Xiaoke Xing

Orchidaceae is a highly valuable horticultural and medicinal plant family worldwide; however, large-scale propagation and conservation remain severely limited. Orchid seeds depend on symbiotic fungi for germination, and pre-symbiotic communication is essential for establishing a successful association, a process that remains poorly understood. In this study, exudates were collected from the germination-promoting fungus Ceratobasidium sp. GS2 and applied to seeds of the terrestrial orchid Gymnadenia conopsea to investigate downstream responses. Multi-omics approaches, including RNA-seq, metabolomics, and phylogenetic analysis, combined with biological validation, revealed that the exudates elicited transcriptional and physiological responses in G. conopsea seeds, potentially promoting dormancy release. Exposure to fungal exudates increased the levels of brassinosteroids, cytokinins, and fatty acids in seeds. Exogenous hormone application confirmed that brassinosteroids and cytokinins promote fungal colonization and facilitate symbiotic seed germination. Phylogenetic analysis revealed the conservation of symbiotic genes in partially mycoheterotrophic orchids, and functional characterization confirmed the role of GcRAM2 in G. conopsea . These findings provide new insights into the mechanisms underlying orchid mycorrhizal symbiosis.

Orchids rely on symbiotic microorganisms for nutrient acquisition throughout their life cycle, from seed germination to plant maturity. In the terrestrial orchid Gymnadenia conopsea , beneficial Pseudomonas species have been previously identified as associated with seed germination and enriched in protocorms. Yet, the specific metabolites that mediate this microbial recruitment remain unknown. In this study, integrated transcriptomic and metabolomic analyses revealed that seed imbibition activates the phenylpropanoid biosynthesis along with starch and sucrose metabolism pathways, resulting in increased secretion of trehalose and sinapyl alcohol. These metabolites were found to attract Pseudomonas sp. and facilitate their colonisation. We further assessed the effects of these metabolites in the presence of the germination‐promoting fungus Ceratobasidium sp. GS2, with or without Pseudomonas . Our results indicated that trehalose enhanced protocorm development when combined with the fungus, and this effect was significantly strengthened by the addition of Pseudomonas . In contrast, sinapyl alcohol promoted protocorm development only when both the fungus and Pseudomonas were present. These findings uncover a metabolite‐mediated synergy that coordinates beneficial microbes to orchestrate early development in G. conopsea , advancing our understanding of metabolite‐fungus‐bacteria interactions and benefiting cultivation practices.

Orchids critically rely on mycorrhizal fungi for seed germination and seedling development, but the extent to which the fungus benefits from the orchid is less clear. Recent work in arbuscular mycorrhizae has suggested that plants can provide fatty acids (FAs) to fungi, but empirical evidence in orchids remains limited. Here, we combine lipidomic and transcriptomic analyses to test the hypothesis that the germination-promoting fungus Ceratobasidium sp. GS2 receives carbon in the form of FAs from Gymnadenia conopsea seeds during symbiotic germination. Confocal and transmission electron microscopy confirmed the potential of FA transfer from seeds to the fungus. Symbiosis resulted in significant changes in the lipid composition of the fungus, with increased concentrations of FAs in the external mycelium. RNA-seq showed upregulation of genes associated with FA synthesis in seeds and downregulation of de novo FA synthesis genes in fungi 12 d post-symbiosis, indicating that the increased amounts of FAs in the fungus may originate from the seeds. These results indicate that FAs absorbed by hyphae in the colonized inner cortex cells support hyphal growth, providing evidence for directional carbon flow from the orchid seeds to the fungus and supporting a “give now and get now” model of mutualism in orchid-fungus symbioses.

Symbiotic germination in orchids is a complex biological process driven by a unique dependence on mycorrhizal fungi. It is generally assumed that, due to a lack of endosperm, orchids fully rely on fungi for carbon sources. However, orchid seed embryos store substantial nutrient reserves, but the metabolic functions of these reserves and their roles in establishing symbiosis during germination remain unclear. Here, we used time‐series transcriptomics and ultrastructural morphological analyses to investigate the early stages of symbiotic germination in Gymnadenia conopsea, a terrestrial orchid widely distributed across Eurasia. We identify three distinct phases during early seed germination (seed imbibition, fungal invasion and symbiotic establishment) that correspond with pronounced changes in gene expression and energy metabolism. During imbibition, lipid metabolism was already active, leading the oleosomes to fuse into large lipid vacuoles, whereas carbohydrate metabolism became dominant after fungal invasion, with lipid droplets appearing within fungal hyphae. Based on this transcriptomic and morphological evidence, we propose a potential model in which lipid vacuoles facilitate lipid transfer from the seeds to the fungi during the initial invasion, after which the fungi supply carbohydrates to the seeds for further development.

Orchids represent an important component of biodiversity in many ecosystems worldwide, notwithstanding their seed germination and distribution may to a large extent be determined and influenced by mycorrhizal fungi. While it is commonly assumed that widespread orchids are mycorrhizal generalists, the degree to which mycorrhizal diversity supports seed germination remains relatively underexplored. In this study, we investigated the role of a variety of Ceratobasidium fungi in supporting seed germination of the widespread terrestrial orchid Gymnadenia conopsea across China. Twelve Ceratobasidium strains isolated from G. conopsea and other orchids were examined for their ability to support germination of G. conopsea seeds collected from twelve sites across China. Of the twelve tested strains, six were able to support seed germination, while the remaining six strains showed no activity. Compatible strains showed a broad phylogenetic breadth, indicating the G. conopsea is capable of initiating associations with a diverse array of Ceratobasidium fungi. However, the six compatible strains differed in their ability to support protocorm formation. Moreover, germination success of seeds collected from different sites differed among Ceratobasidium strains. Seeds from northern China had a significantly higher number of compatible strains (average 5.6) than seeds from southwestern China (average 3.5). Our results suggest that G. conopsea is not only a mycorrhizal generalist in the adult stage but also in the seed germination stage, at least towards Ceratobasidium fungi. However, the significant strain-provenance interactions indicate regional differences in orchid-fungus interactions. These findings are important for improving local population restoration programs and germplasm conservation of this widespread and endangered orchid species.

Fungi and bacteria often occupy very similar niches; they interact closely with each other, and bacteria can provide direct or indirect benefits to plants that form mutualistic interactions with fungi. In orchids, successful seed germination largely depends on compatible mycorrhizal fungi, but whether and how bacteria contribute to seed germination and protocorm development remains largely unknown. Here, we performed field and laboratory experiments to assess the potential role of bacteria in mediating seed germination and protocorm development in the terrestrial orchid Gymnadenia conopsea . Our results suggested that bacterial and fungal communities differ between developmental stages in the germination process. The diversity of bacterial and fungal communities and their interaction network in germinating seeds (Stage 1) differed significantly from those in later developmental stages (Stages 2–5). Pseudomonas gradually became the dominant bacterial group as the protocorms matured and showed a positive association with Ceratobasidiaceae fungi. Seed germination tests in vitro demonstrated that co-inoculation of Ceratobasidium sp. GS2 with Pseudomonas isolates significantly improved protocorm growth and development, suggesting that the observed increase in Pseudomonas abundance during protocorm development directly or indirectly improves the growth of germinating seeds. Overall, our findings indicate that bacteria may exert non-negligible effects on seed germination of orchids and, therefore, offer valuable perspectives for future strategies for conservation and cultivating orchid species. IMPORTANCE: It is well known that orchid seeds depend on mycorrhizal fungi to supply the necessary nutrients that support germination in natural environments. Apart from fungi, bacteria may also be involved in the germination process of orchid seeds, but so far, their role has not been intensively studied. This research provides evidence that bacterial community composition changes during seed germination of the terrestrial orchid Gymnadenia conopsea . Interestingly, in vitro experiments showed that Pseudomonas spp., which were the most dominant bacteria in the later germination stages, improved protocorm growth. These results suggest that bacteria contribute to the germination of orchid seeds, which may open new perspectives to apply bacteria as a biofertilizer in the introduction and restoration of G . conopsea populations.

The rhizosphere, as the “frontline” of plant life, connects plant roots, rhizosphere microorganisms, and surrounding soil, plays a crucial role in plant growth and health, particularly in sustainable agriculture. Despite the well-established contribution of plant-microbe interactions to plant health, the specific molecular mechanisms remain insufficiently understood. This review aims to summarize the physiological adjustments and signal modulation that both plants and microorganisms undergo within this unique ecological niche to ensure successful colonization. By analyzing key processes such as chemotaxis, root attachment, immune evasion, and biofilm formation, we uncover how plants precisely modulate root exudates to either recruit or repel specific microorganisms, thereby shaping their colonization patterns. These findings provide new insights into the complexity of plant-microbe interactions and suggest potential directions for future research in sustainable agriculture.

Almost all orchids rely on mycorrhizal fungus to support their seed germination. To date, the effect of active components in mycorrhizal fungus on orchid seed germination largely remains unknown. In this study, we aimed to investigate the impact of active components found in mycorrhizal fungus on orchid seed germination. Specifically, we focused on a terrestrial orchid Gymnadenia conopsea and its host-specific seed germination supporting fungus Ceratobasidium GS2. In total, several steroids (1–7) were isolated from this fungus. Notably, compounds 1, 2, 4, and 5 exhibited significant enhancements in protocorm volume. Moreover, compounds 1–6 demonstrated strong promotion of protocorm differentiation. These findings suggest that steroids may play a crucial role in the symbiotic germination of G. conopsea seeds. Future studies should continue to explore the specific mechanisms through which these steroids exert their effects, contributing to our understanding of orchid biology and mycorrhizal interaction.