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Paphiopedilum armeniacum, Paphiopedilum wenshanense and Paphiopedilum emersonii are critically endangered wild orchids. Their populations are under severe threat, with a dramatic decline in the number of their natural distribution sites. Ex situ conservation and artificial breeding are the keys to maintaining the population to ensure the success of ex situ conservation and field return in the future. The habitat characteristics and soil nutrient information of the last remaining wild distribution sites of the three species were studied. ITS high-throughput sequencing was used to reveal the composition and structure of the soil fungal community, analyze its diversity and functional characteristics, and reveal its relationship with soil nutrients. The three species preferred to grow on low-lying, ventilated and shaded declivities with good water drainage. There were significant differences in soil alkali-hydrolyzed nitrogen and available phosphorus among the three species. There were 336 fungal species detected in the samples. On average, there were different dominant groups in the soil fungal communities of the three species. The functional groups of soil fungi within their habitats were dominated by saprophytic fungi and ectomycorrhizae, with significant differences in diversity and structure. The co-occurrence network of habitat soil fungi was mainly positive. Soil pH significantly affected soil fungal diversity within their habitats of the three paphiopedilum species. The study confirmed that the dominant groups of soil fungi were significantly correlated with soil nutrients. The three species exhibit comparable habitat inclinations, yet they display substantial variations in the composition, structure, and diversity of soil fungi. The fungal functional group is characterized by a rich presence of saprophytic fungi, a proliferation of ectomycorrhizae, and a modest occurrence of orchid mycorrhizae. The symbiotic interactions among the soil fungi associated with these three species are well-coordinated, enhancing their resilience against challenging environmental conditions. There is a significant correlation between soil environmental factors and the composition of soil fungal communities, with pH emerging as a pivotal factor regulating fungal diversity. Our research into the habitat traits and soil fungal ecosystems of the three wild Paphiopedilum species has established a cornerstone for prospective ex situ conservation measures and the eventual reestablishment of these species in their native landscapes., (© 2024. The Author(s).)

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.)

Background and Aims: The earliest-diverging orchid lineage, Apostasioideae, consists only of two genera: Apostasia and Neuwiedia. Previous reports of Apostasia nipponica indicated a symbiotic association with an ectomycorrhiza-forming Ceratobasidiaceae clade and partial utilization of fungal carbon during the adult stage. However, the trophic strategy of Neuwiedia throughout its development remains unidentified. To further improve our understanding of mycoheterotrophy in the Apostasioideae, this study focused on Neuwiedia malipoensis examining both the mycorrhizal association and the physiological ecology of this orchid species across various development stages., Methods: We identified the major mycorrhizal fungi of N. malipoensis protocorm, leafy seedling and adult stages using molecular barcoding. To reveal nutritional resources utilized by N. malipoensis, we compared stable isotope natural abundances (δ13C, δ15N, δ2H, δ18O) of different developmental stages with those of autotrophic reference plants., Key Results: Protocorms exhibited an association with saprotrophic Ceratobasidiaceae rather than ectomycorrhiza-forming Ceratobasidiaceae and the 13C signature was characteristic of their fully mycoheterotrophic nutrition. Seedlings and adults were predominantly associated with saprotrophic fungi belonging to the Tulasnellaceae. While 13C and 2H stable isotope data revealed partial mycoheterotrophy of seedlings, it is unclear to what extent the fungal carbon supply is reduced in adult N. malipoensis. However, the 15N enrichment of mature N. malipoensis suggests partially mycoheterotrophic nutrition. Our data indicated a transition in mycorrhizal partners during ontogenetic development with decreasing dependency of N. malipoensis on fungal nitrogen and carbon., Conclusions: The divergence in mycorrhizal partners between N. malipoensis and A. nipponica indicates different resource acquisition strategies and allows various habitat options in the earliest-diverging orchid lineage, Apostasioideae. While A. nipponica relies on the heterotrophic carbon gain from its ectomycorrhizal fungal partner and thus on forest habitats, N. malipoensis rather relies on own photosynthetic carbon gain as an adult, allowing it to establish in habitats as widely distributed as those where Rhizoctonia fungi occur., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Main Conclusion: In this review, we have discussed the untapped potential of orchid endophytic bacteria as a valuable reservoir of bioactive metabolites, offering significant contributions to plant growth promotion and disease protection in the context of sustainable agriculture. Orchidaceae is one of the broadest and most diverse flowering plant families on Earth. Although the relationship between orchids and fungi is well documented, bacterial endophytes have recently gained attention for their roles in host development, vigor, and as sources of novel bioactive compounds. These endophytes establish mutualistic relationships with orchids, influencing plant growth, mineral solubilization, nitrogen fixation, and protection from environmental stress and phytopathogens. Current research on orchid-associated bacterial endophytes is limited, presenting significant opportunities to discover new species or genetic variants that improve host fitness and stress tolerance. The potential for extracting bioactive compounds from these bacteria is considerable, and optimization strategies for their sustainable production could significantly enhance their commercial utility. This review discusses the methods used in isolating and identifying endophytic bacteria from orchids, their diversity and significance in promoting orchid growth, and the production of bioactive compounds, with an emphasis on their potential applications in sustainable agriculture and other sectors., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Three novel mycelium-forming actinobacteria, designated OC33-EN06 T , OC33-EN07 T , and OC33-EN08 T , were isolated from wild orchid ( Aerides multiflora Roxb), collected from a hill evergreen forest in Northern Thailand. Strains OC33-EN06 T and OC33-EN07 T showed the highest 16S rRNA gene similarity with Actinomycetospora lutea TT00-04 T , 99.17 and 99.45%, respectively. Strain OC33-EN08 T showed high similarity with four species, namely ' Actinomycetospora termitidis Odt1-22 T ' (99.37%), Actinomycetospora chiangmaiensis DSM 45062 T (99.02%), Actinomycetospora corticicola 014-5 T (99.02%), and Actinomycetospora soli SF1 T (98.81%). Comparative genome analysis of OC33-EN06 T , OC33-EN07 T , and OC33-EN08 T with the closely related type strains showed that average nucleotide identity (ANI) based on blast, ANI based on MUMmer, and average amino acid identity values were less than 95% and the digital DNA-DNA hybridization values were less than 70%, all below the thresholds for species demarcation. The digital G+C content of OC33-EN06 T , OC33-EN07 T , and OC33-EN08 T were 74.5, 74, and 74 mol%, respectively. These three strains developed bud-like chains of non-motile cylindrical spores with a smooth surface. The cell-wall peptidoglycan contained meso -diaminopimelic acid. The whole-cell sugars contained ribose, arabinose, and galactose. The predominant menaquinone was MK-8(H 4 ). The phospholipid profile included phosphatidylcholine, phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylinositol. Based on comparative analysis of genotypic, phenotypic and chemotaxonomic data, strains OC33-EN06 T (=TBRC 18349 T =NBRC 116543 T ), OC33-EN07 T (=TBRC 18350 T =NBRC 116544 T ), and OC33-EN08 T (=TBRC 18318 T =NBRC 116542 T ) represent the type strains of three novel species of the genus Actinomycetospora for which the names Actinomycetospora aeridis sp. nov., Actinomycetospora flava sp. nov., and Actinomycetospora aurantiaca sp. nov., are proposed.

This study characterized and identified the antimicrobial compounds from an endophytic fungus (Fusarium incarnatum (C4)) isolated from the orchid, Cymbidium sp. Chromatographic techniques were employed to separate the bioactive compounds from the crude extracts of F. incarnatum (C4). Following bio-guided fractionation, two fractionated extracts (fractions 1 and 2) of F. incarnatum (C4) exhibited antibacterial and antifungal activities against Bacillus cereus (MIC: 0.156 mg/mL) and Ganoderma boninense (MIC: 0.3125 mg/mL), respectively. The active fractions were discovered to comprise of a variety of bioactive compounds with pharmacological importance (alkaloids, flavonoids, phenolic compounds, terpenoids, peptides and fatty acids). Liquid chromatography mass-spectrometry (LCMS) analysis detected the presence of antibacterial (kanzonol N, rifaximin, linoleic acid (d4), cannabisativine, docosanedioic acid, and stearamide) and antifungal components (3-methyl-quinolin-2-ol, prothiocarb, kanzonol N, peganine, 5Z-tridecene, and tetronasin) in fractions 1 and 2, respectively, which may have contributed to the antimicrobial effects. Findings from this study highlighted the important potential of fungal endophytes from medicinal hosts as producers of antimicrobials and antibiotics., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

This study profiled the various endophytic fungi isolated from the orchid Cymbidium sp. and their L-asparaginase production and antioxidant potential. The L-asparaginase production was first screened through qualitative plate screening then quantified by the Nesslerization method. The antioxidant potential was quantified via the 2,2-diphenyl-1-picrylhydrazyl assay. A total of 30 endophytic fungi were isolated and all fungal isolates exhibited various degrees of radical scavenging activities (45.28% to 76.4%). Isolate Lasiodiplodia theobromae (C11) had the highest antioxidant capacity, represented by the lowest IC 50 value (5.75 mg/mL) and highest ascorbic acid equivalent antioxidant capacity value (12.17 mg/g). Additionally, 16 isolates produced L-asparaginase (53.33%), which includes primarily species of Fusarium proliferatum, Fusarium fujikuroi, Fusarium incarnatum, and Fusarium oxysporum. A new isolate has also been discovered from Cymbidium orchid, Buergenerula spartinae (C28), which showed the highest L-asparaginase activity (1.736 unit/mL). These findings supported the postulation that medicinal species of Orchidaceae such as Cymbidium sp. harbor endophytes that are producers of L-asparaginase and antioxidants with various potential applications., (© 2023. Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i.)

The minute 'dust seeds' of some terrestrial orchids preferentially germinate and develop as mycoheterotrophic protocorms near conspecific adult plants. Here we test the hypothesis that mycorrhizal mycelial connections provide a direct pathway for transfer of recent photosynthate from conspecific green orchids to achlorophyllous protocorms. Mycelial networks of Ceratobasidium cornigerum connecting green Dactylorhiza fuchsii plants with developing achlorophyllous protocorms of the same species were established on oatmeal or water agar before the shoots of green plants were exposed to 14 CO 2 . After incubation for 48 h, the pattern of distribution of fixed carbon was visualised in intact entire autotrophic/protocorm systems using digital autoradiography and quantified in protocorms by liquid scintillation counting. Both methods of analysis revealed accumulation of 14 C above background levels in protocorms, confirming that autotrophic plants supply carbon to juveniles via common mycorrhizal networks. Despite some accumulation of plant-fixed carbon in the fungal mycelium grown on oatmeal agar, a greater amount of carbon was transferred to protocorms growing on water agar, indicating that the polarity of transfer may be influenced by sink strength. We suggest this transfer pathway may contribute significantly to the pattern and processes determining localised orchid establishment in nature, and that 'parental nurture' via common mycelial networks may be involved in these processes., (© 2024 The Authors. New Phytologist © 2024 New Phytologist Foundation.)

Despite being the second largest family of flowering plants, orchids represent community structure variation in plant-microbial associations, contributes to niche partitioning in metacommunity assemblages. Yet, mycorrhizal communities and interactions remain unknown for orchids that are highly specialized or even obligated in their associations with their mycorrhizal partners. In this study, we sought to compare orchid mycorrhizal fungal (OMF) communities of three co-occurring hemiepiphytic Vanilla species (V. hartii, V. pompona, and V. trigonocarpa) in tropical forests of Costa Rica by addressing the identity of their OMF communities across species, root types, and populations, using high-throughput sequencing. Sequencing the nuclear ribosomal internal transcribed spacer (nrITS) yielded 299 fungal Operational Taxonomic Units (OTUs) from 193 root samples. We showed distinct segregation in the putative OMF (pOMF) communities of the three coexisting Vanilla hosts. We also found that mycorrhizal communities associated with the rare V. hartii varied among populations. Furthermore, we identified Tulasnellaceae and Ceratobasidiaceae as dominant pOMF families in terrestrial roots of the three Vanilla species. In contrast, the epiphytic roots were mainly dominated by OTUs belonging to the Atractiellales and Serendipitaceae. Furthermore, the pOMF communities differed significantly across populations of the widespread V. trigonocarpa and showed patterns of distance decay in similarity. This is the first report of different pOMF communities detected in roots of wild co-occurring Vanilla species using high-throughput sequencing, which provides evidence that three coexisting Vanilla species and their root types exhibited pOMF niche partitioning, and that the rare and widespread Vanilla hosts displayed diverse mycorrhizal preferences., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Bletilla striata (Thunb.) Reichb. f. (B. striata) is a perennial herbaceous plant in the Orchidaceae family known for its diverse pharmacological activities, such as promoting wound healing, hemostasis, anti-inflammatory effects, antioxidant properties, and immune regulation. Nevertheless, the microbe-plant-metabolite regulation patterns for B. striata remain largely undetermined, especially in the field of rhizosphere microbes. To elucidate the interrelationships between soil physics and chemistry and rhizosphere microbes and metabolites, a comprehensive approach combining metagenome analysis and targeted metabolomics was employed to investigate the rhizosphere soil and tubers from four provinces and eight production areas in China., Results: Our study reveals that the core rhizosphere microbiome of B. striata is predominantly comprised of Paraburkholderia, Methylibium, Bradyrhizobium, Chitinophaga, and Mycobacterium. These microbial species are recognized as potentially beneficial for plants health. Comprehensive analysis revealed a significant association between the accumulation of metabolites, such as militarine and polysaccharides in B. striata and the composition of rhizosphere microbes at the genus level. Furthermore, we found that the soil environment indirectly influenced the metabolite profile of B. striata by affecting the composition of rhizosphere microbes. Notably, our research identifies soil organic carbon as a primary driving factor influencing metabolite accumulation in B. striata., Conclusion: Our fndings contribute to an enhanced understanding of the comprehensive regulatory mechanism involving microbe-plant-metabolite interactions. This research provides a theoretical basis for the cultivation of high-quality traditional Chinese medicine B. striata., (© 2024. The Author(s).)

Quantifying the abundances of fungi is key to understanding natural variation in mycorrhizal communities in relation to plant ecophysiology and environmental heterogeneity. High-throughput metabarcoding approaches have transformed our ability to characterize and compare complex mycorrhizal communities. However, it remains unclear how well metabarcoding read counts correlate with actual read abundances in the sample, potentially limiting their use as a proxy for species abundances. Here, we use droplet digital PCR (ddPCR) to evaluate the reliability of ITS2 metabarcoding data for quantitative assessments of mycorrhizal communities in the orchid species Neottia ovata sampled at multiple sites. We performed specific ddPCR assays for eight families of orchid mycorrhizal fungi and compared the results with read counts obtained from metabarcoding. Our results demonstrate a significant correlation between DNA copy numbers measured by ddPCR assays and metabarcoding read counts of major mycorrhizal partners of N. ovata, highlighting the usefulness of metabarcoding for quantifying the abundance of orchid mycorrhizal fungi. Yet, the levels of correlation between the two methods and the numbers of false zero values varied across fungal families, which warrants cautious evaluation of the reliability of low-abundance families. This study underscores the potential of metabarcoding data for more quantitative analyses of mycorrhizal communities and presents practical workflows for metabarcoding and ddPCR to achieve a more comprehensive understanding of orchid mycorrhizal communities., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

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.)

In nature, mycorrhizal association with soil-borne fungi is indispensable for orchid species. Compatible mycorrhizal fungi form endo-mycorrhizal structures in orchid cells, and the fungal structures are digested in orchid cells to be supplied to orchids as nutrition. Because orchid seeds lack the reserves for germination, they keep receiving nutrition through mycorrhizal formation from seed germination until nonphotosynthetic young seedlings develop leaves and become photoautotrophic. Seeds of all orchids germinate with the help of their own fungal partners, and therefore, specific partnership has been acquired in a long evolutionary history between orchids and fungi. Assuming that horizontal transmission of viruses may occur in such a close relationship, we are focusing on viruses that infect orchids and their mycorrhizal fungi. We prepared aseptically germinated orchid plants (i.e., fungi-free plants) together with pure-cultured fungal isolates and conducted transcriptome analyses (RNA-seq) by next-generation sequencing (NGS) approach. To reconstruct virus-related sequences that would have been present in the RNA sample of interest, de novo assembly process is required using short read sequences obtained from RNA-seq. In the previous version of our protocol (see Viral Metagenomics, first edition 2018), virus searches were conducted using contig sets constructed by a single assembler, but this time we devised a method to construct more reliable contigs using multiple assemblers and again reinvestigated that viruses could be detected. Because the virus detection efficiency and number of detected virus species clearly differed depending on the assembly pipeline and the number of the input data, multiple methods should be used to identify viral infection, if possible., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Partial mycoheterotrophic i.e., mixotrophic, plants are the species which partially depend on mycorrhizal fungi for its nutrients. Although some of these plants are known to show plasticity in the degree of fungal dependence induced by the changes in light condition, the genetic background of this plasticity is largely unsolved. Here, we investigated the relationships between environmental conditions and nutrient sources based on 13 C and 15 N enrichment in mixotrophic orchid Cymbidium goeringii . We also shaded them for 2 months and evaluated the effect of light condition on the nutrient sources based on the abundance of 13 C and 15 N and the gene expressions by RNA-seq based de novo assembly. The shading had no effect on isotope enrichment, possibly because of the translocation of carbon and nitrogen from the storage organs. Gene expression analysis showed the upregulation of genes involved in jasmonic acid response in leaves of the shaded plants, which suggests that the jasmonic acid played an important role in regulation of degree of dependence against the mycorrhizal fungi. Our results suggest that mixotrophic plants might be controlling their dependency against the mycorrhizal fungi by a common mechanism with the autotrophic plants.

Similar to other orchid species, Paphiopedilum hirsutissimum (Lindl.ex Hook.) Stein, relies on nutrients provided by mycorrhizal fungus for seed germination and seedling development in the wild owing to a lack of endosperm in its seeds. Therefore, obtaining suitable and specialized fungi to enhance seed germination, seedling formation, and further development is considered a powerful tool for orchid seedling propagation, reintroduction, and species conservation. In this study, we investigated the diversity, abundance, and frequency of endophytic fungal strains in the root organs of P. hirsutissimum . One family and five genera of the fungi were isolated and identified through rDNA-ITS sequencing. The ability of isolated fungi to germinate in vitro from the seeds of this species was evaluated, and the development of P. hirsutissimum protocorm has been described. The findings showed that the treatments inoculated with endophytic fungal DYXY033 may successfully support the advanced developmental stage of seedlings up to stage 5. In addition, scanning electron microscopy (SEM) revealed that the mycelium of this strain began to invade from either end of the seeds up to the embryo, extending rapidly from the inside to the outside. Its lengthening resulted in the bursting of the seed coat to form protocorms, which developed into seedlings. The results showed that DYXY033 has a high degree of mycobiont specificity under in vitro symbiotic seed germination conditions and is a representative mycorrhizal fungus with ecological value for the species. In summary, this strain may particularly be significant for the protection of P. hirsutissimum species that are endangered in China. In the long run, it may also contribute to global efforts in reintroducing orchid species and in realizing in situ restorations of threatened orchid populations.

Species of the subgenus Brachypetalum are the most primitive, most ornamental and most threatened group in the Orchid. This study revealed the ecological characteristics, soil nutrient characteristics and soil fungal community structure of habitats of the subgenus Brachypetalum in Southwest China. Lays a foundation for research on the wild populations and conservation Brachypetalum . The results showed that species of the subgenus Brachypetalum preferred a cool and humid environment, grew in scattered or aggregated form in narrow negative terrain, mainly in humic soil. The soil physical and chemical properties and soil enzyme activity indexes of the habitats were significantly different among different species, and the soil properties of different distribution points of the same species also varied greatly. There were significant differences in the soil fungal community structure among the habitats of different species. Basidiomycetes and ascomycetes were the main fungi in habitats of subgenus Brachypetalum species, and their relative abundance varied among different species. The functional groups of soil fungi were mainly symbiotic fungi and saprophytic fungi. LEfSe analysis found that there were different numbers and species of biomarkers in the habitats of subgenus Brachypetalum species, indicating that the habitat preference characteristics of each species in subgenus Brachypetalum were reflected in the fungal community. It was found that environmental factors had an impact on the changes in soil fungal communities in the habitats of subgenus Brachypetalum species, with climatic factors having the highest explanation rate (20.96%). Soil properties were significantly positively or negatively correlated with a variety of dominant soil fungal groups. Conclusions: The results of this study lay the foundation for the study of the habitat characteristics of wild populations of subgenus Brachypetalum and provides data to support in situ and ex situ conservation in the future.

Orchids (Orchidaceae) are dependent on mycorrhizal fungi for germination and to a varying extent as adult plants. We isolated fungi from wild plants of the critically endangered terrestrial orchid Thelymitra adorata and identified them using a multi-region barcoding approach as two undescribed Tulasnella species, one in each of phylogenetic group II and III (OTU1) of the Tulasnellaceae. Using symbiotic propagation methods, we investigated the role of Tulasnella identity (species and isolate) and age post isolation, on the fungus's ability and efficacy in germinating T. adorata. The group II isolate did not support germination. Seed germination experiments were conducted using either (i) three different isolates of OTU1, (ii) 4- and 12-week-old fungal cultures (post isolation) of a single isolate of OTU1, and (iii) T. subasymmetrica which is widespread and known to associate with other species of Thelymitra. Culture age and fungal species significantly (P < 0.05) affected the time to germination and percentage of seed germination, with greater and faster germination with 4-week-old cultures. Tulasnella subasymmetrica was able to germinate T. adorata to leaf stage, although at slightly lower germination percentages than OTU1. The ability of T. adorata to germinate with T. subasymmetrica may allow for translocation sites to be considered outside of its native range. Our findings on the age of Tulasnella culture affecting germination may have applications for improving the symbiotic germination success of other orchids. Furthermore, storage of Tulasnella may need to take account of the culture age post-isolation, with storage at - 80 °C as soon as possible recommended, post isolation., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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.)

Orchids depend on mycorrhizal fungi to germinate from seed. While multiple orchid mycorrhizal (OrM) taxa are often found associated with adult orchids, the relative contribution of particular OrM taxa to germination and early orchid development is poorly understood. We isolated 28 OrM fungi associated with the Mediterranean orchid Anacamptis papilionacea and tested the efficiency of five isolates on germination and early development, four belonging to the Tulasnella calospora species complex and one belonging to Ceratobasidium. Co-cultures of varying two-way and three-way combinations of OrM isolates were used in vitro to compare the simultaneous effect on seed germination rate with monocultures. We then tested whether, when given initial priority over other fungi, particular OrM taxa were more effective during the early stages of development. Seedlings germinated with different isolates were transferred to a growth chamber, and either the same or different isolate was added 45 days later. After 3 months, the number of roots, length of the longest root, and tuber area were measured. All OrM fungi resulted in seed germination; however, lower germination rates were associated with the Ceratobasidium isolate compared to the tulasnelloid isolates. There was significant decreased germination in co-culture experiments when the Ceratobasidium isolate was added. Despite being associated with reduced germination rates, the addition of the Ceratobasidium isolate to the seedlings germinated with tulasnelloid strains resulted in significant increased tuber size. Although A. papilionacea associates with many OrM taxa, these results show that OrM fungi may play different roles during orchid germination and early development. Even when given initial priority, other fungi may colonize developing orchids and interact to influence early orchid development., (© 2023. The Author(s).)

Network analysis is an effective tool to describe and quantify the ecological interactions between plants and root-associated fungi. Mycoheterotrophic plants, such as orchids, critically rely on mycorrhizal fungi for nutrients to survive, so investigating the structure of those intimate interactions brings new insights into the plant community assembly and coexistence. So far, there is little consensus on the structure of those interactions, described either as nested (generalist interactions), modular (highly specific interactions) or of both topologies. Biotic factors (e.g., mycorrhizal specificity) were shown to influence the network structure, while there is less evidence of abiotic factor effects. By using next-generation sequencing of the orchid mycorrhizal fungal (OMF) community associated to with plant individuals belonging to 17 orchid species, we assessed the structure of four orchid-OMF networks in two European regions under contrasting climatic conditions (Mediterranean vs. Continental). Each network contained four to 12 co-occurring orchid species, including six species shared among the regions. All four networks were both nested and modular, and fungal communities were different between co-occurring orchid species, despite multiple sharing of fungi across some orchids. Co-occurring orchid species growing in Mediterranean climate were associated with more dissimilar fungal communities, consistent with a more modular network structure compared to the Continental ones. OMF diversity was comparable among orchid species since most orchids were associated with multiple rarer fungi and with only a few highly dominant ones in the roots. Our results provide useful highlights into potential factors involved in structuring plant-mycorrhizal fungus interactions in different climatic conditions., (© 2023 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Background: Mycoheterotrophs, acquiring organic carbon and other nutrients from mycorrhizal fungi, have evolved repeatedly with substantial plastid genome (plastome) variations. To date, the fine-scale evolution of mycoheterotrophic plastomes at the intraspecific level is not well-characterized. A few studies have revealed unexpected plastome divergence among species complex members, possibly driven by various biotic/abiotic factors. To illustrate evolutionary mechanisms underlying such divergence, we analyzed plastome features and molecular evolution of 15 plastomes of Neottia listeroides complex from different forest habitats., Results: These 15 samples of Neottia listeroides complex split into three clades according to their habitats approximately 6 million years ago: Pine Clade, including ten samples from pine-broadleaf mixed forests, Fir Clade, including four samples from alpine fir forests and Fir-willow Clade with one sample. Compared with those of Pine Clade members, plastomes of Fir Clade members show smaller size and higher substitution rates. Plastome size, substitution rates, loss and retention of plastid-encoded genes are clade-specific. We propose to recognized six species in N. listeroides complex and slightly modify the path of plastome degradation., Conclusions: Our results provide insight into the evolutionary dynamics and discrepancy of closely related mycoheterotrophic orchid lineages at a high phylogenetic resolution., (© 2023. The Author(s).)

The first comprehensive species distribution models for orchid, its fungal symbionts and pollinator are presented. To evaluate impact of global warming on these organisms three different projections and four various climate change scenarios were analysed. The niche modelling was based on presence-only records of Limodorum abortivum, two species of Russula and three insects pollinating orchid (Anthophora affinis, Bombus terrestris, Rhodanthidium septemdentatum). Two sets of orchid predictions were examined-the first one included only climatic data and the second one was based on climate data and data on future distribution of orchid fungal symbionts. Overall, a poleward range shift is predicted to occur as a result of climate change and apparently global warming will be favorable for L. abortivum and its potential geographical range will expand. However, due to the negative effect of global warming on fungal symbionts of L. abortivum, the actual extension of the suitable niches of the orchid will be much limited. Considering future possibility of cross-pollination, the availability of A. affinis for L. abortivum will decrease and this bee will be available in the worst case scenarios only for 21% of orchid populations. On the other hand, the overlap of orchid and the buff-tailed bumblebee will increase and as much as 86.5% of plant populations will be located within B. terrestris potential range. Also the availability of R. septemdentatum will be higher than currently observed in almost all analysed climate change projections. This study showed the importance of inclusion of ecological factors in species distribution models as the climate data itself are not enough to estimate the future distribution of plant species. Moreover, the availability of pollen vectors which is crucial for long-term survival of orchid populations should be analysed in context of climate changes., (© 2023. The Author(s).)

Orchids commonly rely on mycorrhizal fungi to obtain the necessary resources for seed germination and growth. Whereas most photosynthetic orchids typically associate with so-called rhizoctonia fungi to complete their life cycle, there is increasing evidence that other fungi may be involved as well and that the mycorrhizal communities associated with orchids may be more diverse. Coexisting orchid species also tend to associate with different fungi to reduce competition for similar resources and to increase long-term population viability. However, few studies have related the mycorrhizal communities in the rhizosphere to communities found in the roots of closely related coexisting orchid species. In this study, we used high-throughput sequencing to investigate the diversity and community composition of orchid mycorrhizal fungi in the roots and the rhizosphere of four Cypripedium species growing in forests in Northeast China. The results showed that the investigated Cypripedium species associated with a wide variety of fungi including members of Tulasnellaceae, Psathyrellaceae, and Herpotrichiellaceae, whereas members of Russulaceae, Cortinariaceae, Thelephoraceae, and Herpotrichiellaceae showed high abundance in rhizosphere soils. The diversity of fungi detected in the rhizosphere soil was much higher than that in the roots. The observed variation in fungal communities in Cypripedium roots was not related to forest site or orchid species. On the other hand, variation in mycorrhizal communities of rhizosphere soil was significantly related to sampling site. These results indicate that orchid mycorrhizal communities in the rhizosphere display considerable variation among sites and that orchids use only a subset of the locally available fungi. Future studies focusing on the fine-scale spatial distribution of orchid mycorrhizal fungi and more detailed assessments of local environmental conditions will provide novel insights into the mechanisms explaining variation of fungal communities in both orchid roots and the rhizosphere., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Mycorrhizal symbiosis has been related to the coexistence and community assembly of coexisting orchids in few studies despite their obligate dependence on mycorrhizal partners to establish and survive. In hyper-diverse environments like tropical rain forests, coexistence of epiphytic orchids may be facilitated through mycorrhizal fungal specialization (i.e., sets of unique and dominant mycorrhizal fungi associated with a particular host species). However, information on the role of orchid mycorrhizal fungi (OMF) in niche differentiation and coexistence of epiphytic orchids is still scarce. In this study, we sought to identify the variation in fungal preferences of four co-occurring epiphytic orchids in a tropical rainforest in Costa Rica by addressing the identity and composition of their endophytic fungal and OMF communities across species and life stages. We show that the endophytic fungal communities are formed mainly of previously recognized OMF taxa, and that the four coexisting orchid species have both a set of shared mycorrhizal fungi and a group of fungi unique to an orchid species. We also found that adult plants keep the OMF of the juvenile stage while adding new mycobionts over time. This study provides evidence for the utilization of specific OMF that may be involved in niche segregation, and for an aggregation mechanism where adult orchids keep initial fungal mycobionts of the juvenile stage while adding others., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Seed germination and seedling development in nearly all orchid species rely on a symbiotic relationship with mycorrhizal fungi; however, this is not the case with all mycorrhizal fungi. This study aims to provide an understanding about the important role of mycorrhiza in seed germination and growth of Paphiopedilum barbigerum . Therefore, we isolated and identified endophytic fungi from the roots of wild P. barbigerum . The beneficial mycorrhizal fungi Epulorhiza sp. FQXY019 and Tulasnella calospora FQXY017 were screened by seed symbiotic germination tests and found to promote seed germination. However, only the seeds inoculated with FQXY019 progressed from the seed germination to rooting stage. This shows that mycorrhizal fungi and P. barbigerum have a specific relation at different growth phases. In addition, we selected FQXY019 and inoculated it into MS medium, B5 medium, OMA medium, and PDA medium. The results showed that FQXY019 co-cultured on PDA significantly promoted the increase in seedling fresh weight, leaf length, and root length ( p < .01). Furthermore, it significantly promoted the root number and leaf number of seedlings compared with those co-cultured on MS, B5, and OMA media and control ( p < .05). Thus, this study demonstrated the promoting effect of Epulorhiza sp. FQXY019 on seed germination and seedling development, making it an alternative method for the artificial propagation of P. barbigerum.

An endophytic Pseudomonas fluorescens (BsEB-1) was obtained from the roots of Bletilla striata . We investigated its growth-promoting properties and observed the impact of its inoculation on both the growth and polysaccharide content of Bletilla striata tubers. It was found that BsEB-1 possessed three growth-promoting activities: phosphate-solubilizing, produced indoleacetic acid (IAA) and siderophores, but had no nitrogen-fixing activity. BsEB-1 could rapidly attach to the root hairs of Bletilla striata tissue culture seedlings and endophytically colonize the region of maturation in the roots. It also significantly promoted the rooting and transplant survival rate of the seedlings, as well as the growth and expansion of the tubers, but did not increase their polysaccharide content. Pseudomonas fluorescens BsEB-1 exhibits potential for applications in the artificial planting of Bletilla striata.

Fungi with orchid roots have been increasingly proven to play important roles in orchid growth, spatial distribution, and coexistence of natural communities. Here, we used 454 amplicon pyrosequencing with two different primer combinations to investigate the spatial variations in the community of OMF and endophytic fungi associates within the roots of four co-occurring Habenaria species. The results showed that all investigated Habenaria species were generalists and the different fungi communities may contribute to the spatial separation of the four Habenaria species. Firstly, the fungal OTUs identified in the roots of the four species overlapped but their presence differed amongst species and numerous distinct OMF families were unique to each species. Second, NMDS clustering showed samples clustered together based on associated species and PERMANOVA analyses indicated that fungi communities in the roots differed significantly between the Habenaria species, both for all endophytic fungi communities and for OMF communities. Third, the network structure of epiphytic fungi was highly specialized and modular but demonstrated lowly connected and anti-nested properties. However, it calls for more soil nutrition and soil fungal communities' studies to elucidate the contribution of habitat-specific adaptations in general and mycorrhizal divergence.

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.)

Isolates of three endornavirus species were identified co-infecting an unidentified species of Ceratobasidium , itself identified as a symbiont from within the roots of a wild plant of the terrestrial orchid Pterostylis vittata in Western Australia. Isogenic lines of the fungal isolate lacking all three mycoviruses were derived from the virus-infected isolate. To observe how presence of endornaviruses influenced gene expression in the fungal host, we sequenced fungus-derived small RNA species from the virus-infected and virus-free isogenic lines and compared them. The presence of mycoviruses influenced expression of small RNAs. Of the 3272 fungus-derived small RNA species identified, the expression of 9.1% (300 of 3272) of them were up-regulated, and 0.6% (18 of 3272) were down-regulated in the presence of the viruses. Fourteen novel micro-RNA-like RNAs (Cer-milRNAs) were predicted. Gene target prediction of the differentially expressed Cer-milRNAs was quite ambiguous; however, fungal genes involved in transcriptional regulation, catalysis, molecular binding, and metabolic activities such as gene expression, DNA metabolic processes and regulation activities were differentially expressed in the presence of the mycoviruses.

While many Australian terrestrial orchids have highly specialized mycorrhizal associations, we tested the hypothesis that the geographically widespread orchid genus Cryptostylis associates with a diversity of fungal species. Using fungal isolation and molecular approaches, we investigated the mycorrhizal associations of five Australian Cryptostylis species (27 sites sampled) and included limited sampling from three Asiatic Cryptostylis species (two sites). Like related orchid genera, Tulasnellaceae formed the main fungal associations of the Cryptostylis species we sampled, although some ectomycorrhizal, ericoid and saprotrophic fungi were detected infrequently. Each species of Australian Cryptostylis associated with three to seven Tulasnella Operational Taxonomic Units (OTUs), except for C. hunteriana where only one Tulasnella OTU was detected. In total, eleven Tulasnella OTUs associated with Australian Cryptostylis. The Asiatic Cryptostylis associated with four different Tulasnella OTUs belonging to the same lineage as the Australian species. While five Tulasnella OTUs (T. australiensis, T. prima, T. warcupii, T. densa, and T. punctata) were used by multiple species of Australian Cryptostylis, the most commonly used OTU differed between orchid species. The association with different Tulasnella fungi by Cryptostylis species co-occurring at the same site suggests that in any given environmental condition, Cryptostylis species may intrinsically favour different fungal OTUs., Competing Interests: Declaration of competing interest The authors whose names are listed immediately below certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript., (Copyright © 2022 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

In nature, orchid seed germination and seedling development depend on compatible mycorrhizal fungi. Mycorrhizal generalist and specificity affect the orchid distribution and rarity. Here, we investigated the specificity toward fungi in the rare D. huoshanense by mycorrhizal fungal isolation and symbiotic germination in vitro. Twenty mycorrhizal fungal strains were isolated from the roots of adult Dendrobium spp. (six and 12 strains from rare D. huoshanense and widespread D. officinale, respectively, and two strains from D. nobile and D. moniliforme, respectively) and 13 strains belong to Tulasnellaceae and seven strains belong to Serendipitaceae. Germination trials in vitro revealed that all 20 tested fungal strains can stimulate seed germination of D. huoshanense, but only nine strains (~ 50%) can support it up to the seedling stage. This finding indicates that generalistic fungi are important for early germination, but only a few can maintain a symbiosis with host in seedling stage. Thus, a shift of the microbial community from seedling to mature stage probably narrows the D. huoshanense distribution range. In addition, to further understand the relationship between the fungal capability to promote seed germination and fungal enzyme activity, we screened the laccase and pectase activity. The results showed that the two enzymes activities of fungi cannot be directly correlated with their germination-promoting activities. Understanding the host specificity degree toward fungi can help to better interpret the limited geographic distribution of D. huoshanense and provides opportunities for in situ and ex situ conservation and reintroduction programs., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching mycoheterotrophic plants (i.e., plants that obtain carbon from fungi), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their developmental strategies and their relationships with hyphae, the source of nutrients. Another important role of symbiotic fungi is related to the germination of orchid seeds; all Orchidaceae species are mycoheterotrophic during germination and seedling stage (initial mycoheterotrophy), even the ones that photosynthesize in adult stages. Due to the lack of nutritional reserves in orchid seeds, fungal symbionts are essential to provide substrates and enable germination. Analyzing germination stages by structural perspectives can also answer important questions regarding the fungi interaction with the seeds. Different imaging techniques can be applied to unveil fungi endophytes in plant tissues, as are proposed in this article. Freehand and thin sections of plant organs can be stained and then observed using light microscopy. A fluorochrome conjugated to wheat germ agglutinin can be applied to the fungi and co-incubated with Calcofluor White to highlight plant cell walls in confocal microscopy. In addition, the methodologies of scanning and transmission electron microscopy are detailed for mycoheterotrophic orchids, and the possibilities of applying such protocols in related plants is explored. Symbiotic germination of orchid seeds (i.e., in the presence of mycorrhizal fungi) is described in the protocol in detail, along with possibilities of preparing the structures obtained from different stages of germination for analyses with light, confocal, and electron microscopy.

Background and Aims: Many terrestrial orchids have an obligate dependence on their mycorrhizal associations for nutrient acquisition, particularly during germination and early seedling growth. Though important in plant growth and development, phosphorus (P) nutrition studies in mixotrophic orchids have been limited to only a few orchid species and their fungal symbionts. For the first time, we demonstrate the role of a range of fungi in the acquisition and transport of inorganic P to four phylogenetically distinct green-leaved terrestrial orchid species (Diuris magnifica, Disa bracteata, Pterostylis sanguinea and Microtis media subsp. media) that naturally grow in P-impoverished soils., Methods: Mycorrhizal P uptake and transfer to orchids was determined and visualized using agar microcosms with a diffusion barrier between P source (33P orthophosphate) and orchid seedlings, allowing extramatrical hyphae to reach the source., Key Results: Extramatrical hyphae of the studied orchid species were effective in capturing and transporting inorganic P into the plant. Following 7 d of exposure, between 0.5 % (D. bracteata) and 47 % (D. magnifica) of the P supplied was transported to the plants (at rates between 0.001 and 0.097 fmol h-1). This experimental approach was capable of distinguishing species based on their P-foraging efficiency, and highlighted the role that fungi play in P nutrition during early seedling development., Conclusions: Our study shows that orchids occurring naturally on P-impoverished soils can obtain significant amounts of inorganic P from their mycorrhizal partners, and significantly more uptake of P supplied than previously shown in other green-leaved orchids. These results provide support for differences in mycorrhiza-mediated P acquisition between orchid species and fungal symbionts in green-leaved orchids at the seedling stage. The plant-fungus combinations of this study also provide evidence for plant-mediated niche differentiation occurring, with ecological implications in P-limited systems., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Orchid seeds depend on colonization by orchid mycorrhizal (OM) fungi for their germination; therefore, the orchids and OM fungi have long maintained a close relationship (e.g., formation of the hyphal mass structure, peloton) during their evolution. In the present study, we isolated new partitiviruses from OM fungi; partitivirus were separately found in different subcultures from the same fungi. Partitiviruses have been believed to lack an RNA silencing suppressor (RSS), which is generally associated with viral pathogenicity, because most partitiviruses isolated so far are latent in both plants and fungi. However, we found that the coat protein (CP) of our partitiviruses indeed had RSS activity, which differed among the virus isolates from OM fungi; one CP showed RSS activity in both plants and fungi, while another CP showed no activity. The family Partitiviridae include viruses isolated from plants and fungi, and it has been suggested that these viruses may occasionally be transmitted between plant and fungal hosts. Given that there are several reports showing that viruses can adapt to nonhost using strong RSS, we here discussed the idea that partitiviruses may be better able to migrate between the orchid and fungus probably through the pelotons formed in the orchid cells, if host RNA silencing is suppressed by partitivirus RSS., (© 2022. The Author(s).)

Background: Symbiotic associations of endophytic fungi have been proved by possessing an ability to produce hormones and metabolites for their host plant. Members of the Orchidaceae are obligate mycorrhizal species but a non-mycorrhizal association needs more investigation for their ability to promote plant growth and produce plant growth hormones. In the present study, endophytic fungi were isolated from the roots of Dendrobium longicornu Lindl., to investigate the root colonizing activity and role in plant growth and development., Results: Among 23 fungal isolates were identified both by morphological and molecular technique as Penicillium sp., Fusarium sp., Coniochaeta sp., Alternaria sp., and Cladosporium sp. The dominate species were Coniochaeta sp. and Cladosporium sp. The dominant species as per the isolation was Coniochaeta sp. These fungal strains were screened for growth-promoting activity of Cymbidium aloifolium (plantlet) consider as cross genus interaction and Dendrobium longicornu (protocorms) as a host plant in in-vitro condition. Importantly, Cladosporium sp., and Coniochaeta sp. showed successful colonization and peloton formation with roots of C. aloifolium. Moreover, it also enhanced acclimatization of plantlets. Fungal elicitors from nine fungal isolates enhanced the growth of the in vitro grown protocorms of D. longicornu. Key bioactive compounds detected in the fungal colonized plant extract were 2H-pyran-2-one, Cyclopropanecarboxylic acid, Oleic Acid and d-Mannitol, which may have a potential role in plant-microbe interaction. All fungal endophytes were able to synthesize the indole acetic acid (IAA) in presence of tryptophan. Moreover, fungal extract DLCCR7 treated with DL-tryptophan yielded a greater IAA concentration of 43 μg per ml than the other extracts. The iaaM gene involved in IAA synthesis pathway was amplified using iaaM gene primers successfully from Alternaria sp., Cladosporium sp., and Coniochaeta sp., Conclusions: Hence, this study confirms the production of IAA by endophytes and demonstrated their host as well as cross-genus plant growth-promoting potential by producing metabolites required for the growth of the plant., (© 2022. The Author(s).)

Background: To illustrate the molecular mechanism of mycoheterotrophic interactions between orchids and fungi, we assembled chromosome-level reference genome of Gastrodia menghaiensis (Orchidaceae) and analyzed the genomes of two species of Gastrodia., Results: Our analyses indicated that the genomes of Gastrodia are globally diminished in comparison to autotrophic orchids, even compared to Cuscuta (a plant parasite). Genes involved in arbuscular mycorrhizae colonization were found in genomes of Gastrodia, and many of the genes involved biological interaction between Gatrodia and symbiotic microbionts are more numerous than in photosynthetic orchids. The highly expressed genes for fatty acid and ammonium root transporters suggest that fungi receive material from orchids, although most raw materials flow from the fungi. Many nuclear genes (e.g. biosynthesis of aromatic amino acid L-tryptophan) supporting plastid functions are expanded compared to photosynthetic orchids, an indication of the importance of plastids even in totally mycoheterotrophic species., Conclusion: Gastrodia menghaiensis has the smallest proteome thus far among angiosperms. Many of the genes involved biological interaction between Gatrodia and symbiotic microbionts are more numerous than in photosynthetic orchids., (© 2022. The Author(s).)

To improve our understanding of the origin and evolution of mycoheterotrophic plants, we here present the chromosome-scale genome assemblies of two sibling orchid species: partially mycoheterotrophic Platanthera zijinensis and holomycoheterotrophic Platanthera guangdongensis. Comparative analysis shows that mycoheterotrophy is associated with increased substitution rates and gene loss, and the deletion of most photoreceptor genes and auxin transporter genes might be linked to the unique phenotypes of fully mycoheterotrophic orchids. Conversely, trehalase genes that catalyse the conversion of trehalose into glucose have expanded in most sequenced orchids, in line with the fact that the germination of orchid non-endosperm seeds needs carbohydrates from fungi during the protocorm stage. We further show that the mature plant of P. guangdongensis, different from photosynthetic orchids, keeps expressing trehalase genes to hijack trehalose from fungi. Therefore, we propose that mycoheterotrophy in mature orchids is a continuation of the protocorm stage by sustaining the expression of trehalase genes. Our results shed light on the molecular mechanism underlying initial, partial and full mycoheterotrophy., (© 2022. The Author(s).)

The endophytic microbiome in orchid plants is rich and diverse; however, few studies have analyzed the endophytic microbiome of Cymbidium plants in different tissues and soils. This study implemented the Illumina Miseq technology to investigate the diversity of endophytic fungi in different tissues of wild Cymbidium goeringii. The results demonstrated that different tissue samples harbor a rich fungal endophytic community, and those fungi could be classified into 4 phyla, at least 145 families, and 185 genera. The endophytic fungal community diversity differed among the orchid tissues and soils, and some fungal taxa were clearly concentrated in certain orchid tissues, with more operational taxonomic units (OTUs) being detected. Investigation of mycorrhizal associations showed that 43 (about 3.8%) of the total 1137 OTUs could be assigned as Orchidaceae mycorrhizal fungi (OMF), while about 96.2% the OTUs were non-mycorrhizal fungi. Among the OMFs, OTUs of the ectomycorrhizal fungi Russulaceae and Thelephoraceae families were the most abundant, with different richness in the soil, followed by Tulasnellaceae and Ceratobasidiaceae, which were dominant in the root communities of C. goeringii. In the seeds, the absolutely dominant family was Nectriaceae, and the common OMFs Ceratobasidiaceae (five OTUs) and Tulasnellaceae (one OTU) were also detected in the seeds. Two Tulasnella spp. isolates from the roots of wild C. goeringii could effectively promote seed germination and rhizome formation of wild C. goeringii, and these strains might be particularly important in the practice of conservation for many endangered C. goeringii in China., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Some plants abandoned photosynthesis and developed full dependency on fungi for nutrition. Most of the so-called mycoheterotrophic plants exhibit high specificity towards their fungal partners. We tested whether natural rarity of mycoheterotrophic plants and usual small and fluctuating population size make their populations more prone to genetic differentiation caused by restricted gene flow and/or genetic drift. We also tested whether these genetic characteristics might in turn shape divergent fungal preferences. We studied the mycoheterotrophic orchid Epipogium aphyllum, addressing the joint issues of genetic structure of its populations over Europe and possible consequences for mycorrhizal specificity within the associated fungal taxa. Out of 27 sampled E. aphyllum populations, nine were included for genetic diversity assessment using nine nuclear microsatellites and plastid DNA. Population genetic structure was inferred based on the total number of populations. Individuals from 17 locations were included into analysis of genetic identity of mycorrhizal fungi of E. aphyllum based on barcoding by nuclear ribosomal DNA. Epipogium aphyllum populations revealed high genetic diversity (uHe = 0.562) and low genetic differentiation over vast distances (F ST  = 0.106 for nuclear microsatellites and F ST  = 0.156 for plastid DNA). Bayesian clustering analyses identified only two genetic clusters, with a high degree of admixture. Epipogium aphyllum genets arise from panmixia and display locally variable, but relatively high production of ramets, as shown by a low value of rarefied genotypic richness (R r  = 0.265). Epipogium aphyllum genotype control over partner selection was negligible as (1) we found ramets from a single genetic individual associated with up to 68% of the known Inocybe spp. associating with the plant species, (2) and partner identity did not show any geographic structure. The absence of mosaicism in the mycorrhizal specificity over Europe may be linked to preferential allogamous habit of E. aphyllum and significant gene flow, which tend to promote host generalism., (© 2022. The Author(s).)

Tulasnella (Tulasnellaceae) is a genus of fungus that can form mycorrhizal associations with orchids (Orchidaceae). Here we used molecular phylogenetic analyses and morphological characteristics of pure cultures across four different media to support the description of five new Tulasnella species associated with commonly occurring and endangered Australian orchids. Tulasnella nerrigaensis associates with Calochilus; T. subasymmetrica and T. kiataensis with Thelymitra; and T. korungensis and T. multinucleata with Pyrorchis and Rimacola respectively. The newly described species were primarily delimited by analyses of five loci: nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS), C14436 (adenosine triphosphate [ATP] synthase), C4102 (glutamate synthase), C3304 (ATP helicase), and mt large subunit 16S rDNA (mtLSU). Tulasnella subasymmetrica is introduced for some isolates previously identified as T. asymmetrica , and this latter species is characterized from multilocus sequencing of a new isolate that matches ITS sequences from the ex-type culture. Morphological differences between the new species are slight. Tulasnella multinucleata has 6-12 nuclei per hyphal compartment which is the first instance of multinucleate rather than binucleate or trinucleate hyphal compartments in Tulasnella . The formal description of these species of Tulasnella will aid in future evolutionary and ecological studies of orchid-fungal interactions.

Background: As in most land plants, the roots of orchids (Orchidaceae) associate with soil fungi. Recent studies have highlighted the diversity of the fungal partners involved, mostly within Basidiomycotas. The association with a polyphyletic group of fungi collectively called rhizoctonias (Ceratobasidiaceae, Tulasnellaceae and Serendipitaceae) is the most frequent. Yet, several orchid species target other fungal taxa that differ from rhizoctonias by their phylogenetic position and/or ecological traits related to their nutrition out of the orchid roots (e.g. soil saprobic or ectomycorrhizal fungi). We offer an evolutionary framework for these symbiotic associations., Scope: Our view is based on the 'Waiting Room Hypothesis', an evolutionary scenario stating that mycorrhizal fungi of land flora were recruited from ancestors that initially colonized roots as endophytes. Endophytes biotrophically colonize tissues in a diffuse way, contrasting with mycorrhizae by the absence of morphological differentiation and of contribution to the plant's nutrition. The association with rhizoctonias is probably the ancestral symbiosis that persists in most extant orchids, while during orchid evolution numerous secondary transitions occurred to other fungal taxa. We suggest that both the rhizoctonia partners and the secondarily acquired ones are from fungal taxa that have broad endophytic ability, as exemplified in non-orchid roots. We review evidence that endophytism in non-orchid plants is the current ecology of many rhizoctonias, which suggests that their ancestors may have been endophytic in orchid ancestors. This also applies to the non-rhizoctonia fungi that were secondarily recruited by several orchid lineages as mycorrhizal partners. Indeed, from our review of the published literature, they are often detected, probably as endophytes, in extant rhizoctonia-associated orchids., Conclusion: The orchid family offers one of the best documented examples of the 'Waiting Room Hypothesis': their mycorrhizal symbioses support the idea that extant mycorrhizal fungi have been recruited among endophytic fungi that colonized orchid ancestors., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Cremastra appendiculata is a rare terrestrial orchid with a high market value as an ornamental and medicinal plant. However, the species depends entirely on fungi for seed germination under natural conditions. In a previous study, we have successfully isolated and identified the mycorrhizal fungus Coprinellus disseminatus which was able to induce the germination of C. appendiculata seeds. We then speculated that C. disseminatus may do so by breaking the testa imposed dormancy of the seeds. In this study, biochemical and transcriptomic analyses were used to characterize the germination of C. appendiculata seeds, collected at different stages of germination, as affected by C. disseminatus., Results: The lignocellulose in the seeds coat of C. appendiculata was degraded by the mycorrhizal fungus resulting in facilitated absorption of water. The rate of decline in lignin content was 67 and 73% at 6 and 12 days after sowing, respectively. The water content increased from 13 to 90% during symbiosis. A total of 15,382 genes showing significantly different levels of expression (log 2 FPKM≥2.0, Qvalue≤0.05) were successfully identified among all libraries, where the highest number of DEGs was shared between 6 days versus 0 day after symbiotic germination. Gene annotation results suggested that 15 key genes related water-status, such as DHN gene family and Xero 1 were down-regulated. The genes zeaxanthin epoxidase ZEP, 9-cis-epoxycarotenoid dioxygenase NCED3 and β-carotene hydroxylase involved in the biosynthesis of abscisic acid (ABA) were significantly down-regulated in 6 days as compared to 0 day after symbiotic germination., Conclusions: This work demonstrates that mycorrhizal fungus C. disseminatus can stimulate C. appendiculata seeds germination through a mechanism of breaking the testa imposed dormancy and inducing water absorption of the embryo., (© 2022. The Author(s).)

Many of the Orchidaceae species are threatened due to environmental changes and over exploitation for full fill global demands. The main objective of this article was critically analyzed the recent global distribution of Orchidaceae diversity, its disease patterns, microbial disease identification, detection, along with prevention and challenges. Critical analysis findings revealed that Orchidaceae growth and developments were affected indirectly or directly as a result of complex microbial ecological interactions. Studies have identified many species associated with orchids, some are pathogenic and cause symptoms such as soft rot, brown rot, brown spot, black rot, wilt, foliar, root rot, anthracnose, leaf spot. The review was provided the comprehensive data to evaluate the identification and detection of microbial disease, which is the most important challenge for sustainable cultivation of Orchidaceae diversity. Furthermore, this article is the foremost of disease triggering microbes, orchid relations, and assimilates various consequences that both promoted the considerate and facts of such disease multipart, and will permit the development of best operative disease management practices.

Background: Pogoniopsis schenckii Cogn. is a mycoheterotrophic orchid that can be used as a model to understand the influence of mycoheterotrophy at different stages of the reproductive cycle. We aimed to verify the presence of endophytic and epiphytic fungi at each stage of the reproductive process and investigated how the breeding system may relate to genetic structure and diversity of populations. In this study we performed anatomical and ultrastructural analyses of the reproductive organs, field tests to confirm the breeding system, and molecular analysis to assess genetic diversity and structure of populations., Results: During the development of the pollen grain, embryo sac and embryogenesis, no fungal infestation was observed. The presence of endophytic fungal hyphae was observed just within floral stems and indehiscent fruit. Beyond assuring the presence of fungus that promote seed germination, specific fungi hyphae in the fruit may affect other process, such as fruit ripening. As other mycoheterotrophic orchids, P. schenckii is autogamous, which may explain the low genetic diversity and high genetic structure in populations., Conclusions: We discuss an interesting interaction: fungal hyphae in the indehiscent fruit. These fungal hyphae seem to play different roles inside fruit tissues, such as acting in the fruit maturation process and increasing the proximity between fungi and plant seeds even before dispersion occurs. As other mycoheterotrophic orchids, P. schenckii is autogamous, which may explain the low genetic diversity and high genetic structure in populations. Altogether, our findings provide important novel information about the mechanisms shaping ecology and evolution of fragmented populations of mycoheterotrophic plant., (© 2021. The Author(s).)

Aims: The aim of this study was to assess the effects of beneficial micro-organisms on the growth, nutrient accumulation and root-associated fungal species composition of pot orchids grown in the greenhouse., Methods and Results: A greenhouse pot experiment was conducted to investigate the beneficial effects of a mycorrhizal fungus, Epulorhiza repens isolate ML01, an endophytic fungus, Umbelopsis nana isolate ZH3A-3 and a mixed commercial inoculum Rem, alone or in combination. Nested PCR assays showed that both isolates ML01 and ZH3A-3 can successfully establish in inoculated soil. All the inoculants significantly increased the plant total dry weight of Cymbidium hybridum 'Golden Boy', whereas only co-inoculation with the endophytic fungus ZH3A-3 and the Rem enhanced the fresh weight and height of host plants. The mycorrhizal fungus positively affected P, K, Ca, Mg content in shoots and Zn content in roots, while the endophytic fungus improved N, P, Ca accumulation in shoots and roots. Co-inoculation with the Rem and ML01 improved root to shoot translocation of Fe and Zn. In addition, inoculation with ZH3A-3, ML01+Rem and ZH3A-3+Rem decreased the relative frequency of Fusarium sp. on orchid roots. Trichoderma sp. were isolated from the roots treated with ML01, ML01+Rem and ZH3A-3+Rem., Conclusions: Both mycorrhizal and endophytic fungi had the potential to create favourable microflora in the orchid roots and stimulate the growth of transplanted plantlets under greenhouse condition., Significance and Impact of the Study: The newly isolated endophytic strain ZH3A-3 showed significant application value in orchid production., (© 2020 The Society for Applied Microbiology.)

Epiphytic orchids exhibit varying degrees of phorophyte tree specificity. We performed a pilot study to investigate why epiphytic orchids prefer or avoid certain trees. We selected two orchid species, Panisea uniflora and Bulbophyllum odoratissimum co-occurring in a forest habitat in southern China, where they showed a specific association with Quercus yiwuensis and Pistacia weinmannifolia trees, respectively. We analysed a number of environmental factors potentially influencing the relationship between orchids and trees. Difference in bark features, such as water holding capacity and pH were recorded between Q. yiwuensis and P. weinmannifolia, which could influence both orchid seed germination and fungal diversity on the two phorophytes. Morphological and molecular culture-based methods, combined with metabarcoding analyses, were used to assess fungal communities associated with studied orchids and trees. A total of 162 fungal species in 74 genera were isolated from bark samples. Only two genera, Acremonium and Verticillium, were shared by the two phorophyte species. Metabarcoding analysis confirmed the presence of significantly different fungal communities on the investigated tree and orchid species, with considerable similarity between each orchid species and its host tree, suggesting that the orchid-host tree association is influenced by the fungal communities of the host tree bark.

Background: A plant growth-promoting endophytic bacterium PVL1 isolated from the leaf of Vanda cristata has the ability to colonize with roots of plants and protect the plant. PVL1 was isolated using laboratory synthetic media. 16S rRNA gene sequencing method has been employed for identification before and after root colonization ability., Results: Original isolated and remunerated strain from colonized roots were identified as Bacillus spp. as per EzBiocloud database. The presence of bacteria in the root section of the plantlet was confirmed through Epifluorescence microscopy of colonized roots. The in-vitro plantlet colonized by PVL1 as well as DLMB attained higher growth than the control. PVL1 capable of producing plant beneficial phytohormone under in vitro cultivation. HPLC and GC-MS analysis suggest that colonized plants contain Indole Acetic Acid (IAA). The methanol extract of Bacillus spp., contains 0.015 μg in 1 μl concentration of IAA. PVL1 has the ability to produce antimicrobial compounds such as ethyl iso-allocholate, which exhibits immune restoring property. One-way ANOVA shows that results were statistically significant at P ≤ 0.05 level., Conclusions: Hence, it has been concluded that Bacillus spp. PVL1 can promote plant growth through secretion of IAA during root colonization and ethyl iso-allocholate to protect plants from foreign infections. Thus, this study supports to support Koch's postulates of bacteria establishment.