Orchid mycorrhizae are symbiotic associations between orchid plant roots and fungi, in which fungal hyphae grow within living plant cells and form intracellular pelotons [17, 18]. Orchid mycorrhizal fungi (OMFs) are considered to belong to the so-called rhizoctonia aggregate, a polyphyletic group of fungi belonging to Tulasnellaceae, Ceratobasidiaceae, and Serendipitaceae . Orchids are highly dependent on mycorrhizae for survival and growth: OMFs not only supply carbohydrates to facilitate the growth of nonphotosynthetic protocorms during the seed germination stage but also provide nutrients and growth factors to plants, conferring plant metal tolerance and inhibiting the development of pathogens [17, 20–23].
Mycorrhizal technologies and microbial fertilizers could simplify orchid seedling production and promote plant growth and have been considered important aids to the sustainable development of the Dendrobium industry in China [6, 24, 25]. As the most popular medicinal plant species in the genus Dendrobium, D. officinale has received much research attention regarding its mycorrhizal symbionts . Traditionally, to obtain the optimal source of fungal mycobionts for symbiotic germination or plant growth, fungi have been isolated and screened from the roots of mature wild plants [26, 27]. Among the 28 fungal species reported in 11 D. officinale studies, only five fungal species in one study were obtained from protocorms via ex situ seed baiting , while the other 23 fungi in the remaining 10 studies were isolated from the roots of wild mature plants, including D. officinale and other orchid species (Additional file 2). The 28 fungal species/strains, originally obtained from different orchid species and belonging to a wide range of taxonomic groups (17 OMFs and 11 non-OMFs), were found to have positive effects on the growth of D. officinale seedlings (Additional file 2). Seemingly, a randomly obtained fungus could promote seedling growth in D. officinale; however, all of the above fungi were tested in vitro, and there have been no reports on the successful application of these fungi in practice so far. Theoretically, a high diversity of fungi may associate with the roots of an adult orchid plant, an orchid will utilize different OMFs at different life history stages, and the mycorrhizal symbionts may change in different developmental stages [14, 15, 29, 30]. It is unclear whether the fungi involved in the seedling stage remain until the plants reach adulthood in D. officinale.
The in situ/ex situ seed baiting technique has been suggested as an effective and easy way to obtain seed germination-enhancing fungi [31, 32]. Using this method, we successfully obtained efficient germination-enhancing fungi for different Dendrobium species [9–12], as well as other terrestrial orchids [33, 34]. This led to the development of the idea of using seedling-trap experiments to capture seedling growth-promoting fungi in the current study. After in vitro-produced seedlings of D. officinale were transplanted for more than one year in their original habitats, newly established roots of well-growing plants were sampled six times across one year in different seasons (Additional file 1). In total, five OMFs and one non-OMFs were obtained and identified (Table 1). Tulasnella species TPYD-1 and TPYD-2 were the dominant OMFs with a higher frequency of isolation than other OMFs, and TPYD-1 was present in all six samples, while TPYD-2 was present in five samples (Additional file 1). The three fungi TPYD-1, TPYD-2 and TPYD-3 with high isolation frequencies were closely related species, as they were clustered together in the phylogenetic tree (Fig. 2). Fusarium oxysporum TPYD-6 was also dominant, with a high isolation frequency and presence among the samples (Additional file 1).
In this study, to test and screen the fungi that could be used in real practice for growth of D. officinale seedlings, in vitro-produced seedlings were moved to mixed cultivation substrates in open environments, in which seedlings normally undergo an acclimatization stage. This could be a possible reason why the three fungi TPYD-1, TPYD-2 and TPYD-3 started to colonize seedling roots after a long period of 120 d. However, the corresponding increases in pelotons in the roots and in the five measured indices in all three fungal treatments at 150 and 180 d suggested that all three fungi could promote seedling growth in D. officinale but showed different efficiencies (Fig. 3). At 180 d after incubation, the longest root length, plant height, fresh weight and dry weight of seedlings in the TPYD-2 treatment were significantly longer or higher than those in the other two fungal treatments and the control treatment (Fig. 3). TPYD-2 could be selected as an ideal OMF for seedling growth in the restoration-friendly cultivation of D. officinale.
Tulasnellaceae is regarded as one of the main fungal families of orchid mycorrhizae, and many of these species are found to support seed germination and seedling growth in Dendrobium species [9, 10, 19, 24]. The other two Tulasnella species, TPYD-1 and TPYD-3, were closely related to TPYD-2 but showed less pronounced effects on seedling growth in D. officinale. Seedlings of D. officinale might need a longer time to acclimatize to fungal infection , or the two fungi might have other ecological functions [21, 22]. In the current study, we focused on the effects of fungi obtained from seedling baiting on seedling growth, but it would also be worthwhile to explore whether these fungi could effectively promote seed germination in D. officinale. In another study, we conducted comparisons of symbiotic germination of seeds inoculated with the TPYD-2 obtained in this study and six other fungal strains isolated from protocorms of D. officinale via in situ seed baiting. At 90 d after incubation, the percentage of seedlings in the LQ treatment was 70.09 ± 3.2%, while no protocorms or seedlings were found in the TPYD-2 treatment (Wang et al. unpublished data). The results also suggested that D. officinale could associate with different fungi in different life stages.
In addition to OMFs, other root-associated nonmycorrhizal endophytes have also been recorded and identified from a wide range of orchid species [36, 37]. In this study, Fusarium oxysporum TPYD-6 was isolated from all 6 samples with high isolation rates. Fusarium species have been reported to be associated with different orchid species [33, 38]. Interestingly, although Fusarium species have been reported as pathogens in many orchid species , especially F. oxysporum, which was reported to cause wilt disease in D. officinale , other studies have suggested that Fusarium species can stimulate seed germination in the terrestrial orchid Cypripedium reginae  and enhance resistance to pathogens and promote plant growth in Dendrobium species [42, 43]. In a recent study, F. oxysporum KB-3, obtained from the roots of Bletilla striata, was considered an OMF because it could promote seed germination of B. striata, establish colonization and produce coiled hyphal structures within the cortical cells in the roots of B. striata and Dendrobium candidum . As noted in many studies, the border between endophytic and mycorrhizal fungi could be difficult to define, and some fungus-plant interactions can easily shift from mutualism to parasitism depending on the plant’s physiology and environmental conditions [45, 46, 47]. For the current study, it is worth exploring the effect of F. oxysporum TPYD-6 alone as well as the possible synergistic effect of TPYD-6 with other OMFs on the growth of D. officinale seedlings.