Orchidaceae is one of the most diverse plant families consisting of approximately 30,000 orchid species worldwide and majority of these are rare in nature because of their high eco-specialization strategies. To better strategize for the long term in-situ conservation of orchid species diversity, it is important to delineate these highly specialized ecological strategies in orchids by identifying them and then investigating their functional mechanisms. While orchid mycorrhizal fungus (OMF) surveys are common in orchids, higher fitness and biomass of orchid seedlings germinated with dual inoculations of OMF, and endophytic helper bacteria (EHB) shed new light on the importance of bacterial symbionts in orchid niche. However, simultaneous surveys of fungal and bacterial symbiont diversity in orchid species are not available that can guide the research on tripartite interaction among orchids, OMF and bacteria. Thus, an objective of our study is to understand the effect of EHB, OMF, and intrahyphal bacterial diversity of symbionts in the germination of the rare disjunctly populated species, Platanthera chapmanii, Roots of 15 individuals in 2018, and 16 in 2019 were collected from Texas, and 14 from Florida in 2019. Endophytic bacteria, OMF, and their IHB were isolated from fungal pelotons and cortical tissue of orchid roots. Fungi and bacteria were identified through sequencing of nrITS and 16S barcoding regions. Intrahyphal bacteria were confirmed using scanning electron microscopy of the host OMF fungus.
Results/Conclusions
Out of ninety resulting sequences, those above 200 bp were quality filtered and clustered at 97% into operational taxonomic units (OTU) belonging to 15 bacterial OTU, and 23 fungal OTU. The 15 bacterial OTU were distributed evenly amongst 15 individuals from Texas and eight from the Floridian population. The 23 fungal OTU were unevenly distributed, largely concentrated amongst 13 Texan individuals. Only two fungal OUT were represented from the Floridan individuals. Electron scanning microscopy of external hyphae allowed for the confirmation of the novel isolation of six intrahyphal bacteria from orchid mycorrhizal fungi from the families Tulasnellaceae and Ceratobasidiaceae. This investigation of mycorrhizal fungal, endophytic bacterial, and IHB diversity of P.chapmanii will guide the development of more effective germination and conservation approaches for vulnerable or disjunct orchid populations. This project aims to further provide guidelines and methodology in the isolation of intrahyphal bacteria from orchid mycorrhizal partners, essential in future delineation of mechanisms within fungal host, and function within host plant.