonectomycorrhizal Sebacinales Clade B, a group of rhizoctonia symbionts of green orchids, regardless of the habitat or growth stage. Few additional rhizoctonias from Ceratobasidiaceae and Tulasnellaceae, and ectomycorrhizal fungi were detected. Isotope abundances did not detect carbon gain from the ectomycorrhizal fungi, suggesting a usual nutrition of rhizoctonia-associated green orchids. Considering associations of related partially or fully mycoheterotrophic species such as Neottia camtschatea or N. nidus-avis with ectomycorrhizal Sebacinales Clade A, we propose that the genus Neottia displays a mycorrhizal preference for Sebacinales and that the association with nonectomycorrhizal Sebacinales Clade B is likely ancestral. Such a change in preference for mycorrhizal associates differing in ecology within the same fungal taxon is rare among orchids. Moreover, the existence of rhizoctonia-associated Neottia spp. challenges the shift to ectomycorrhizal fungi as an ancestral pre-adaptation to mycoheterotrophy in the whole Neottieae.

In the dryland Pacific Northwest wheat cropping systems, no-till is becoming more prevalent as a way to reduce soil erosion and fuel inputs. Tillage can have a profound effect on microbial communities and soilborne fungal pathogens, such as Rhizoctonia. We compared the fungal communities in long-term no-till (NT) plots adjacent to conventionally tilled (CT) plots, over three years at two locations in Washington state and one location in Idaho, US. We used pyrosequencing of the fungal ITS gene and identified 422 OTUs after rarefication. Fungal richness was higher in NT compared to CT, in two of the locations. Humicola nigrescens, Cryptococcus terreus, Cadophora spp. Hydnodontaceae spp., and Exophiala spp. were more abundant in NT, while species of Glarea, Coniochaetales, Mycosphaerella tassiana, Cryptococcus bhutanensis, Chaetomium perlucidum, and Ulocladium chartarum were more abundant in CT in most locations. Other abundant groups that did not show any trends were Fusarium, Mortierella, Penicillium, Aspergillus, and Macroventuria. Plant pathogens such as Rhizoctonia (Ceratobasidiaceae) were not abundant enough to see tillage differences, but Microdochium bolleyi, a weak root pathogen, was more abundant in NT. Our results suggest that NT fungi are better adapted at utilizing intact, decaying roots as a food source and may exist as root endophytes. CT fungi can utilize mature plant residues that are turned into the soil with tillage as pioneer colonizers, and then produce large numbers of conidia. But a larger proportion of the fungal community is not affected by tillage and may be niche generalists.

Orchidaceae is one of the most species-rich angiosperm families, and all orchids are fully dependent on fungi for their seed germination and their life cycle. The level of specificity of the association between orchid species and fungi can be related to the number of co-occurring orchid species. To investigate orchid mycorrhizal associations in adult-photosynthetic orchids, 16 Mediterranean orchid species belonging to 4 genera (Anacamptis, Ophrys, Orchis, and Serapias) at 11 different sites were subjected to DNA-based analysis. Eighteen operational taxonomic units representing two fungal families, Tulasnellaceae and Ceratobasidiaceae, were identified. All examined orchid species associated with different mycorrhizal fungi. Interestingly, there was a positive correlation between number of orchid species and number of mycorrhizal. Monospecific populations showed a lower number of fungi, while sympatric populations had a higher number of mycorrhizal fungi. Our results showed that Mediterranean orchid...

Summary The distribution and abundance of orchid populations depend on a suite of biological and ecological factors, including seed production and dispersal, availability of mycorrhizal fungi and appropriate environmental conditions, with the weighting of these factors depending on the spatial scale considered. Disentangling the factors determining successful orchid establishment represents a major challenge, involving seed germination experiments, molecular techniques and assessment of environmental conditions. Identification of fungi from large-scale surveys of mycorrhizal associations in a range of orchid species has shown that mycorrhizal fungi may be widespread and occur in varied habitats. Further, a meta-analysis of seed introduction experiments revealed similar seed germination in occupied and unoccupied habitat patches. Orchid rarity was also unrelated to mycorrhizal specificity. Nonetheless, seed germination within sites appears to depend on both biotic and abiotic conditions. In the few cases that have been examined, coexisting orchids have distinct mycorrhizal communities and show strong spatial segregation, suggesting that mycorrhizal fungi are important drivers of niche partitioning and contribute to orchid coexistence. A broader investigation of orchid mycorrhizal fungus distribution in the soil, coupled with fungus and recruitment mapping, is needed to translate fungal abundance to orchid population dynamics and may lead to better orchid conservation.

Summary Because orchids are dependent on mycorrhizal fungi for germination and establishment of seedlings, differences in the mycorrhizal communities associating with orchids can be expected to mediate the abundance, spatial distribution and coexistence of terrestrial orchids in natural communities. We assessed the small-scale spatial distribution of seven orchid species co-occurring in 25 × 25 m plots in two Mediterranean grasslands. In order to characterize the mycorrhizal community associating with each orchid species, 454 pyrosequencing was used. The extent of spatial clustering was assessed using techniques of spatial point pattern analysis. The community of mycorrhizal fungi consisted mainly of members of the Tulasnellaceae, Thelephoraceae and Ceratobasidiaceae, although sporadically members of the Sebacinaceae, Russulaceae and Cortinariaceae were observed. Pronounced differences in mycorrhizal communities were observed between species, whereas strong clustering and significant segregation characterized the spatial distribution of orchid species. However, spatial segregation was not significantly related to phylogenetic dissimilarity of fungal communities. Our results indicate that co-occurring orchid species have distinctive mycorrhizal communities and show strong spatial segregation, suggesting that mycorrhizal fungi are important factors driving niche partitioning in terrestrial orchids and may therefore contribute to orchid coexistence.

Fungi from the Ceratobasidiaceae family have important ecological roles as pathogens, saprotrophs, non-mycorrhizal endophytes, orchid mycorrhizal and ectomycorrhizal symbionts, but little is known about the distribution and evolution of these nutritional modes. All public ITS sequences of Ceratobasidiaceae were downloaded from databases, annotated with ecological and taxonomic metadata, and tested for the non-random phylogenetic distribution of nutritional modes. Phylogenetic analysis revealed six main clades within Ceratobasidiaceae and a poor correlation between molecular phylogeny and morphological–cytological characters traditionally used for taxonomy. Sequences derived from soil (representing putative saprotrophs) and orchid mycorrhiza clustered together, but remained distinct from pathogens. All nutritional modes were phylogenetically conserved in the Ceratobasidiaceae based on at least one index. Our analyses suggest that in general, autotrophic orchids form root symbiosis with available Ceratobasidiaceae isolates in soil. Ectomycorrhiza-forming capability has evolved twice within the Ceratobasidiaceae and it had a strong influence on the evolution of mycoheterotrophy and host specificity in certain orchid taxa.

Nervilia nipponica is a tuberous terrestrial orchid that has a highly restricted distribution within common secondary evergreen forest communities in central and western Japan. Such a limited occurrence could be attributable to a requirement for a specific mycorrhizal fungus. As part of a broader examination of this hypothesis, we sought to elucidate the mycorrhizal associations of N. nipponica. Seventy-five samples of mycorrhizae from forty individuals were collected at ten populations throughout the orchid's range in Japan. The identity of mycorrhizal fungi was investigated by sequencing two genetic markers (nrDNA ITS and nrDNA 28S LSU) and their relationships were assessed via phylogenetic analyses. The most frequently encountered mycorrhizal fungi consisted of four closely related Agaricomycetes that infected an average of 78.7 % of individuals per population. All four formed a discrete, monophyletic clade with low sequence homology to other fungi registered in GenBank, indicating that they belong to a novel, unnamed family. Two additional fungal groups, belonging to Ceratobasidiaceae and "Group B" Sebacinales, were found in 22.0 and 21.5 % of individuals per population, respectively. The orchid probably uses these two groups opportunistically, because they were found at lower densities and always in combination with the unidentified Agaricomycete. These findings suggest that a group of novel Agaricomycete fungi constitutes the dominant mycobiont of N. nipponica.

Orchid species are critically dependent on mycorrhizal fungi for completion of their life cycle, particularly during the early stages of their development when nutritional resources are scarce. As such, orchid mycorrhizal fungi play an important role in the population dynamics, abundance, and spatial distribution of orchid species. However, less is known about the ecology and distribution of orchid mycorrhizal fungi. In this study, we used 454 amplicon pyrosequencing to investigate ecological and geographic variation in mycorrhizal associations in fourteen species of the orchid genus Dactylorhiza. More specifically, we tested the hypothesis that variation in orchid mycorrhizal communities resulted primarily from differences in habitat conditions where the species were growing. The results showed that all investigated Dactylorhiza species associated with a large number of fungal OTUs, the majority belonging to the Tulasnellaceae, Ceratobasidiaceae and Sebacinales. Mycorrhizal specificity was low, but significant variation in mycorrhizal community composition was observed between species inhabiting different ecological habitats. Although several fungi had a broad geographic distribution, Species Indicator Analysis revealed some fungi that were characteristic for specific habitats. Overall, these results indicate that orchid mycorrhizal fungi may have a broad geographic distribution, but that their occurrence is bounded by specific habitat conditions.

In epiphytic orchids, distinctive groups of fungi are involved in the symbiotic association. However, little is known about the factors that determine the mycorrhizal community structure. Here, we analyzed the orchid mycorrhizal fungi communities associated with three sympatric Cymbidieae epiphytic tropical orchids (Cyrtochilum flexuosum, Cyrtochilum myanthum, and Maxillaria calantha) at two sites located within the mountain rainforest of southern Ecuador. To characterize these communities at each orchid population, the ITS2 region was analyzed by Illumina MiSeq technology. Fifty-five mycorrhizal fungi operational taxonomic units (OTUs) putatively attributed to members of Serendipitaceae, Ceratobasidiaceae and Tulasnellaceae were identified. Significant differences in mycorrhizal communities were detected between the three sympatric orchid species as well as among sites/populations. Interestingly, some mycorrhizal OTUs overlapped among orchid populations. Our results suggested that populations of studied epiphytic orchids have site-adjusted mycorrhizal communities structured around keystone fungal species. Interaction with multiple mycorrhizal fungi could favor orchid site occurrence and co-existence among several orchid species. © 2016 Springer-Verlag Berlin Heidelberg

Orchids are a main component of the diversity of vascular plants in Ecuador with approximately 4000 species representing about 5.3% of the orchid species described worldwide. More than a third of these species are endemics. As orchids, in contrast to other plants, depend on mycorrhizal fungi already for seed germination and early seedling establishment, availability of appropriate fungi may strongly influence distribution of orchid populations. It is currently debated if green orchids depend on specific mycobionts or may be equally promoted by a broad spectrum of mycorrhizal fungi, discussion mostly based on data from temperate regions. Here we summarize results obtained from broad scale investigations in the tropical mountain rain forest of Ecuador revealing associations with members of Serendipitaceae (Sebacinales), Tulasnellaceae, Ceratobasidiaceae (Cantharellales), and Atractiellales. Recent molecular data show that these worldwide spread fungal groups have broad ecological implications and are specifically suited as mycorrhizal fungi of green orchids. We found that main fungal partners and different levels of specificity among orchids and their mycobionts in the tropical mountain forests correspond to findings in other biomes despite the large ecological differences. Orchids are a main component of the diversity of vascular plants in Ecuador with approximately 4000 species representing about 5.3% of the orchid species described worldwide. More than a third of these species are endemics. As orchids, in contrast to other plants, depend on mycorrhizal fungi already for seed germination and early seedling establishment, availability of appropriate fungi may strongly influence distribution of orchid populations. It is currently debated if green orchids depend on specific mycobionts or may be equally promoted by a broad spectrum of mycorrhizal fungi, discussion mostly based on data from temperate regions. Here we summarize results obtained from broad scale investigations in the tropical mountain rain forest of Ecuador revealing associations with members of Serendipitaceae (Sebacinales), Tulasnellaceae, Ceratobasidiaceae (Cantharellales), and Atractiellales. Recent molecular data show that these worldwide spread fungal groups have broad ecological implications and are specifically suited as mycorrhizal fungi of green orchids. We found that main fungal partners and different levels of specificity among orchids and their mycobionts in the tropical mountain forests correspond to findings in other biomes despite the large ecological differences.&nbsp

Made available in DSpace on 2018-12-11T16:41:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-04-01 National Institute of Food and Agriculture Phylogenetic relationships of Rhizoctonia fungi within the order Cantharellales were studied using sequence data from portions of the ribosomal DNA cluster regions ITS-LSU, rpb2, tef1, and atp6 for 50 taxa, and public sequence data from the rpb2 locus for 165 taxa. Data sets were analysed individually and combined using Maximum Parsimony, Maximum Likelihood, and Bayesian Phylogenetic Inference methods. All analyses supported the monophyly of the family Ceratobasidiaceae, which comprises the genera Ceratobasidium and Thanatephorus. Multi-locus analysis revealed 10 well-supported monophyletic groups that were consistent with previous separation into anastomosis groups based on hyphal fusion criteria. This analysis coupled with analyses of a larger sample of 165 rpb2 sequences of fungi in the Cantharellales supported a sister relationship between the Botryobasidiaceae and Ceratobasidiaceae and a sister relationship of the Tulasnellaceae with the rest of the Cantharellales. The inclusion of additional sequence data did not clarify incongruences observed in previous studies of Rhizoctonia fungi in the Cantharellales based on analyses of a single or multiple genes. The diversity of ecological and morphological characters associated with these fungi requires further investigation on character evolution for re-evaluating homologous and homoplasious characters. Instituto de Ecología A.C. Red de Biodiversidad y Sistemática, Carretera Antigua a Coatepec No. 351 Department of Plant Pathology North Carolina State University Center for Integrated Fungal Research, Campus Box 7251 CBS Fungal Biodiversity Centre, Uppsalalaan 8 Department of Microbial Ecology Netherlands Institute of Ecology (NIOO/KNAW), Droevendaalsesteeg 10 UNESP Faculdade de Ciências Agronômicas, CP 237 Department of Biology Duke University, Durham UNESP Faculdade de Ciências Agronômicas, CP 237 National Institute of Food and Agriculture: 2010-34500-21676

* 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.

Dendrobium is a large genus of tropical epiphytic orchids. Some members of this genus are in danger of extinction across China. To investigate orchid mycorrhizal associations of the genus Dendrobium, plants from two Dendrobium species (Dendrobium officinale and Dendrobium fimbriatum) were collected from two habitats in Guangxi Province, China, and clone libraries were constructed to identify the mycorrhizal fungi of individual plants. A low and high degree of specificity was observed in D. officinale and D. fimbriatum, respectively. Phylogenetic analysis revealed that the majority of Dendrobium mycorrhizal fungi are members of the Tulasnellaceae, but, in some plants, members of the Ceratobasidiaceae and Pluteaceae were also found. In D. officinale, individual plants associated with more than three fungi simultaneously, and, in some cases, associations with five fungi at the same time. One fungus was shared by individual plants of D. officinale collected from the two habitats. In D. fimbriatum, only one fungal partner was found in each population, and this fungus differed between populations. The two species of Dendrobium sampled from the same habitat did not share any fungal taxa. These results provide valuable information for conservation of these orchid species.

Characterizing the architecture of bipartite networks is increasingly used as a framework to study biotic interactions within their ecological context and to assess the extent to which evolutionary constraint shape them. Orchid mycorrhizal symbioses are particularly interesting as they are viewed as more beneficial for plants than for fungi, a situation expected to result in an asymmetry of biological constraint. This study addressed the architecture and phylogenetic constraint in these associations in tropical context. We identified a bipartite network including 73 orchid species and 95 taxonomic units of mycorrhizal fungi across the natural habitats of Reunion Island. Unlike some recent evidence for nestedness in mycorrhizal symbioses, we found a highly modular architecture that largely reflected an ecological barrier between epiphytic and terrestrial subnetworks. By testing for phylogenetic signal, the overall signal was stronger for both partners in the epiphytic subnetwork. Moreover, in the subnetwork of epiphytic angraecoid orchids, the signal in orchid phylogeny was stronger than the signal in fungal phylogeny. Epiphytic associations are therefore more conservative and may co-evolve more than terrestrial ones. We suggest that such tighter phylogenetic specialization may have been driven by stressful life conditions in the epiphytic niches. In addition to paralleling recent insights into mycorrhizal networks, this study furthermore provides support for epiphytism as a major factor affecting ecological assemblage and evolutionary constraint in tropical mycorrhizal symbioses.

Summary 1. Understanding the many factors that affect community composition and coexistence of species in natural environments is one of the central goals in ecology. As germination and establishment of seedlings in orchids are crucially dependent on mycorrhizal availability, the diversity and spatial distribution of orchid mycorrhizal fungi are likely factors that contribute to orchid coexistence. 2. In this study, we combined molecular identification techniques with seed germination experiments and spatial point pattern analyses to investigate to what extent differences in mycorrhizal association patterns affected spatial variation in seed germination and the above-ground distribution of three co-occurring terrestrial orchid species (Anacamptis morio, Gymnadenia conopsea and Orchis mascula). 3. The three species associated with a different set of mycorrhizal fungi, except A. morio and G. conopsea, which shared one fungal associate. The number of fungal lineages detected associating with each species also differed between species, being highest in G. conopsea, which associated with five lineages, and restricted to a single lineage in O. mascula. 4. Seed germination experiments showed that below-ground seed germination was restricted to locations where orchid individuals were most abundant, and quickly declined with increasing distance from the nearest above-ground congener. Spatial point pattern analyses indicated significant fine-scale spatial clustering that was highest in O. mascula and lowest in G. conopsea. Moreover, the spatial distributions of the three species were independent from each other, except for A. morio and G. conopsea. 5. Synthesis. Our results support previous findings that co-occurring orchid species tend to use different mycorrhizal partners. Although the possibility that variation in local environmental conditions affected seed germination could not be completely ruled out, our results suggest that the presence of specific mycorrhizal fungi contributed, at least partly, to the spatial distribution and coexistence of the investigated orchid species.

Epiphytic orchids have received considerable study, yet little has been published on their germination requirements in situ involving mycorrhizal fungi. Such research has been hampered by the small, dust-like size of seeds and leafless seedlings (protocorms) which are difficult to pinpoint on natural substrates, especially those on arboreal substrates (tree limbs). We report a novel seed sowing and retrieval method, modified from one applied to terrestrial orchids, used in the acquisition of epiphytic orchid protocorms from the Florida Panther National Wildlife Refuge. Seeds from two epiphytic orchid species (Epidendrum amphistomum A. Richard, E. nocturnum Jacquin) were placed in separate nylon mesh packets secured within 35 mm plastic slide mounts, and affixed to tree bark using gutter mesh and a staple gun. To confirm that the embryos were viable, some seeds were also sown on asymbiotic media in the laboratory which subsequently germinated after 52 days incubation. Of 60 packets distributed among 18 tree limb sites, one packet - harboring seeds of E. amphistomum affixed to pop ash (Fraxinus caroliniana Mill.) on a moss substrate - harbored protocorms after 267 days. Using molecular markers, a fungus assignable to the Ceratobasidiaceae, appears to be the mycorrhizal associate of these protocorms suggesting that this fungus may be associated with the germination process in situ.