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• All orchids rely on mycorrhizal fungi for organic carbon, at least during early development. In fact, orchid seed germination leads to the formation of a protocorm, a heterotrophic postembryonic structure colonized by intracellular fungal coils, thought to be the site of nutrient transfer. The molecular mechanisms underlying mycorrhizal interactions and metabolic changes induced by this symbiosis in both partners remain mostly unknown. • We studied plant–fungus interactions in the mycorrhizal association between the Mediterranean orchid Serapias vomeracea and the basidiomycete Tulasnella calospora using nontargeted metabolomics. Plant and fungal metabolomes obtained from symbiotic structures were compared with those obtained under asymbiotic conditions. • Symbiosis induced substantial metabolomic alterations in both partners. In particular, structural and signaling lipid compounds increased markedly in the external fungal mycelium growing near the symbiotic protocorms, whereas chito-oligosaccharides were identified uniquely in symbiotic protocorms. • This work represents the first description of metabolic changes occurring in orchid mycorrhiza. These results – combined with previous transcriptomic data – provide novel insights on the mechanisms underlying the orchid mycorrhizal association and open intriguing questions on the role of fungal lipids in this symbiosis.

Orchid seed germination in nature is an extremely complex physiological and ecological process involving seed development and mutualistic interactions with a restricted range of compatible mycorrhizal fungi. The impact of the fungal species' partner on the orchids' transcriptomic and metabolic response is still unknown. In this study, we performed a comparative transcriptomic analysis between symbiotic and asymbiotic germination at three developmental stages based on two distinct fungi ( Tulasnella sp. and Serendipita sp.) inoculated to the same host plant, Dendrobium officinale . Differentially expressed genes (DEGs) encoding important structural proteins of the host plant cell wall were identified, such as epidermis-specific secreted glycoprotein, proline-rich receptor-like protein, and leucine-rich repeat (LRR) extensin-like protein. These DEGs were significantly upregulated in the symbiotic germination stages and especially in the protocorm stage (stage 3) and seedling stage (stage 4). Differentially expressed carbohydrate-active enzymes (CAZymes) in symbiotic fungal mycelium were observed, they represented 66 out of the 266 and 99 out of the 270 CAZymes annotated in Tulasnella sp. and Serendipita sp., respectively. These genes were speculated to be involved in the reduction of plant immune response, successful colonization by fungi, or recognition of mycorrhizal fungi during symbiotic germination of orchid seed. Our study provides important data to further explore the molecular mechanism of symbiotic germination and orchid mycorrhiza and contribute to a better understanding of orchid seed biology.

Mycorrhizal fungi have been shown to promote seed germination and seedling growth in Orchidaceae plants. In the present study, a mycorrhizal fungus designated as BJ1 was isolated from the roots of Bletilla striata (Thunb.) Reiehb.f. Fluorescence staining and morphological analysis revealed that this fungus exhibited characteristics highly similar to those of Tulasnella . Subsequently, the strain was confirmed as a new strain of Tulasnella through sequencing and phylogenetic analysis of four loci: the internal transcribed spacer region ITS1-ITS4 (ITS), ATP synthase (C14436), glutamate synthase (C4102), and ATP deconjugase (C3304). Additionally, we investigated the in vitro biological activity of strain BJ1 and its effects on germination and growth of B. striata seeds. The results indicated that BJ1 is capable of producing plant cell-degrading enzymes, including pectinase and protease. Furthermore, it demonstrates an ability to solubilize inorganic phosphorus and synthesize indoleacetic acid (IAA). Nevertheless, it does not exhibit laccase activity or possess the capacity to produce siderophores, nor can it solubilize organic phosphorus. Microscopic observations revealed that strain BJ1 mainly colonizes the base of the B. striata protocorm, thereby enhancing seed germination, growth, and expansion. Notably, by the fourth week of germination, 74.23% of seeds in the symbiotic group had developed to stage 5, a significantly higher proportion compared to 50.43% in the non-symbiotic group. Additionally, the length, width, and fresh weight of seeds in the symbiotic group were 2.2 times, 1.8 times, and 3.7 times greater than those in the non-symbiotic group, respectively. Furthermore, by adding L-tryptophan as a substrate during co-cultivation with BJ1, there was a significant enhancement in IAA synthesis capability; this also led to a marked acceleration in the symbiotic germination process of B. striata seeds. These results suggest that strain BJ1 holds significant potential for application in the artificial propagation of B. striata seedlings. It can enhance propagation efficiency and improve seedling quality, thereby playing a crucial role in the conservation and sustainable development of germplasm resources of endangered orchids.

MAIN CONCLUSION : Orchid mycorrhiza has been often interpreted as an antagonistic relationship. Our data on mycorrhizal protocorms do not support this view as plant defence genes were not induced, whereas some nodulin-like genes were significantly up-regulated. Orchids fully depend on symbiotic interactions with specific soil fungi for seed germination and early development. Germinated seeds give rise to a protocorm, a heterotrophic organ that acquires nutrients, including organic carbon, from the mycorrhizal partner. It has long been debated if this interaction is mutualistic or antagonistic. To investigate the molecular bases of the orchid response to mycorrhizal invasion, we developed a symbiotic in vitro system between Serapias vomeracea, a Mediterranean green meadow orchid, and the rhizoctonia-like fungus Tulasnella calospora. 454 pyrosequencing was used to generate an inventory of plant and fungal genes expressed in mycorrhizal protocorms, and plant genes could be reliably identified with a customized bioinformatic pipeline. A small panel of plant genes was selected and expression was assessed by real-time quantitative PCR in mycorrhizal and non-mycorrhizal protocorm tissues. Among these genes were some markers of mutualistic (e.g. nodulins) as well as antagonistic (e.g. pathogenesis-related and wound/stress-induced) genes. None of the pathogenesis or wound/stress-related genes were significantly up-regulated in mycorrhizal tissues, suggesting that fungal colonization does not trigger strong plant defence responses. In addition, the highest expression fold change in mycorrhizal tissues was found for a nodulin-like gene similar to the plastocyanin domain-containing ENOD55. Another nodulin-like gene significantly more expressed in the symbiotic tissues of mycorrhizal protocorms was similar to a sugar transporter of the SWEET family. Two genes coding for mannose-binding lectins were significantly up-regulated in the presence of the mycorrhizal fungus, but their role in the symbiosis is unclear.

Orchids rely strictly on mycorrhizal symbioses for nutrition, including carbon, from seed germination to protocorm growth. However, whether orchids establish symbiotic compatibility selectively with specific mycorrhizal fungi during this early growth period remains to be determined for most species. We examined pair-wise symbiotic compatibility between three terrestrial orchid species, Bletilla striata , Pecteilis radiata , and Spiranthes australis , and five closely related strains of Tulasnella , using a procedure we recently developed for detailed and quantitative evaluation of mycorrhizal symbiosis during post-germination growth. Our analyses revealed significant differences among colonization effects of the Tulasnella strains on the hosts, both at seed germination and during post-germination growth . The effects of the mycorrhizal associations tended to differ by stage, suggesting a variety of determinants for symbiotic compatibility. One Tulasnella strain exhibited high compatibility across the three distantly related host species, implying that inherent properties of mycorrhizal fungi can influence their symbiotic compatibility with a wide range of orchid species.

Abstract Background and Aims An understanding of mycorrhizal variation, orchid seed germination temperature and the effect of co-occurring plant species could be critical for optimizing conservation translocations of endangered plants with specialized mycorrhizal associations. Methods Focusing on the orchid Thelymitra epipactoides, we isolated mycorrhizal fungi from ten plants within each of three sites; Shallow Sands Woodland (SSW), Damp Heathland (DH) and Coastal Heathland Scrub (CHS). Twenty-seven fungal isolates were tested for symbiotic germination under three 24 h temperature cycles: 12 °C for 16 h–16 °C for 8 h, 16 °C for 16 h–24 °C for 8 h or 27 °C constant. Fungi were sequenced using the internal transcribed spacer (ITS), nuclear large subunit 1 (nLSU1), nLSU2 and mitochondrial large rRNA gene (mtLSU). Orchids were grown to maturity and co-planted with each of ten associated plant species in a glasshouse experiment with tuber width measured at 12 months after co-planting. Key Results Two Tulasnella fungal lineages were isolated and identified by phylogenetic analyses, operational taxonomic unit 1 (OTU1) and ‘T. asymmetrica’. Fungal lineages were specific to sites and did not co-occur. OTU1 (from the SSW site) germinated seed predominantly at 12–16 °C (typical of autumn–winter temperature) whereas ‘T. asymmetrica’ (from the DH and CHS sites) germinated seed across all three temperature ranges. There was no difference in the growth of adult orchids germinated with different OTUs. There was a significant reduction in tuber size of T. epipactoides when co-planted with six of the commonly co-occurring plant species. Conclusions We found that orchid fungal lineages and their germination temperature can change with habitat, and established that translocation sites can be optimized with knowledge of co-occurring plant interactions. For conservation translocations, particularly under a changing climate, we recommend that plants should be grown with mycorrhizal fungi tailored to the recipient site.

This article is a Commentary on Fochi et al., 213: 365–379.

Multispecies assemblages often consist of a complex network of interactions. Describing the architecture of these networks is a first step in understanding the stability and persistence of these species-rich communities. Whereas a large body of research has been devoted to the description of above-ground interactions, much less attention has been paid to below-ground interactions, probably because of difficulties to adequately assess the nature and diversity of interactions occurring below the ground. In this study, we used 454 amplicon pyrosequencing to investigate the architecture of the network between mycorrhizal fungi and 20 orchid species co-occurring in a species-rich Mediterranean grasslands. We found 100 different fungal operational taxonomic units (OTUs) known to be mycorrhizal in orchids, most of which were members related to the genera Ceratobasidium and Tulasnella. The network of interactions was significantly compartmentalized (M = 0.589, P = 0.001), but not significantly nested (N = 0.74, NODF = 10.58; P > 0.05). Relative nestedness was negative (N* = −0.014), also suggesting the existence of isolated groups of interacting species. Compartmentalization is a typical feature of ecological systems showing high interaction intimacy, and may reflect strong specialization between orchids and fungi resulting from physiological, physical or spatial constraints.

Mycorrhizal specificity, i.e., the range of fungi allowing mycorrhizal partnerships, differs among orchid species, but that at early developmental stages is unclear. We investigated whether mycorrhizal specificity during seed germination and seedling development differs among three Dendrobium species, D. officinale, D. okinawense and D. moniliforme, in vitro. Nine mycorrhizal fungal strains were obtained from the roots of these species and cultured with a seed of each Dendrobium species. Five to eight fungal strains stimulated seed germination, whereas one to four fungal isolates significantly promoted protocorm development in the three species. To evaluate effects on leafy seedling growth, seedlings obtained from asymbiotic culture were cultured with nine fungal isolates. D. officinale and D. okinawense showed specificity for a single Serendipitaceae or Tulasnellaceae isolate, whereas D. moniliforme exhibited specificity for three isolates of Serendipitaceae and Tulasnellaceae. Therefore, the three Dendrobium species had a growth bottleneck from seed germination to the protocorm stage, and mycorrhizal specificity of protocorm growth and seedling development in vitro varied among the species. Our findings imply divergent mycorrhizal specificity in Dendrobium species at early developmental stages. This study provides insights into the diversity of orchid mycorrhizal specificity, as well as valuable information for conservation of endangered orchids.

Orchids are highly dependent on their mycorrhizal fungal partners for nutrient supply, especially during early developmental stages. In addition to organic carbon, nitrogen (N) is probably a major nutrient transferred to the plant because orchid tissues are highly N-enriched. We know almost nothing about the N form preferentially transferred to the plant or about the key molecular determinants required for N uptake and transfer. We identified, in the genome of the orchid mycorrhizal fungus Tulasnella calospora, two functional ammonium transporters and several amino acid transporters but found no evidence of a nitrate assimilation system, in agreement with the N preference of the free-living mycelium grown on different N sources. Differential expression in symbiosis of a repertoire of fungal and plant genes involved in the transport and metabolism of N compounds suggested that organic N may be the main form transferred to the orchid host and that ammonium is taken up by the intracellular fungus from the apoplatic symbiotic interface. This is the first study addressing the genetic determinants of N uptake and transport in orchid mycorrhizas, and provides a model for nutrient exchanges at the symbiotic interface, which may guide future experiments.