Integrating Mycorrhizal Fungi into Orchid Reintroduction: Case Studies, Challenges, and Future Directions

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

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.

Specificity and localised distribution of mycorrhizal fungi in the soil may contribute to co-existence of orchid species

In nature, germination of orchid seeds and early plant development rely on a symbiotic association with orchid mycorrhizal (ORM) fungi. These fungi provide the host with the necessary nutrients and facilitate the transition from embryos to protocorms. Despite recent advances in omics technologies, our understanding of this symbiosis remains limited, particularly during the initial stages of the interaction. To address this gap, we employed transcriptomics and metabolomics to investigate the early responses occurring in the mycorrhizal fungus Tulasnella sp. isolate SV6 when co-cultivated with orchid seeds of Serapias vomeracea. The integration of data from gene expression and metabolite profiling revealed the activation of some fungal signalling pathways before the establishment of the symbiosis. Prior to seed contact, an indole-related metabolite was produced by the fungus, and significant changes in the fungal lipid profile occurred throughout the symbiotic process. Additionally, the expression of plant cell wall-degrading enzymes (PCWDEs) was observed during the pre-symbiotic stage, as the fungus approached the seeds, along with changes in amino acid metabolism. Thus, the dual-omics approach employed in this study yielded novel insights into the symbiotic relationship between orchids and ORM fungi and suggest that the ORM fungus responds to the presence of the orchid seeds prior to contact.

Orchids require mycorrhizal fungi for germination of seed and growth of seedlings. The colonisation of bauxite-mine rehabilitation areas by orchids is therefore dependent on the availability of both seed and mycorrhizal fungi. Orchid mycorrhizal fungi baiting trials were carried out in rehabilitation areas that were 1, 10 and 26 years old (established in 2001, 1992 and 1976) and adjacent unmined jarrah forest areas at Jarrahdale, Western Australia. Fungal baits consisted of buried six-chambered nylon-mesh packets containing seed of six jarrah forest orchid taxa, Caladenia flava subsp. flava R.Br., Disa bracteata Sw., Microtis media subsp. media R.Br., Pterostylis recurva Benth., Pyrorchis nigricans (R.Br.) D.L.Jones & M.A.Clem. and Thelymitra crinita Lindl. Detection of orchid mycorrhizal fungi was infrequent, especially at the youngest rehabilitation sites where only mycorrhizal fungi associated with P. recurva were detected. Mycorrhizal fungi of the other orchid taxa were widespread but sparsely distributed in older rehabilitation and forest areas. Detection of mycorrhizal fungi varied between taxa and baiting sites for the two survey years (2002 and 2004). Caladenia flava subsp. flava and T. crinita mycorrhizal fungi were the most frequently detected. The presence of C. flava mycorrhizal fungi was correlated with leafy litter cover and maximum depth, and soil moisture at the vegetation type scale (50 × 5 m belt transects), as well as tree and litter cover at the microhabitat scale (1-m2 quadrats). The presence of T. crinita mycorrhizal fungi was positively correlated with soil moisture in rehabilitation areas and low shrub cover in forest. The frequency of detection of orchid mycorrhizal fungi both at rehabilitated sites (15–25% of baits) and in unmined forest (15–50% of baits) tended to increase with rehabilitation age as vegetation recovered. The failure of some orchid taxa to reinvade rehabilitation areas is unlikely to be entirely due to absence of the appropriate mycorrhizal fungi. However, since the infrequent detection of fungi suggests that they occur in isolated patches of soil, the majority of dispersed orchid seeds are likely to perish, especially in recently disturbed habitats.

Orchid mycorrhizal (OM) fungi form mutualistic relationships with orchids, facilitating the uptake of minerals from the soil in exchange for sugars and lipids provided by the plant. In this study, we explored the role of the DoSWEET14 sugar transporter in Dendrobium officinale during OM symbiosis. Transcriptome sequencing revealed significant changes in gene expression in mycorrhizal roots, particularly the upregulation of DoSWEET14. Subcellular localization studies confirmed that DoSWEET14 is localized to the plasma membrane, suggesting its involvement in nutrient exchange between the orchid and OM fungi. Complementation assays using a yeast strain deficient in hexose transport demonstrated that DoSWEET14 has broad substrate specificity, efficiently transporting multiple monosaccharides. Additionally, overexpression of DoSWEET14 in Arabidopsis increased sugar content without affecting biomass, supporting its role in carbohydrate transport and storage during symbiosis. These findings suggest that DoSWEET14 plays a key role in regulating carbon allocation and stabilizing the mutualistic relationship between Dendrobium officinale and OM fungi.

Variation in nutrient-acquisition patterns by mycorrhizal fungi of rare and common orchids explains diversification in a global biodiversity hotspot

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.

Changes in orchid populations and endophytic fungi with rainfall and prescribed burning in Pterostylis revoluta in Victoria, Australia.

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

Little is known about the soil factors influencing root-associated fungal communities in Orchidaceae. Limited evidence suggests that soil nutrients may modulate the association with orchid mycorrhizal fungi (OMF), but their influence on non-mycorrhizal fungi remains unexplored. To study how nutrient availability affects mycorrhizal and non-mycorrhizal fungi associated with the orchid Bipinnula fimbriata, we conducted a metagenomic investigation within a large population with variable soil conditions. Additionally, we tested the effect of phosphorus (P) addition on fungal communities and mycorrhizal colonization. Soil P negatively correlated with the abundance of OMF, but not with the abundance of non-mycorrhizal fungi. After fertilization, increments in soil P negatively affected mycorrhizal colonization; however, they had no effect on OMF richness or composition. The abundance and richness of pathotrophs were negatively related to mycorrhizal colonization and then, after fertilization, the decrease in mycorrhizal colonization correlated with an increase in pathogen richness. Our results suggest that OMF are affected by soil conditions differently from non-mycorrhizal fungi. Bipinnula fimbriata responds to fertilization by altering mycorrhizal colonization rather than by switching OMF partners in the short term, and the influence of nutrients on OMF is coupled with indirect effects on the whole fungal community and potentially on plant's health.

As orchids rely on their mycorrhizal fungi for nutrient supply, their spatial range is dependent on the distribution of orchid mycorrhizal (OM) fungi. We addressed possible correlations between mycorrhizal specificity and the geographic distribution of orchids and OM fungi in three populations of the rare sister species Orchis patens and O. canariensis. Metabarcoding of the fungal ITS2 region indicated that, although adult plants of either species were colonized by several ceratobasidioid, tulasnelloid, sebacinoid and serendipitoid fungi, the mycobiont spectra were dominated by Tulasnella helicospora (which occurred in 100% of examined plants with high read numbers), which is a globally distributed fungus. In vitro assays with a T. helicospora isolate obtained from O. patens indicated the effectiveness of this OM fungus at germinating seeds of its native host. At a local scale, higher read numbers for T. helicospora were found in soil samples collected underneath O. patens roots than at locations unoccupied by the orchid. Although these findings suggest that the geographical pattern of the main fungal symbiont does not limit the distribution of O. patens and O. canariensis at this scale, the actual causal link between orchid and OM fungal occurrence/abundance still needs to be better understood.

Dendrobium officinale is a medicinal herbal plant with important health care value and high demand. Due to its slow growth and scarcity in nature, its yield depends on intensified cultivation while biotic and abiotic stresses were important factors that causes production loss. Orchidaceae can form association with rhizoctonias collectively, and studies have found that some orchids showed a high level of strain-species specificity to orchid mycorrhizal fungi (OMF), yet the specificity of OMF on D. officinale needs to explored. In this study, the effects on D. officinale of four OMF isolated from Cymbidium were tested. The obviously higher mass yield of the treated plants in medium and pots indicated the growth promotion effect of the fungi. Furthermore, an abiotic stress test indicated stronger drought tolerance among the treated plants. For the biotic stress test, two root rot pathogens, Fusarium solani and Fusarium graminearum , were isolated and identified from root rot of D. officinale. In an in vitro inhibition test, the four OMF could resist the growth of these pathogens. In vivo studies showed that these four OMF could improve the survival rate and fresh weight and decrease the root rot rate of pathogen-inoculated seedlings. The four OMF namely; Hyphomycete sp., Umbelopsis sp., Ceratorhiza sp. and Ceratorhiza sp. are compatible strains for improving the growth rate of D. officinale by increasing its environmental stress tolerance, providing an effective way to supply resources through artificial reproduction.

Dendrobium officinale is a medicinal herbal plant with important health care value and high demand. Due to its slow growth and scarcity in nature, its yield depends on intensified cultivation while biotic and abiotic stresses were important factors that causes production loss. Orchidaceae can form association with rhizoctonias collectively, and studies have found that some orchids showed a high level of strain-species specificity to orchid mycorrhizal fungi (OMF), yet the specificity of OMF on D. officinale needs to explored. In this study, the effects on D. officinale of four OMF isolated from Cymbidium were tested. The obviously higher mass yield of the treated plants in medium and pots indicated the growth promotion effect of the fungi. Furthermore, an abiotic stress test indicated stronger drought tolerance among the treated plants. For the biotic stress test, two root rot pathogens, Fusarium solani and Fusarium graminearum , were isolated and identified from root rot of D. officinale. In an in vitro inhibition test, the four OMF could resist the growth of these pathogens. In vivo studies showed that these four OMF could improve the survival rate and fresh weight and decrease the root rot rate of pathogen-inoculated seedlings. The four OMF namely; Hyphomycete sp., Umbelopsis sp., Ceratorhiza sp. and Ceratorhiza sp. are compatible strains for improving the growth rate of D. officinale by increasing its environmental stress tolerance, providing an effective way to supply resources through artificial reproduction.

Dendrobium officinale is a medicinal herbal plant with important health care value and high demand. Due to its slow growth and scarcity in nature, its yield depends on intensified cultivation while biotic and abiotic stresses were important factors that causes production loss. Orchidaceae can form association with rhizoctonias collectively, and studies have found that some orchids showed a high level of strain-species specificity to orchid mycorrhizal fungi (OMF), yet the specificity of OMF on D. officinale needs to explored. In this study, the effects on D. officinale of four OMF isolated from Cymbidium were tested. The obviously higher mass yield of the treated plants in medium and pots indicated the growth promotion effect of the fungi. Furthermore, an abiotic stress test indicated stronger drought tolerance among the treated plants. For the biotic stress test, two root rot pathogens, Fusarium solani and Fusarium graminearum , were isolated and identified from root rot of D. officinale. In an in vitro inhibition test, the four OMF could resist the growth of these pathogens. In vivo studies showed that these four OMF could improve the survival rate and fresh weight and decrease the root rot rate of pathogen-inoculated seedlings. The four OMF namely; Hyphomycete sp., Umbelopsis sp., Ceratorhiza sp. and Ceratorhiza sp. are compatible strains for improving the growth rate of D. officinale by increasing its environmental stress tolerance, providing an effective way to supply resources through artificial reproduction.