ABSTRACT Symbiosis between orchid seeds and mycorrhizal fungi has been reported to be a determining factor in the success of germination and protocorm development in vitro. The aim of this study was to isolate and identify by molecular analysis the mycorrhizal fungus associated with Cyrtopodium paludicolum, and to evaluate its efficiency in facilitating seed germination and development. Germination experiments were carried out using a fungus isolated from C. paludicolum (CH01) and Epidendrum secundum (M65), which has been successfully used a number of times in symbiotic germination. The experiments were conducted in a completely randomized design with treatments of CH01, M65 as well as under asymbiotic conditions. The mycobiont CH01 was successfully isolated from Cyrtopodium paludicolum and identified as Tulasnella sp. Treatments with both fungi reached a higher germination percentage than under asymbiotic conditions, indicating no specificity in the relationship between Cyrtopodium paludicolum and the fungi. The results presented have the potential to advance research into the propagation and conservation of C. paludicolum, a native of the Cerrado biome.

Cyrtopodium glutiniferum is an endemic orchid of Brazil with potential medicinal and ornamental applications. As mycorrhizal fungi are essential for the initiation of the orchid life cycle, the aim of this study was to determine the strains of mycorrhizal fungi suitable for seed germination and protocorm development of C. glutiniferum and to characterize the symbiotic development of protocorms. Seeds of C. glutiniferum were inoculated with nine mycorrhizal fungi, Epulorhiza spp., Ceratorhiza spp., Rhizoctonia sp., originally isolated from Brazilian neotropical orchids. Only Epulorhiza isolates promoted seed germination and protocorm development. Three Epulorhiza isolates (M1, M6 = E. epiphytica, M20 = Epulorhiza sp.) promoted protocorm development until leaf production at 63 days. The protocorms are comprised of parenchyma cells delimited by a unistratified epidermis; the parenchyma cells of the upper part of the protocorms are smaller than those located more towards the base. Intact and digested pelotons were observed inside of protocorms implying that the seedlings were capable of mycotrophy. Additionally, the development of a bud primordium only occurred after colonization by fungus. This study suggests that C. glutiniferum has a preference for strains of Epulorhiza and that fungus digestion is essential to protocorm development.

In nature, orchid seeds obtain the nutrients necessary for germination by degrading intracellular fungal structures formed after colonization of the embryo by mycorrhizal fungi. Protocols for asymbiotic germination of orchid seeds typically use media with high concentrations of soluble carbohydrate and minerals. However, when reintroduced into the field, seedlings obtained via asymbiotic germination have lower survival rates than do seedlings obtained via symbiotic germination. Tree fern fiber, the ideal substrate for orchid seedling acclimatization, is increasingly scarce. Here, we evaluated seed germination and protocorm development of Cyrtopodium glutiniferum Raddi cultivated in asymbiotic media (Knudson C and Murashige & Skoog) and in oatmeal agar (OA) medium inoculated with the mycorrhizal fungus Epulorhiza sp., using non-inoculated OA medium as a control. We also evaluated the performance of tree fern fiber, pine bark, eucalyptus bark, corncob and sawdust as substrates for the acclimatization of symbiotically propagated plants. We determined germination percentages, protocorm development and growth indices at 35 and 70 days of cultivation. Relative growth rates and the effects of substrates on mycorrhizal formation were calculated after 165 days of cultivation. Germination efficiency and growth indices were best when inoculated OA medium was used. Corncob and pine bark showed the highest percentages of colonized system roots. The OA medium inoculated with Epulorhiza sp. shows potential for C. glutiniferum seedling production. Corncob and pine bark are promising substitutes for tree fern fiber as substrates for the acclimatization of orchid seedlings.