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Orchidaceae has diversified in tree canopies and accounts for 68% of vascular epiphytes. Differences in mycorrhizal communities among epiphytic orchids can reduce species competition for mycorrhizal fungi and contribute to niche partitioning, which may be a crucial driver of the unusual species diversification among orchids. Mycorrhizal specificity-the range of fungi allowing mycorrhizal partnerships-was evaluated by assessment of mycorrhizal communities in the field (ecological specificity) and symbiotic cultures in the laboratory (physiological specificity) for three epiphytic orchids inhabiting Japan. Mycorrhizal communities were assessed with co-existing individuals growing within 10 cm of each other, revealing that ecological specificity varied widely among the three species, ranging from dominance by a single Ceratobasidiaceae fungus to diverse mycobionts across the Ceratobasidiaceae and Tulasnellaceae. In vitro seed germination tests revealed clear differences in physiological specificity among the three orchids, and that the primary mycorrhizal partners contributed to seed germination. In vitro compatibility ranges of three orchids strongly reflect the mycorrhizal community composition of wild populations. This suggests that differences in in situ mycorrhizal communities are not strongly driven by environmental factors, but are primarily due to physiological differences among orchid species. This study shows that the symbiotic strategy among the epiphytic orchid species varies from specialized to generalized association, which may contribute to biotic niche partitioning., (© 2023. The Author(s) under exclusive licence to The Botanical Society of Japan.)

Orchids are crucial for the horticulture industry. Mycorrhizal fungi benefit crops by improving nutrition, plant growth, and disease resistance. However, the mycorrhizal association of horticultural hybrid orchids is poorly understood. To address this, we investigated mycorrhizal colonization in the entire root system and assessed the mycorrhizal community using a Dendrobium cultivar, D . Stardust 'Firebird', obtained from three nurseries. Additionally, we isolated and tested mycorrhizal fungi in symbiotic culture to assess their role in the seed germination and growth of Dendrobium species. All plants were colonized by mycorrhizal fungi, with a higher colonization rate in mature than in juvenile plants. Molecular identification of mycorrhizal fungi by Sanger and high-throughput sequencing revealed that the cultivar was associated with a phylogenetically diverse group of fungi, including mycorrhizal fungi from Tulasnellaceae, and several wood-decaying fungi. The Tulasnellaceae isolates significantly enhanced the seed germination of three Dendrobium species and increased the survival rate and growth of asymbiotic seedlings of D. moniliforme . This study is the first comprehensive examination of mycorrhizal associations in horticultural orchid hybrids, providing valuable insights for commercial production.

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., (© 2020 Botanical Society of America.)

We performed in-vitro germination tests on seeds from five Gastrodia orchids ( G. confusa , G. elata var. elata , G. elata var. pallens , G. nipponica , and G. pubilabiata ) using one Marasmiaceae and two Mycena isolates. Mycena sp. 1 promoted germination of all five Gastrodia orchids, with root and/or tuber formation observed in G. confusa , G. nipponica , and G. pubilabiata . No additional growth was observed in the other two orchids. Mycena sp. 2 induced G. confusa , G. elata var. elata , and G. nipponica germination, whereas Marasmiaceae sp. 1 induced G. nipponica and G. pubilabiata germination. Phylogenetic analyses indicated that the two Mycena isolates represent distinct lineages within the Mycenaceae . Mycena sp. 1 and Marasmiaceae sp. 1 are closely related to Mycena abramsii and Marasmiellus rhizomorphogenus , respectively. Our results imply that Mycena and marasmioid fungi play important roles in early development in Gastrodia species, and that Mycena fungi in particular may be common mycobionts of Gastrodia species. Root and/or tuber development was observed with four plant-fungus combinations, implying that these associations persist throughout the life cycle, whereas G. elata var. elata may require different associates over time. Our findings will contribute to elucidating the mycorrhizal associations of mycoheterotrophic orchids throughout their life cycle., (2022, by The Mycological Society of Japan.)

Primer bias toward Tulasnellaceae fungi during PCR is a known issue with metabarcoding analyses for the assessment of orchid mycorrhizal communities. However, this bias had not been evaluated for the fungal communities of epiphytic orchids, which account for 69% of all orchid species diversity. We compared the mycorrhizal communities detected using two primer pairs, a fungal universal primer pair (ITS86F/ITS4) and Tulasnella -specific primer pair (5.8STulngs/ITS4-Tul2), using a mock community of fungal isolates from epiphytic orchids and also environmental samples, including orchid roots and a tree bark tip from the host tree of an epiphytic orchid collected. The detected mycorrhizal communities differed widely depending on the primer pairs used. The fungal universal primer pair successfully identified Ceratobasidiaceae and Serendipitaceae fungi but did not reflect Tulasnellaceae diversity. Tulasnellaceae fungi were mainly detected using the Tulasnella -specific primer pair. These tendencies were observed in both the mock community and environmental samples. These results strongly suggest that the use of a Tulasnella -specific primer in combination with a fungal universal primer is essential for assessing the mycorrhizal communities of orchids through metabarcoding analysis, especially in epiphytic orchids. Our study contributes to further understanding of the diversity of mycorrhizal fungi in orchids., (2021, by The Mycological Society of Japan.)

Mycoheterotrophic plants (MHPs) are leafless, achlorophyllous, and completely dependent on mycorrhizal fungi for their carbon supply. Mycorrhizal symbiosis is a mutualistic association with fungi that is undertaken by the majority of land plants, but mycoheterotrophy represents a breakdown of this mutualism in that plants parasitize fungi. Most MHPs are associated with fungi that are mycorrhizal with autotrophic plants, such as arbuscular mycorrhizal (AM) or ectomycorrhizal (ECM) fungi. Although these MHPs gain carbon via the common mycorrhizal network that links the surrounding autotrophic plants, some mycoheterotrophic lineages are associated with saprotrophic (SAP) fungi, which are free-living and decompose leaf litter and wood materials. Such MHPs are dependent on the forest carbon cycle, which involves the decomposition of wood debris and leaf litter, and have a unique biology and evolutionary history. MHPs associated with SAP fungi (SAP-MHPs) have to date been found only in the Orchidaceae and likely evolved independently at least nine times within that family. Phylogenetically divergent SAP Basidiomycota, mostly Agaricales but also Hymenochaetales, Polyporales, and others, are involved in mycoheterotrophy. The fungal specificity of SAP-MHPs varies from a highly specific association with a single fungal species to a broad range of interactions with multiple fungal orders. Establishment of symbiotic culture systems is indispensable for understanding the mechanisms underlying plant-fungus interactions and the conservation of MHPs. Symbiotic culture systems have been established for many SAP-MHP species as a pure culture of free-living SAP fungi is easier than that of biotrophic AM or ECM fungi. Culturable SAP-MHPs are useful research materials and will contribute to the advancement of plant science.

To evaluate the nutritional modes of orchids associated with 'rhizoctonia' fungi, analyses of hydrogen (δ 2 H), carbon (δ 13 C), and nitrogen (δ 15 N) stable isotope ratios are usually adopted. However, previous studies have not fully accounted for exchangeable hydrogens, which could affect these evaluations. Here, we performed standard δ 13 C, δ 15 N, and δ 2 H analyses on bulk samples. Additionally, we conducted δ 2 H analysis on α-cellulose and cellulose nitrate samples to investigate whether the heterogeneity of exchangeable hydrogens among plant species influences the assessment of nutritional modes. The δ 2 H of orchids were consistently higher than those of surrounding autotrophic plants, irrespective of the three pretreatments. Although the rhizoctonia-associated orchid exhibited lower δ 13 C, its δ 2 H was higher than those of the autotrophs. Notably, among all response variables, δ 15 N and δ 2 H exhibited high abilities for discriminating the nutritional modes of rhizoctonia-associated orchids. These results indicate that a time-efficient bulk sample analysis is an effective method for evaluating plant nutritional modes, as the heterogeneity of exchangeable hydrogens does not significantly impact the estimation. Using δ 15 N and δ 2 H benefits the assessment of partial mycoheterotrophy among rhizoctonia-associated orchids., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Mycoheterotrophic plants (MHPs) rely on their mycorrhizal fungus for carbon and nutrient supply, thus a shift in mycobionts may play a crucial role in speciation. This study aims to explore the mycorrhizal diversity of two closely related and sympatric fully MHPs, Monotropastrum humile var. humile (Mhh) and M. humile var. glaberrimum (Mhg), and determine their mycorrhizal associations. A total of 1,108,710 and 1,119,071 ectomycorrhizal fungal reads were obtained from 31 Mhh and 31 Mhg, and these were finally assigned to 227 and 202 operational taxonomic units, respectively. Results show that sympatric Mhh and Mhg are predominantly associated with different fungal genera in Russulaceae. Mhh is consistently associated with members of Russula, whereas Mhg is associated with members of Lactarius. Associating with different mycobionts and limited sharing of fungal partners might reduce the competition and contribute to their coexistence. The ectomycorrhizal fungal communities are significantly different among the five forests in both Mhh and Mhg. The distinct mycorrhizal specificity between Mhh and Mhg suggests the possibility of different mycobionts triggered ecological speciation between sympatric species., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Mycorrhizal symbiosis between plants and fungi is ubiquitous, and has been played key roles in plant terrestrialization and diversification. Although arbuscular mycorrhizal (AM) symbioses with Glomeromycotina fungi have long been recognized as both ancient and widespread symbionts, recent studies showed that Mucoromycotina fungi were also ancestral symbionts and would thus be expected to co-exist with many land plants. To explore whether Mucoromycotina colonize fern gametophytes, we subjected fungal associations with gametophytes of two distantly related ferns, Angiopteris lygodiifolia (Marattiales) and Osmunda japonica (Osmundales), to molecular analysis. Direct PCR amplification from intracellular hyphal coils was also performed. We detected Mucoromycotina sequences in the gametophytes of A. lygodiifolia and O. japonica at rates of 41% (7/17) and 50% (49/98) of gametophytes, respectively, and assigned them to 10 operational taxonomic units of Endogonales lineages. In addition, we used AM fungal-specific primers and detected Glomeromycotina sequences in all individuals examined. The results suggest that Glomeromycotina and Mucoromycotina colonized fern gametophytes simultaneously. We found that Mucoromycotina were present in fern gametophytes of Marratiales and Osmundales, which implies that a variety of fern taxa have Mucoromycotina associations.

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.

Arbuscular (AM) and orchid (OrM) mycorrhiza are the most widespread mycorrhizal symbioses among flowering plants, formed by distinct fungal and plant species. They are both endosymbioses because the fungal hyphae can enter inside the plant cell to develop intracellular fungal structures that are surrounded by the plant membrane. The symbiotic plant-fungus interface is considered to be the major site of nutrient transfer to the host plant. We summarize recent data on nutrient transfer in OrM and compare the development and function of the arbuscules formed in AM and the pelotons formed in OrM in order to outline differences and conserved traits. We further describe the unexpected similarities in the form and function of the intracellular mycorrhizal fungal structures observed in orchids and in the roots of mycoheterotrophic plants forming AM. We speculate that these similarities may be the result of convergent evolution of mycorrhizal types in mycoheterotrophic plants and highlight knowledge gaps and new research directions to explore this scenario., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

Although the absence of normal leaves is often considered a sign of full heterotrophy, some plants remain at least partially autotrophic despite their leafless habit. Leafless orchids with green stems and capsules probably represent a late evolutionary stage toward full mycoheterotrophy and serve as valuable models for understanding the pathways leading to this nutritional strategy. In this study, based on molecular barcoding and isotopic analysis, we explored the physiological ecology of the leafless orchid Eulophia zollingeri, which displays green coloration, particularly during its fruiting phase. Although previous studies had shown that E. zollingeri, in its adult stage, is associated with Psathyrellaceae fungi and exhibits high 13 C isotope signatures similar to fully mycoheterotrophic orchids, it remained uncertain whether this symbiotic relationship is consistent throughout the orchid's entire life cycle and whether the orchid relies exclusively on mycoheterotrophy for its nutrition during the fruiting season. Our study has demonstrated that E. zollingeri maintains a specialized symbiotic relationship with Psathyrellaceae fungi throughout all life stages. However, isotopic analysis and chlorophyll data have shown that the orchid also engages in photosynthesis to meet its carbon needs, particularly during the fruiting stage. This research constitutes the first discovery of partial mycoheterotrophy in leafless orchids associated with saprotrophic non-rhizoctonia fungi., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

We have investigated whether mycobiont identity and environmental conditions affect morphology and physiology of the chlorophyllous orchid: Cremastra variabilis. This species grows in a broad range of environmental conditions and associates with saprotrophic rhizoctonias including Tulasnellaceae and saprotrophic non-rhizoctonian fungi from the family Psathyrellaceae. We cultured the orchid from seeds under aseptic culture conditions and subsequently inoculated the individuals with either a Tulasnellaceae or a Psathyrellaceae isolate. We observed underground organ development of the inoculated C. variabilis plants and estimated their nutritional dependency on fungi using stable isotope abundance. Coralloid rhizome development was observed in all individuals inoculated with the Psathyrellaceae isolate, and 1-5 shoots per seedling grew from the tip of the coralloid rhizome. In contrast, individuals associated with the Tulasnellaceae isolate did not develop coralloid rhizomes, and only one shoot emerged per plantlet. In darkness, δ 13 C enrichment was significantly higher with both fungal isolates, whereas δ 15 N values were only significantly higher in plants associated with the Psathyrellaceae isolate. We conclude that C. variabilis changes its nutritional dependency on fungal symbionts depending on light availability and secondly that the identity of fungal symbiont influences the morphology of underground organs., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The climbing orchid Erythrorchis altissima is the largest mycoheterotroph in the world. Although previous in vitro work suggests that E. altissima has a unique symbiosis with wood-decaying fungi, little is known about how this giant orchid meets its carbon and nutrient demands exclusively via mycorrhizal fungi. In this study, the mycorrhizal fungi of E. altissima were molecularly identified using root samples from 26 individuals. Furthermore, in vitro symbiotic germination with five fungi and stable isotope compositions in five E. altissima at one site were examined. In total, 37 fungal operational taxonomic units (OTUs) belonging to nine orders in Basidiomycota were identified from the orchid roots. Most of the fungal OTUs were wood-decaying fungi, but underground roots had ectomycorrhizal Russula. Two fungal isolates from mycorrhizal roots induced seed germination and subsequent seedling development in vitro. Measurement of carbon and nitrogen stable isotope abundances revealed that E. altissima is a full mycoheterotroph whose carbon originates mainly from wood-decaying fungi. All of the results show that E. altissima is associated with a wide range of wood- and soil-inhabiting fungi, the majority of which are wood-decaying taxa. This generalist association enables E. altissima to access a large carbon pool in woody debris and has been key to the evolution of such a large mycoheterotroph., (© 2018 John Wiley & Sons Ltd.)

Background: Mycoheterotrophic plants are one of the most difficult plant groups to conserve because they are entirely dependent on symbiotic fungi. Establishment of viable culture systems would greatly aid their conservation. We describe a simple culture system for the mycoheterotrophic orchid, Gastrodia pubilabiata, that does not require laboratory facilities. The orchid is symbiotic with leaf-litter-decomposing fungi., Results: Gastrodia pubilabiata seeds were incubated in plastic boxes or glass bottles filled with leaf litter collected from the natural habitat of the species. Seed germination was observed after 35 days and seedling development followed. Fungal isolates from seedlings were identified as Mycenaceae (Basidiomycota), a leaf-litter-decomposing mycorrhizal fungus of Gastrodia species., Conclusion: Our method can be used to conserve endangered mycoheterotrophic plants associated with leaf litter-decomposing fungi efficiently, and can also serve as a model system for physiological and molecular studies of such plants.

The evolution of mycoheterotrophy has been accompanied by extreme reductions in plant leaf size and photosynthetic capacity. Partially mycoheterotrophic plants, which obtain carbon from both photosynthesis and their mycorrhizal fungi, include species with leaves of normal size and others that are tiny-leaved. Thus, plant species may lose their leaves in a gradual process of size reduction rather than through a single step mutation. Little is known about how the degree of mycoheterotrophy changes during reductions in leaf size. We compared the degree of mycoheterotrophy among five Japanese Cephalanthera species, four with leaves of normal size (Cephalanthera falcata, Cephalanthera erecta, Cephalanthera longibracteata and Cephalanthera longifolia), one with tiny leaves (Cephalanthera subaphylla), and one albino form of C. falcata (as reference specimens for fully mycoheterotrophic plants). The levels of mycoheterotrophy were determined by stable isotope natural abundance analysis. All Cephalanthera species were relatively enriched in 13 C and 15 N in comparison with surrounding autotrophic plants. Cephalanthera subaphylla was strongly enriched in 13 C and 15 N to levels similar to the albinos. Species with leaves of normal size were significantly less enriched in 13 C than C. subaphylla and the albinos. Thus, C. subaphylla was strongly mycoheterotrophic, obtaining most of its carbon from mycorrhizal fungi even though it has tiny leaves; species with leaves of normal size were partially mycoheterotrophic. Hence, during the evolutionary pathway to full mycoheterotrophy, some plant species appear to have gained strong mycoheterotrophic abilities before completely losing foliage leaves.

Premise of the Study: Since mycoheterotrophic plants (MHPs) completely depend on their mycorrhizal fungi for carbon, selection of fungal partners has an important role in the speciation of MHPs. However, the causes and mechanisms of mycobiont changes during speciation are not clear. We tested fungal partner shifts and changes in mycorrhizal specificity during speciation of three closely related MHPs-Gastrodia confusa (Gc), G. pubilabiata (Gp), and G. nipponica (Gn) (Orchidaceae)-and correlations between these changes and the vegetation types where each species grows., Methods: We investigated the diversity of mycobionts of the three species by sequencing nrDNA ITS, and the sequence data were subjected to test changes in fungal specificity and fungal partner shifts among the three species. Furthermore, we conducted multivariate analysis to test for differences in mycobiont communities of vegetation types where each species grows., Key Results: Two saprobic Basidiomycota, Marasmiaceae and Mycenaceae, were dominant fungal partners of the three species, and Gn was simultaneously associated with the ectomycorrhizal Russulaceae and Sebacinaceae. Although mycobiont composition differed among the three species, they also sometimes shared identical fungal species. Multivariate analysis revealed that mycobiont communities of the three species in bamboo thickets differed significantly from those in other vegetation types., Conclusions: Fungal partner shifts are not necessarily associated with the evolution of MHPs, and fungal specificity of Gc and Gp was significantly higher than that of Gn, implying that the specificity fluctuates during speciation. Further, Gc exclusively inhabits bamboo thickets, which suggests that adaptation to particular fungi specific to bamboo thickets triggered speciation of this species., (© 2016 Botanical Society of America.)

To determine the mycorrhizal status of pteridophyte gametophytes in diverse taxa, the mycorrhizal colonization of wild gametophytes was investigated in terrestrial cordate gametophytes of pre-polypod leptosporangiate ferns, i.e., one species of Osmundaceae (Osmunda banksiifolia), two species of Gleicheniaceae (Diplopterygium glaucum, Dicranopteris linearis), and four species of Cyatheales including tree ferns (Plagiogyriaceae: Plagiogyria japonica, Plagiogyria euphlebia; Cyatheaceae: Cyathea podophylla, Cyathea lepifera). Microscopic observations revealed that 58 to 97% of gametophytes in all species were colonized with arbuscular mycorrhizal (AM) fungi. Fungal colonization was limited to the multilayered midrib (cushion) tissue in all gametophytes examined. Molecular identification using fungal SSU rDNA sequences indicated that the AM fungi in gametophytes primarily belonged to the Glomeraceae, but also included the Claroideoglomeraceae, Gigasporaceae, Acaulosporaceae, and Archaeosporales. This study provides the first evidence for AM fungal colonization of wild gametophytes in the Plagiogyriaceae and Cyatheaceae. Taxonomically divergent photosynthetic gametophytes are similarly colonized by AM fungi, suggesting that mycorrhizal associations with AM fungi could widely occur in terrestrial pteridophyte gametophytes.

Petrosaviaceae is a monocotyledonous plant family that comprises two genera: the autotrophic Japonolirion and the mycoheterotrophic Petrosavia. Accordingly, this plant family provides an excellent system to examine specificity differences in mycobionts between autotrophic and closely related mycoheterotrophic plant species. We investigated mycobionts of Japonolirion osense, the sole species of the monotypic genus, from all known habitats of this species by molecular identification and detected 22 arbuscular mycorrhizal (AM) fungal phylotypes in Archaesporales, Diversisporales, and Glomerales. In contrast, only one AM fungal phylotype in Glomerales was predominantly detected from the mycoheterotrophic Petrosavia sakuraii in a previous study. The high mycobiont diversity in J. osense and in an outgroup plant, Miscanthus sinensis (Poaceae), indicates that fungal specificity increased during the evolution of mycohetrotrophy in Petrosaviaceae. Furthermore, some AM fungal sequences of J. osense showed >99% sequence similarity to the dominant fungal phylotype of P. sakuraii, and one of them was nested within a clade of P. sakuraii mycobionts. These results indicate that fungal partners are not necessarily shifted, but rather selected for in the course of the evolution of mycoheterotrophy. We also confirmed the Paris-type mycorrhiza in J. osense.

Orchids and arbuscular mycorrhiza (AM) plants evolved independently and have different structures and fungal partners, but they both facilitate nutrient uptake. Orchid mycorrhiza (OM) supports orchid seed germination, but unlike AM, its role in disease resistance of mature plants is largely unknown. Here, we examined whether OM induces systemic disease resistance against a necrotrophic pathogen in a similar fashion to AM. We investigated the priming effect of mycorrhizal fungi inoculation on resistance of a terrestrial orchid, Bletilla striata, to soft rot caused by Dickeya fangzhongdai. We found that root colonization by a compatible OM fungus primed B. striata seedlings and induced systemic resistance against the infection. Transcriptome analysis showed that priming was mediated by the downregulation of jasmonate and ethylene pathways and that these pathways are upregulated once infection occurs. Comparison with the reported transcriptome of AM fungus-colonized rice leaves revealed similar mechanisms in B. striata and in rice. These findings highlight a novel aspect of commonality between OM and AM plants in terms of induced systemic resistance. [ABSTRACT FROM AUTHOR]

The tiny terrestrial orchids (plant height less than 8 cm) in the genus Corybas dependent on mycorrhizal fungal (OMF) partners for seed germination and seedling development. The OMF community of the Corybas remains poorly understood, although the relevant knowledge is very important for in situ and ex situ conservation of these orchids. In this study, we characterized OMF richness and compositions of three helmet-orchid species, i.e., Corybas geminigibbus, C. himalaicus, and C. shanlinshiensis, from their natural habitats by using Illumina sequencing of the internal transcribed spacer 2 region. We found that fungal colonization was restricted in the rhizomes of the helmet-orchids instead of their tuberoids, and serendipitoid fungi were predominant, while tulasnelloid were absent in the three investigated Corybas species regardless of their altitude. The three Corybas species shared 27 serendipitoid operational taxonomic units that are different to those of their related orchids, the genera of Cyrtostylis and Stigmatodactylus. Corybas shanlinshiensis alone had a range of ectomycorrhizal fungi (mainly russuloid and thelephoroid) broader than C. himalaicus and C. geminigibbus. Our study provides new information about terrestrial orchid–fungi associations and may further contribute to orchid conservation. [ABSTRACT FROM AUTHOR]

Among land plants, which generally exhibit autotrophy through photosynthesis, about 880 species are mycoheterotrophs, dependent on mycorrhizal fungi for their carbon supply. Shifts in nutritional mode from autotrophy to mycoheterotrophy are usually accompanied by evolution of various combinations of characters related to structure and physiology, e.g., loss of foliage leaves and roots, reduction in seed size, degradation of plastid genome, and changes in mycorrhizal association and pollination strategy. However, the patterns and processes involved in such alterations are generally unknown. Hybrids between autotrophic and mycoheterotrophic plants may provide a breakthrough in molecular studies on the evolution of mycoheterotrophy. We have produced the first hybrid between autotrophic and mycoheterotrophic plant species using the orchid group Cymbidium. The autotrophic Cymbidium ensifolium subsp. haematodes and mycoheterotrophic C. macrorhizon were artificially pollinated, and aseptic germination of the hybrid seeds obtained was promoted by sonication. In vitro flowering was observed five years after seed sowing. Development of foliage leaves, an important character for photosynthesis, segregated in the first generation; that is, some individuals only developed scale leaves on the rhizome and flowering stems. However, all of the flowering plants formed roots, which is identical to the maternal parent.

Stable isotope signatures of fungal sporocarps have been instrumental in identifying carbon gains of chlorophyllous orchids from a fungal source. Yet, not all mycorrhizal fungi produce macroscopic sporocarps and frequently fungi of different taxa occur in parallel in orchid roots. To overcome this obstacle, we investigated stable isotope signatures of fungal pelotons extracted from orchid roots and compared these data to the respective orchid and reference plant tissues. Anoectochilus sandvicensis and Epipactis palustris represented specialized or unspecialized rhizoctonia-associated orchids. Epipactis atrorubens and Epipactis leptochila are orchids considered ectomycorrhiza-associated with different preferences for Basidio- and Ascomycota. 13 C enrichment of rhizoctonia pelotons was minor compared with plant tissues and significantly lower than enrichments of pelotons from ectomycorrhizal Epipactis species. 15 N values of pelotons from E. leptochila and E. atrorubens showed similar patterns as known for respective sporocarps of ectomycorrhizal Ascomycota and Basidiomycota, however, with an offset towards lower 15 N enrichments and nitrogen concentrations. Our results suggest an explicit fungal nutrition source of orchids associated with ectomycorrhizal fungi, whereas the low 13 C enrichment in rhizoctonia-associated orchids and fungal pelotons hamper the detection of carbon gains from fungal partners. 15 N isotopic pattern of orchids further suggests a selective transfer of 15 N-enriched protein-nitrogen into orchids., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

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.

Mycorrhizal symbiosis is common among land plants including pteridophytes (monilophytes and lycophytes). In pteridophytes with diplohaplontic life cycle, mycorrhizal formations were mostly reported for sporophytes, but very few for gametophytes. To clarify the mycorrhizal association of photosynthetic gametophytes, field-collected gametophytes of Angiopteris lygodiifolia (Marattiaceae, n = 52) and Osmunda japonica (Osmundaceae, n = 45) were examined using microscopic and molecular techniques. Collected gametophytes were mostly cut into two pieces. One piece was used for light and scanning microscopic observations, and the other for molecular identification of plant species (chloroplast rbcL sequences) and mycorrhizal fungi (small subunit rDNA sequences). Microscopic observations showed that 96 % (50/52) of Angiopteris and 95 % (41/43) of Osmunda gametophytes contained intracellular hyphae with arbuscules and/or vesicles and fungal colonization was limited to the inner tissue of the thick midribs (cushion). Fungal DNA analyses showed that 92 % (48/52) of Angiopteris and 92 % (35/38) of Osmunda have sequences of arbuscular mycorrhizal fungi, which were highly divergent but all belonged to Glomus group A. These results suggest that A. lygodiifolia and O. japonica gametophytes consistently form arbuscular mycorrhizae. Mycorrhizal formation in wild fern gametophytes, based on large-scale sampling with molecular identification of host plant species, was demonstrated for the first time.

Premise of the Study: Mycoheterotrophic plants, which completely depend upon mycorrhizal fungi for their nutrient supply, have unusual associations with fungal partners. The processes involved in shifts in fungal associations during cladogenesis of plant partners from autotrophy to mycoheterotrophy have not been demonstrated using a robust phylogenetic framework., Methods: Consequences of a mycorrhizal shift were examined in Cymbidium (Orchidaceae) using achlorophyllous and sister chlorophyllous species. Fungal associates of the two achlorophyllous mycoheterotrophs (C. macrorhizon and C. aberrans), their close relatives, the chlorophyllous mixotrophs (C. goeringii and C. lancifolium) and an outgroup, the chlorophyllous autotroph C. dayanum, were identified by internal transcribed spacers of the nuclear ribosomal DNA sequences., Key Results: Molecular identification of mycorrhizal fungi revealed: (1) the outgroup autotroph is predominantly dependent on saprobic Tulasnellaceae, (2) the mixotrophs associate with the Tulasnellaceae and ectomycorrhizal groups including the Sebacinales, Russulaceae, Thelephoraceae and Clavulinaceae, and (3) the two mycoheterotrophs are mostly specialized with ectomycorrhizal Sebacinales., Conclusion: Fungal partners in Cymbidium have shifted from saprobic to ectomycorrhizal fungi via a phase of coexistence of both nutritional types of fungi. These three phases correspond to the evolution from autotrophy to mycoheterotrophy via mixotrophy in Cymbidium. We demonstrate that shifts in mycorrhizal fungi correlate with the evolution of nutritional modes in plants. Furthermore, gradual shifts in fungal partners through a phase of coexistence of different types of mycobionts may play a crucial role in the evolution of mycoheterotrophic plants.

Mycorrhizal fungi in roots of the achlorophyllous Petrosavia sakuraii (Petrosaviaceae) were identified by molecular methods. Habitats examined were plantations of the Japanese cypress Chamaecyparis obtusa in Honshu, an evergreen broad-leaved forest in Amami Island in Japan and a mixed deciduous and evergreen forest in China. Aseptate hyphal coils were observed in root cortical cells of P. sakuraii, suggesting Paris-type arbuscular mycorrhiza (AM). Furthermore, hyphal coils that had degenerated to amorphous clumps were found in various layers of the root cortex. Despite extensive sampling of P. sakuraii from various sites in Japan and China, most of the obtained AM fungal sequences of the nuclear small subunit ribosomal RNA gene were nearly identical and phylogenetic analysis revealed that they formed a single clade in the Glomus group A lineage. This suggests that the symbiotic relationship is highly specific. AM fungi of P. sakuraii were phylogenetically different from those previously detected in the roots of some mycoheterotrophic plants. In a habitat in C. obtusa plantation, approximately half of the AM fungi detected in roots of C. obtusa surrounding P. sakuraii belonged to the same clade as that of P. sakuraii. This indicates that particular AM fungi are selected by P. sakuraii from diverse indigenous AM fungi. The same AM fungi can colonize both plant species, and photosynthates of C. obtusa may be supplied to P. sakuraii through a shared AM fungal mycelial network. Although C. obtusa plantations are widely distributed throughout Japan, P. petrosavia is a rare plant species, probably because of its high specificity towards particular AM fungi., (© Springer-Verlag 2011)

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.

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 clade 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 clade 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.

We investigated the physiological ecology of the Asian non-photosynthetic orchid Gastrodia confusa. We revealed its mycorrhizal partners by using molecular identification and identified its ultimate nutritional source by analysing carbon and nitrogen natural stable isotope abundances. Molecular identification using internal transcribed spacer and large subunit nrDNA sequences showed that G. confusa associates with several species of litter- and wood-decomposer Mycena fungi. The carbon and nitrogen isotope signatures of G. confusa were analysed together with photosynthetic plant reference samples and samples of the ectomycorrhizal epiparasite Monotropa uniflora. We found that G. confusa was highly enriched in (13)C but not greatly in (15)N, while M. uniflora was highly enriched in both (13)C and (15)N. The (13)C and (15)N signatures of G. confusa were the closest to those of the fruit bodies of saprotrophic fungi. Our results demonstrate for the first time using molecular and mass-spectrometric approaches that myco-heterotrophic plants gain carbon through parasitism of wood or litter decaying fungi. Furthermore, we demonstrate that, several otherwise free-living non-mycorrhizal, Mycena can be mycorrhizal partners of orchids.

Epipactis helleborine (L.) Crantz, one of the most widespread orchid species, occurs in a broad range of habitats. This orchid is fully myco-heterotrophic in the germination stage and partially myco-heterotrophic in the adult stage, suggesting that a mycorrhizal partner is one of the key factors that determines whether E. helleborine successfully colonizes a specific environment. We focused on the coastal habitat of Japanese E. helleborine and surveyed the mycorrhizal fungi from geographically different coastal populations that grow in Japanese black pine (Pinus thunbergii Parl.) forests of coastal sand dunes. Mycorrhizal fungi and plant haplotypes were then compared with those from inland populations. Molecular phylogenetic analysis of large subunit rRNA sequences of fungi from its roots revealed that E. helleborine is mainly associated with several ectomycorrhizal taxa of the Pezizales, such as Wilcoxina, Tuber, and Hydnotrya. All individuals from coastal dunes were exclusively associated with a pezizalean fungus, Wilcoxina, which is ectomycorrhizal with pine trees growing on coastal dunes. Wilcoxina was not detected in inland forests. Coastal populations were indistinguishable from inland populations based on plant trnL intron haplotypes. Our results indicate that mycorrhizal association with geographically restricted pezizalean ectomycorrhizal fungi is a key control upon this orchid species' distribution across widely different forest habitats.

Because mycoheterotrophic plants fully depend on their mycorrhizal partner for their carbon supply, the major limiting factor for the geographic distribution of these plants may be the presence of their mycorrhizal partner. Although this factor may seem to be a disadvantage for increasing geographic distribution, widespread mycoheterotrophic species nonetheless exist. The mechanism causing the wide distribution of some mycoheterotrophic species is, however, seldom discussed. We identified the mycorrhizal partner of a widespread mycoheterotrophic orchid, Eulophia zollingeri, using 12 individuals from seven populations in Japan, Myanmar, and Taiwan by DNA-based methods. All fungal ITS sequences from the roots closely related to those of Psathyrella candolleana (Coprinaceae) from GenBank accessions and herbarium specimens. These results indicate that E. zollingeri is exclusively associated with the P. candolleana species group. Further, the molecular data support the wide distribution and wide-ranging habitat of this fungal partner. Our data provide evidence that a mycoheterotrophic plant can achieve a wide distribution, even though it has a high mycorrhizal specificity, if its fungal partner is widely distributed.

Dendrobium officinale Kimura & Migo in the genus Dendrobium of Orchidaceae is an important medicinal plant that produces various bibenzyl and phenanthrene derivatives. In some orchids, these derivatives have been reported to increase with fungal infection. Bibenzyl biosynthesis is regulated by bibenzyl synthase (BBS). Although six genes of the BBS family have been registered from D. officinale, their gene regulation mechanisms are unclear. The infection of Dendrobium with mycorrhizal fungi also reportedly increases the expression of genes involved in biosynthesis; however, the effect of mycorrhizal fungi on bibenzyl production is unknown. The present study examined the effects of three mycorrhizal fungi isolated from D. officinale on BBS gene expression and bibenzyl production over time. One of the Tulasnellaceae operational taxonomic units induced BBS gene expression and increased two representative bibenzyls, gigantol and dendrophenol, at specific time points. Furthermore, 19 BBS sequences were cloned from 12 Dendrobium species, and a phylogenetic analysis was performed. The results indicated that repeated BBS gene duplication occurred during the evolution of the genus, and further duplication occurred after speciation. These results suggest that it is possible to optimize metabolite production by selecting suitable symbiotic fungi. [ABSTRACT FROM AUTHOR]

Multi-generational asexual reproduction of Gastrodia elata Bl. will cause seedling species degeneration. Sexual reproduction of Gastrodia elata Bl. seed is an effective method to solve the problem of degeneration. The development of Gastrodia elata Bl. seeds cannot be separated from the germination fungus. However, there are few strains of germination fungus in production, and there is also the problem of species degradation in application for many years. It is very important for the sexual reproduction of Gastrodia elata Bl. to isolate more new strains of excellent germination fungus from the origin. This study used the Gastrodia elata Bl. f. glauca S. chow seeds germination vegetative propagation corms capture method to isolate its symbiotic germination fungus, and comprehensively identified the species of germination fungus by colony morphology, ITS, sporocarps regeneration and germination function, and compared the growth characteristics and germination ability with other germination fungus (Mycena purpureofusca, Mycena dendrobii and Mycena osmundicola). The germination fungus was isolated from the vegetative propagation corms of Gastrodia elata Bl. f. glauca S. chow seeds and named GYGL-1. After comprehensive identification, GYGL-1 was Mycetinis scorodonius. Compared with other germination fungus, GYGL-1 has fast germination speed, vigorous growth, and high germination ability for Gastrodia elata Bl. f. glauca S. chow seeds. Innovated the isolation method of Gastrodia elata Bl. seeds germination fungus, obtained the regenerated sporocarps of the germination fungus, and discovered that Mycetinis scorodonius has a new function of germinating Gastrodia elata Bl. f. glauca S. chow seeds, enriching the resource library of Gastrodia elata Bl. germination fungus. [ABSTRACT FROM AUTHOR]

Basidiomycota is one of the major phyla in the fungal tree of life. The outline of Basidiomycota provides essential taxonomic information for researchers and workers in mycology. In this study, we present a time-framed phylogenomic tree with 487 species of Basidiomycota from 127 families, 47 orders, 14 classes and four subphyla; we update the outline of Basidiomycota based on the phylogenomic relationships and the taxonomic studies since 2019; and we provide notes for each order and discuss the history, defining characteristics, evolution, justification of orders, problems, significance, and plates. Our phylogenomic analysis suggests that the subphyla diverged in a time range of 443–490 Myr (million years), classes in a time range of 312–412 Myr, and orders in a time range of 102–361 Myr. Families diverged in a time range of 50–289 Myr, 76–224 Myr, and 62–156 Myr in Agaricomycotina, Pucciniomycotina, and Ustilaginomycotina, respectively. Based on the phylogenomic relationships and divergence times, we propose a new suborder Mycenineae in Agaricales to accommodate Mycenaceae. In the current outline of Basidiomycota, there are four subphyla, 20 classes, 77 orders, 297 families, and 2134 genera accepted. When building a robust taxonomy of Basidiomycota in the genomic era, the generation of molecular phylogenetic data has become relatively easier. Finding phenotypical characters, especially those that can be applied for identification and classification, however, has become increasingly challenging. [ABSTRACT FROM AUTHOR]

In 2017, two candidate species of Mycena were reported from Japan, with the Japanese names “Togari-sakura-take” and “Mitsuhida-sakura-take”. However, to date, no taxonomic study or formal description has been undertaken for these two species. In the present study, we conducted comprehensive morphological and molecular phylogenetic examinations of “Togari-sakura-take” and “Mitsuhida-sakura-take”, and compared them to known species within the genus Mycena. We performed phylogenetic analyses on a concatenated dataset, including the internal transcribed spacer region of ribosomal RNA, RNA polymerase II largest subunit, and translation elongation factor-1 alpha genes. “Togari-sakura-take” formed a clade with Mycena subulata, which was recently described from China, whereas “Mitsuhida-sakura-take” formed a distinct independent clade. We identified the former as M. subulata based on molecular phylogenetic analyses and morphological observations. However, the Japanese specimens displayed dextrinoid cheilocystidia and caulocystidia as well as the inamyloidity of basidiospores, which differed from the original description of M. subulata based on the materials from China. “Mitsuhida-sakura-take” was characterized by its remarkably dense lamellae and could be distinguished from known Mycena species by the combination of absent pleurocystidia and presence of bowling pin-shaped cheilocystidia. Here, we describe “Mitsuhida-sakura-take” as a new species, named Mycena densilamellata, in the section Calodontes. [ABSTRACT FROM AUTHOR]

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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]

Premise: Approximately 14% of all fern species have physiologically active chlorophyllous spores that are much more short-lived than the more common and dormant achlorophyllous spores. Most chlorophyllous-spored species (70%) are epiphytes and account for almost 37% of all epiphytic ferns. Chlorophyllous-spored ferns are also overrepresented among fern species in habitats with waterlogged soils, of which nearly 60% have chlorophyllous spores. Ferns in these disparate habitat types also have a low incidence of mycorrhizal associations. We therefore hypothesized that autotrophic chlorophyllous spores represent an adaptation of ferns to habitats with scarce mycorrhizal associations., Methods: We evaluated the coevolution of chlorophyllous spores and mycorrhizal associations in ferns and their relation to habitat type using phylogenetic comparative methods., Results: Although we did not find support for the coevolution of spore type and mycorrhizal associations, we did find that chlorophyllous spores and the absence of mycorrhizal associations have coevolved with epiphytic and waterlogged habitats. Transition rates to epiphytic and waterlogged habitats were significantly higher in species with chlorophyllous spores compared to achlorophyllous lineages., Conclusions: Spore type and mycorrhizal associations appear to play important roles in the radiation of ferns into different habitat types. Future work should focus on clarifying the functional significance of these associations., (© 2022 The Authors. American Journal of Botany published by Wiley Periodicals LLC on behalf of Botanical Society of America.)

Epiphytic orchids are commonly found in exposed environments, which plausibly lead to different root fungal community structures from terrestrial orchids. Until recently, few studies have been conducted to show the fungal community structure during the growth of a photosynthetic and epiphytic orchid in its natural growing site. In this study, the Vanda falcata (commonly known as Neofinetia falcata), one of Japan's ornamental orchids, was used to characterize the fungal community structure at different developmental stages. Amplicon sequencing analysis showed that all development stages contain a similar fungal community: Ascomycota dominate half of the community while one-third of the community belongs to Basidiomycota. Rhizoctonia-like fungi, a polyphyletic basidiomycetous fungal group forming mycorrhizas in many orchids, exist even in a smaller portion (around one-quarter) compared to other Basidiomycota members. While ascomycetous fungi exhibit pathogenicity, two Ceratobasidium strains isolated from young and adult plants could initiate seed germination in vitro. It was also found that the colonization of mycorrhizal fungi was concentrated in a part of the root where it directly attaches to the phorophyte bark, while ascomycetous fungi were distributed in the velamen but never colonized cortical cells. Additionally, the root parts attached to the bark have denser exodermal passage cells, and these cells were only colonized by mycorrhizal fungi that further penetrated into the cortical area. Therefore, we confirmed a process that physical regulation of fungal entry to partition the ascomycetes and mycorrhizal fungi results in the balanced mycorrhizal symbiosis in this orchid., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The orchid family (Orchidaceae) is one of the most diverse plant families in the world, but at the same time also contains one of the largest number of rare and endangered species. While conservation actions such as in situ and ex situ conservation and prohibition of international trade of wild orchids have achieved positive results to slow down the current decline of orchids, species with high medicinal or ornamental value may require more advanced measures. Recent pilot studies using novel cultivation techniques have successfully reintroduced endangered epiphytic orchids and facilitated the commercial cultivation of medicinal orchids. Because fungal partners play a key role in in situ symbiotic seed germination and industrial cultivation, we advocate for the development of fungus banks in laboratories engaging in orchid research, where fungi obtained from germinating seeds or seedlings can be studied and used to improve orchid germination under both in vitro and in situ conditions. Furthermore, these fungi could be shared nationally and internationally, enhancing orchid conservation efforts across the globe. Similar to seed banks, the development of fungus banks will reduce the possibility of fungi going extinct and ensure their availability for reintroduction programs and commercial cultivation. With the availability of both a fungus and seed bank, the conservation of threatened orchid species can be significantly enhanced by improving restoration programs and commercial cultivation of medicinal orchids and releasing the pressure on wild populations from harvesting. [ABSTRACT FROM AUTHOR]

The degree of specificity between fully myco-heterotrophic (MH) orchids and mycorrhizal fungi is regarded as high, but some species undergo a mycobiont shift as they transition from juvenile to adult plant. We investigated morphological and physiological aspects of the interaction between the fully MH Cyrtosia septentrionalis and its four known mycobionts to elucidate developmental consequences of variable, life-stage-dependent specificity. Of five randomly sampled germinated seeds co-cultured with each mycobiont, Physisporinus sp. 'TK-10' colonized all, Armillaria gallica and Desarmillaria tabescens colonized one, and A. mellea subsp. nipponica colonized none. Whereas 16.2% of aseptically germinated seeds exhibited onward growth when co-cultured with TK-10, just 1.5-2.6% did so with the other species. Even so, A. gallica colonized and formed internal rhizomorphs within rhizomes established with TK-10, suggesting that this mycobiont can replace and potentially can oust the latter. We infer that the orchid can associate with Armillaria and Desarmillaria mycobionts throughout its life, but that TK-10 enhances early growth. However, because TK-10 has a higher wood-rotting capacity than A. gallica, rapid resource exhaustion may cause nutrient supply shortages. We hypothesize that secondary colonization by Armillaria or Desarmillaria species triggers TK-10 displacement and that this mycobiont shift sustains orchid growth for longer. [ABSTRACT FROM AUTHOR]

Objective: To Identify the endophytic fungal community of the G. skinneri root in urban populations, showing differences in composition between velamen and cortex to better understand the fungus-root interaction in these orchids. Design/Methodology/Approach: We collected tissues from velamen and root from five specimens of G. skinneri growing on urban trees in Tapachula, Chiapas. We extracted DNA, PCR amplified the ITS marker, sequenced on the Illumina platform followed by diversity analyses and taxonomic assignment. Results: We detected 845 OTUs that were assigned to the Fungi kingdom. Velamen and cortex share 403 OTUs, 402 were found exclusively in velamen and 40 in cortex. Besides orchid mycorrhiza forming fungi, we detected other species (e.g Alternaria sp., Beauveria sp., Fusarium sp., Glomus sp. and Tricoderma sp.) that could be involved in root physiology during development and defense against pathogens and predators. Study Limitations/Implications: Metagenomic studies provide substantial amounts of data that go beyond conventional studies. However, the information generated is still limited regarding the role of each endophyte. Findings/Conclusions: Despite these limitations, our work fills a knowledge gap because we detected endophytes that were previously unknown for G. skinneri, leading to new research questions about rootendophyte relationships. [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]

Full myco‐heterotrophic orchid Gastrodia elata Bl. is widely distributed in Northeast Asia, and previous research has not fully investigated the symbiotic fungal community of its early immature tubers. This study utilized Illumina sequencing to compare symbiotic fungal communities in natural G. elata immature tubers and their habitats. LEfSe (Linear Discriminant Analysis Effect Size) was used to screen for Biomarkers that could explain variations among different fungal communities, and correlation analyses were performed among Biomarkers and other common orchid mycorrhizal fungi. Our results illustrate that the symbiotic fungal communities of immature G. elata tubers cannot be simply interpreted as subsets of the environmental fungal communities because some key members cannot be traced back to the environment. The early growth of G. elata was related to a small group of fungi, such as Sebacina, Thelephora, and Inocybe, which were also common mycorrhizal fungi from other orchids. In addition, Mycena, Auricularia, and Cryptococcus were unique fungal partners of G. elata, and many new species have yet to be discovered. Possible symbiotic Mycena should be M. plumipes and its sibling species in this case. Our results provide insight into the symbiotic partner switch and trophic pattern change during the development and maturation of G. elata. [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]

Gastrodia elata Blume, a fully mycoheterotrophic perennial plant of the family Orchidaceae, is a traditional Chinese herb with medicinal and edible value. Interestingly, G. elata requires symbiotic relationships with Mycena and Armillaria strains for seed germination and plant growth, respectively. However, there is no comprehensive summary of the symbiotic mechanism between fungi and G. elata. Here, the colonization and digestion of hyphae, the bidirectional exchange of nutrients, the adaptation of fungi and G. elata to symbiosis, and the role of microorganisms and secondary metabolites in the symbiotic relationship between fungi and G. elata are summarized. We comprehensively and deeply analyzed the mechanism of symbiosis between G. elata and fungi from three perspectives: morphology, nutrition, and molecules. The aim of this review was to enrich the understanding of the mutualistic symbiosis mechanisms between plants and fungi and lay a theoretical foundation for the ecological cultivation of G. elata. [ABSTRACT FROM AUTHOR]

Purpose. Improvement of the gene pool of the cultivated form of eggplant (Solanum melongena L.) through interspecific hybridisation with Solanum aethiopicum L. and gametic breeding; analysis of the interspecific lines for a set of valuable quantitative traits determining the yield structure. Methods. Studies for the comprehensive evaluation of eggplant lines of interspecific origin were carried out during 2021-2023 in protected soil conditions at the experimental base of the Institute of Vegetable and Melon Growing of the National Academy of Agrarian Sciences of Ukraine. Phenological observations and biometric measurements of plants of four lines were carried out. The stability of the manifestation of seven quantitative traits during the research years was determined by the methods of variation statistics [calculation of the mean square deviation (σ), coefficient of variation (V), Pearson's paired linear correlation (rp)]. Results. The analysis identified eggplant lines of interspecific origin that were superior to the standard variety 'Almaz' in terms of the level and stability of quantitative traits. Thus, F6(Pavlotas-20 / 'Almaz')I1 and ВС1[F5 (Pavlotas-20 / 'Almaz')]I1 showed a statistically significant increase in fruit width (by 16.48% and 14.85%, respectively). Meanwhile, line BC2[F5 (Pavlotas-20 / 'Almaz')]I1 exhibited higher productivity (by 18.11%) compared to the standard variety. The correlation analysis shows that the trait "Productivity of one plant" has a strong positive relationship with the trait "Number of fruits per plant" (rp = 0.75) and a strong negative relationship with "Average fruit width" (rp = -0.70) and "Duration of the period from mass germination to technical fruit maturity" (rp = -0.72). The selected line BC2[F5 (Pavlotas-20 / 'Almaz')]I1 exhibited a four-day shorter period from mass germination to technical fruit maturity compared to the standard variety. Conclusions. The line BC2[F5 (Pavlotas-20 / 'Almaz')]I1 was isolated based on the complex of quantitative traits. It underwent an additional stage of gametophytic breeding, resulting in a high adaptive potential to growing conditions. [ABSTRACT FROM AUTHOR]