Symbiosis between Dendrobium catenatum protocorms and Serendipita indica involves the plant hypoxia response pathway.

Mycorrhizae are ubiquitous symbioses established between fungi and plant roots. Orchids, in particular, require compatible mycorrhizal fungi for seed germination and protocorm development. Unlike arbuscular mycorrhizal fungi, which have wide host ranges, orchid mycorrhizal fungi are often highly specific to their host orchids. However, the molecular mechanism of orchid mycorrhizal symbiosis is largely unknown compared to that of arbuscular mycorrhizal and rhizobial symbiosis. Here, we report that an endophytic Sebacinales fungus, Serendipita indica, promotes seed germination and the development of protocorms into plantlets in several epiphytic Epidendroideae orchid species (6 species in 2 genera), including Dendrobium catenatum, a critically endangered orchid with high medicinal value. Although plant-pathogen interaction and high meristematic activity can induce the hypoxic response in plants, it has been unclear whether interactions with beneficial fungi, especially mycorrhizal ones, also involve the hypoxic response. By studying the symbiotic relationship between D. catenatum and S. indica, we determined that hypoxia-responsive genes, such as those encoding alcohol dehydrogenase (ADH), are highly induced in symbiotic D. catenatum protocorms. In situ hybridization assay indicated that the ADH gene is predominantly expressed in the basal mycorrhizal region of symbiotic protocorms. Additionally, the ADH inhibitors puerarin and 4-methylpyrazole both decreased S. indica colonization in D. catenatum protocorms. Thus, our study reveals that S. indica is widely compatible with orchids and that ADH and its related hypoxia-responsive pathway are involved in establishing successful symbiotic relationships in germinating orchids.

What role does the seed coat play during symbiotic seed germination in orchids: an experimental approach with Dendrobium officinale.

BACKGROUND: Orchids require specific mycorrhizal associations for seed germination. During symbiotic germination, the seed coat is the first point of fungal attachment, and whether the seed coat plays a role in the identification of compatible and incompatible fungi is unclear. Here, we compared the effects of compatible and incompatible fungi on seed germination, protocorm formation, seedling development, and colonization patterns in Dendrobium officinale; additionally, two experimental approaches, seeds pretreated with NaClO to change the permeability of the seed coat and fungi incubated with in vitro-produced protocorms, were used to assess the role of seed coat played during symbiotic seed germination. RESULTS: The two compatible fungi, Tulasnella sp. TPYD-2 and Serendipita indica PI could quickly promote D. officinale seed germination to the seedling stage. Sixty-two days after incubation, 67.8 ± 5.23% of seeds developed into seedlings with two leaves in the PI treatment, which was significantly higher than that in the TPYD-2 treatment (37.1 ± 3.55%), and massive pelotons formed inside the basal cells of the protocorm or seedlings in both compatible fungi treatments. In contrast, the incompatible fungus Tulasnella sp. FDd1 did not promote seed germination up to seedlings at 62 days after incubation, and only a few pelotons were occasionally observed inside the protocorms. NaClO seed pretreatment improved seed germination under all three fungal treatments but did not improve seed colonization or promote seedling formation by incompatible fungi. Without the seed coat barrier, the colonization of in vitro-produced protocorms by TPYD-2 and PI was slowed, postponing protocorm development and seedling formation compared to those in intact seeds incubated with the same fungi. Moreover, the incompatible fungus FDd1 was still unable to colonize in vitro-produced protocorms and promote seedling formation. CONCLUSIONS: Compatible fungi could quickly promote seed germination up to the seedling stage accompanied by hyphal colonization of seeds and formation of many pelotons inside cells, while incompatible fungi could not continuously colonize seeds and form enough protocorms to support D. officinale seedling development. The improvement of seed germination by seed pretreatment may result from improving the seed coat hydrophilicity and permeability, but seed pretreatment cannot change the compatibility of a fungus with an orchid. Without a seed coat, the incompatible fungus FDd1 still cannot colonize in vitro-produced protocorms or support seedling development. These results suggest that seed coats are not involved in symbiotic germination in D. officinale.

A novel method to produce massive seedlings via symbiotic seed germination in orchids.

Orchids produce large numbers of dust-like seeds that rely heavily on orchid mycorrhizal fungi (OMFs) for germination. Using OMFs to facilitate orchid proliferation is considered an effective method for orchid conservation but still presents challenges in practice. In this study, orchid seed-fungus complexes, in which orchid seeds and fungal mycelia were embedded together to form granules, were developed as platforms to facilitate seed germination and seedling production. Overall, seedlings were produced by seed-fungus complexes for five orchid species with large variations in the percentages of seedlings produced among species/treatments. For the different fungal treatments in Dendrobium officinale, Sebacinales LQ performed much better than the other fungal strains. At 90 days after sowing, 75.8±2.6% seedlings were produced in the LQ treatment, which was significantly higher than in the Tulasnella sp. JM (22.0±3.0%) and Tulasnella sp. TPYD-2 (5.3±1.0%) treatments, as well as in the LQ and TPYD-2 cocultured treatment (40.4±3.2%), while no seedlings were formed in the Tulasnella sp. SSCDO-5 or control treatments. For the other four orchid species, only one compatible fungus for each species was used, and the percentages of seedlings in epiphytic Dendrobium devonianum (67.2±2.9%) and D. nobile (38.9±2.8%) were much higher than those in terrestrial Paphiopedilum spicerianum (2.9±1.1%) and Arundina graminifolia (6.7±2.1%) at 90 days after sowing. Adding 1% polymer water-absorbent resin to the seed-fungus complexes of D. officinale seeds with fungal strain Sebacinales LQ significantly increased seedling formation, while other additional substances showed negative effects on seedling formation. For the storage of seed-fungus complexes, it is recommended to store the seed-fungus complexes in valve bags at room temperature for a short time and at a low temperature of 4°C for no more than 30 days. As a platform for symbiotic seed germination, the seed-fungus complex can facilitate seed germination, produce seedlings and support subsequent seedling growth, and its seedling productivity depends on seed germination characteristics, seed viability, and the efficiency of fungi. Seed-fungus complexes have great potential to be used as propagules in orchid conservation.

Using Ex Situ Seedling Baiting to Capture Seedling-Associated Mycorrhizal Fungi in Medicinal Orchid Dendrobium officinale.

Using orchid mycorrhizal fungi (OMFs) to facilitate orchid proliferation is considered an effective method of orchid conservation. Based on the success of using in situ seedling baiting to obtain plant growth-promoting fungi in our previous study, in this study, we developed the method of using ex situ seedling baiting to capture seedling-associated fungi from Dendrobium officinale. We collected substrates (e.g., litters, barks and mosses) from six original habitats of D. officinale in different geographical locations in China, and then, transplanted in vitro-produced seedlings of D. officinale into the substrates. After cultivation for 75 days, it was obvious that fungi colonized the seedling roots and formed large numbers of pelotons in all six groups. From these seedling roots, a total of 251 fungal strains, which were divided into 16 OMF and 11 non-OMF species, were successfully isolated. The 16 OMFs included 13 Tulasnella and 3 Serendipitaceae species. The fungal species isolated from the different groups (original habitat sources) were not identical, but the dominant OMFs with high isolation frequencies (more than 10 times) were commonly isolated from more than four original sources. Among the 11 non-OMFs, Fusarium oxysporum TP-18 and Muscodor sp. TP-26 were the dominant endophytes. Fusarium oxysporum is a common endophyte associated with many orchid species, including D. officinale. The results suggest that ex situ seedling baiting is an easy and efficient approach to obtaining seedling-associated fungi for this species and could be performed for other over-collected species, especially orchids for which wild plants have disappeared in the field but their original habitats are known. This approach has great potential for application in OMF studies in the future.