The critically endangered orchid Dendrobium officinale, valued for its medicinal properties, depends on specific seedling-associated mycorrhizal fungi (SAMF) for successful early-stage seedling development. However, conservation efforts are often hindered by difficulties in obtaining suitable SAMF, leading to poor seedling establishment in both natural and cultivated environments. In this study, we explored the growth-promoting effects of SAMF and evaluated the performances of synthetic fungal combinations. Our results demonstrated that mycorrhizal fungi, widely distributed across multiple habitats with high isolation frequencies, significantly promoted the growth of D. officinale, with specific fungi favoring different growth parameters. Tulasnella sp. TP-2 and TP-3 significantly improved stem diameter and plant height by 2.622 mm and 4.621 cm, while Tulasnella sp. TP-8 significantly increased tillering by a factor of 4.47. Additionally, Tulasnella sp. TP-11 and TP-13 markedly increased the number of new leaves (4.45) and new roots (2.688), respectively, identifying them as essential core OMFs for D. officinale seedlings. Contrary to expectations, synthetic fungal combinations composed of core orchid mycorrhizal fungi (core OMFs) did not exhibit synergistic growth-promoting effects. Instead, pronounced offset effects were observed, indicating that interactions between fungi may introduce competition or inhibition, limiting their collective ability to enhance plant growth. Our results confirmed that the core OMFs significantly promoted the growth of D. officinale seedlings. These core OMFs can serve as essential components in specialized microbial fertilizers for D. officinale, improving growth efficiency and yield, and supporting the sustainable development of the D. officinale industry.

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.