Endophytic fungi are integral components of orchid biology, contributing to nutrient acquisition, stress tolerance, and ecological adaptations.
In the present study, root-associated fungal endophytes were isolated and characterized from an epiphytic orchid, Luisia trichorhiza
(Hook.) Blume, collected from the Chamoli district of Uttarakhand, India. Fungal isolates were cultured on Potato Dextrose Agar (PDA)
medium and examined using morphological, anatomical, and molecular approaches. Distinct colony morphologies and micromorphological
traits indicated the presence of two taxonomically different fungal isolates. Molecular identification based on sequencing of the internal
transcribed spacer (ITS) region revealed that isolate LT-1 showed 99.58% sequence similarity with Neocosmospora rubicola, whereas
isolate LT-2 exhibited 94.76% similarity with Thelonectria blackeriella and therefore, the latter was conservatively identified up to the genus
level as Thelonectria sp. The corresponding ITS sequences were deposited in NCBI GenBank under accession numbers PX640217 (LT-
1) and PX735845 (LT-2). Phylogenetic analyses using the Maximum Likelihood method further supported the taxonomic placement of both
these isolates within their respective lineages. Anatomical investigations confirmed that fungal colonisation was restricted to the root
system, with the formation of characteristic intracellular pelotons within cortical cells, particularly in the root hair zone, while aerial tissues
remained uninfected. The coexistence of both common and relatively rare fungal taxa highlighted the complexity of the root-associated
mycobiome of L. trichorhiza. The present findings provide novel insights into orchid-fungal associations and establish a baseline for future
studies on orchid conservation, in vitro symbiotic germination, and biotechnological potential of endophytic fungi.