Ultrastructural studies and molecular characterization of root-associated fungi of Crepidium acuminatum (D. Don) Szlach.: a threatened and medicinally important taxon

Crepidium acuminatum (Orchidaceae) is a threatened medicinal orchid that grows under shady and moist forest floor where light remains for a very short period of time. Mycorrhizal association is known to be essential for seed germination and seedling establishment in a majority of orchids. Identification of fungi that form mycorrhizae with orchids is of crucial importance for orchid conservation. We used both morphological as well as molecular approaches to study this plant–fungal interaction. Scanning electron microscopy showed that fungi grow and proliferate in the middle layers of the cortex. Also, spiral-root hairs were found along with root hairs, which is an unusual observation. Spiral-root hairs provide more surface area for fluid absorption and entrance of colonizers. Further, total root genomic DNA was isolated and fungal internal-transcribed spacer (ITS) regions were polymerase chain reaction (PCR)-amplified using specific primer combinations ITS1F/ITS4 and ITS1/ITS4tul. ITS sequences were obtained and analysed to know the closest sequence matche in the GenBank using BLASTn hosted by NLM-NCBI. Subject sequences were identified to be belonging to three main genera, namely, Tulasnella , Aspergillus and Penicillium . Results indicate that mycorrhizal association is necessary for the growth and development of the plant. In addition, this symbiosis influences the distribution and rarity of this medicinally valuable taxon. Specific fungal partners may lead to an enhanced seed germination rate and increased efficiency of nutrient exchange between both the partners. Hence, knowledge of mycorrhizal fungi is essential for future in vitro germination and seedling establishment programmes, because they rely on fungi for germination. Identification of mycorrhizal fungi can be used for orchid propagation and conservation programmes.