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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.

Aims Previous studies have shown that Dendrobium plants form a specific symbiotic relationship with fungi at differentiation stages during natural seed germination. In order to explore the evolution and adaptation of this symbiotic relationship in interspecific hybrid progenies, this study was to understand whether the strong specificity with symbiotic fungi during seedling formation and differentiation was also an important factor limiting the formation of hybrid progenies in Dendrobium, and the relationship between hybrid progenies of orchids and symbiotic fungi during seed germination stage. Methods The effects of fungi on germination of interspecific hybrid seeds of D. officinale and D. tortile were studied using the highly specific fungi strains Tulasnella SSCDO-5 and Epulorhiza FDd1, which can effectively promote seed germination and seedling formation in D. officinale and D. devonianum, respectively. Important findings The results showed that both SSCDO-5 and FDd1 strains could effectively promote the protocorm and seedling formation of hybrid seeds after 68 days incubation with no significant difference. The SSCDO-5 strain from D. officinale did not show any advantages, and the seedling formation rate of hybrid was lower than that of FDd1 strain from D. devonianum. The seedling formation rate incubation with SSCDO-5 strain was (22.13 ± 6.62)% while with FDd1 strain was (29.53 ± 5.51)%. The specificity of SSCDO-5 strain with D. officinale at seedling formation and development stage was not inherited or expressed in hybrid progenies, indicating that hybridization broke the symbiotic relationship of this specificity, which enabled hybrid progenies to establish new symbiotic relationship with different fungi. Our results do not support the hypothesis that the specificity of symbiotic fungi is an important limiting factor for the formation of hybrid progenies in Dendrobium. We

Background: Orchids are highly dependent on mycorrhizae for survival and growth. Traditionally, to obtain orchid mycorrhizal fungi (OMFs) for plant growth, fungi have been isolated from the roots of mature wild plants; however, the species of OMF may change as the plants undergo different developmental stages. In this study, we developed the idea of using in situ seedling baiting to capture seedling growth-promoting fungi from Dendrobium officinale, an overcollected medicinal orchid in China.Results: In vitro-produced seedlings of D. officinale were transplanted into their original habitats, and newly established roots of well-growing seedlings were sampled for fungal isolation. Roots were sampled at 6 different times over one year, and five Tulasnella species and one Fusarium species were obtained and identified morphologically and molecularly. The ability to promote seedling growth was tested in three Tulasnella species TPYD-1, TPYD-2 and TPYD-3, with high isolation frequencies by inoculation onto in vitro-produced D. officinale seedlings. Although the three fungi were closely related species and clustered together in the phylogenetic tree, they showed different efficiencies in promoting D. officinale seedling growth. Tulasnella TPYD-2 showed a strong ability to promote seedling growth and could be selected for use in restoration plantings of D. officinale.Conclusions: Our results suggest that using an in situ seedling baiting technique could be an efficient way to obtain seedling growth-promoting fungi, and this approach could have broad applications in orchid mycorrhiza studies and orchid conservation.