Abstract
I investigated the culturable endophytic fungi resident in symptom-free leaves of two medicinal Lamiaceae herbs, Ocimum sanctum (Tulsi) and Leucas aspera (Thumbai). Leaves were surface-sterilized, plated on Potato Dextrose Agar (PDA), and incubated at 28 ± 1 °C. Within five days, reproducible colonies emerged from most leaf segments. Nine morphotypes were documented by macromorphology and subculture behavior, consistent with genera such as Aspergillus, Cladosporium, Fusarium, and Diaporthe/Phomopsis. This work provides a clean isolate set for downstream molecular identification and bioactivity screening.
Background
Endophytes are microorganisms that live within healthy plant tissues without causing disease. Medicinal plants frequently host diverse endophyte communities which can synthesize metabolites overlapping with or complementing host chemistry. Prior studies on Lamiaceae herbs report high endophyte diversity in O. sanctum and measurable colonization in L. aspera.
Objectives
- Isolate viable endophytic fungi from healthy leaves of both hosts.
- Establish a sterile culturing workflow on PDA and document emergence times.
- Record macroscopic morphologies and host incidence for distinct morphotypes.
- Prepare clean subcultures for downstream molecular identification.
Materials and Methods
Sampling and Handling
Mature, symptom-free leaves were collected in mid-July 2025 from home gardens in Krishnamurthy Puram (Mysuru, India), transported in sterile, labeled bags, and processed within four hours in a laminar flow hood.
Surface Sterilization and Plating
Leaf segments were processed by 75% ethanol (1 min) → 2% sodium hypochlorite (1 min) → three sterile water rinses; tissues were cut into ~5 × 5 mm squares and placed adaxial side down on PDA (4–6 pieces/plate). Plates were incubated at 28 ± 1 °C and inspected daily for up to five days.
Documentation
Plates and subcultures were photographed; colony color, growth rate, aerial mycelium, and sporulation were logged. Distinct colonies were transferred to fresh PDA for purity and morphotyping.
Results
Extensive emergence was observed from both hosts. Nine distinct morphotypes were reproducibly recognized across subcultures, based on macromorphology and growth behavior.
| # | Putative identity | Representative macromorphology | Host incidence |
|---|---|---|---|
| 1 | Aspergillus niger group | Fast-growing, black conidial heads; biseriate phialides | O. sanctum & L. aspera (more frequent in L. aspera) |
| 2 | Aspergillus flavus complex | Yellow-green penicilli; globose, echinulate conidia | Mainly L. aspera |
| 3 | Cladosporium spp. | Olivaceous-grey, velutinous; ramoconidia; shield-shaped conidia | Both hosts |
| 4 | Diaporthe/Phomopsis spp. | White→grey; concentric black pycnidia after day 7 | Only O. sanctum |
| 5 | Fusarium oxysporum s.l. | Pale salmon sporodochia; sickle-shaped macroconidia | Both hosts (more frequent in L. aspera) |
| 6 | Fusarium solani s.l. | Cottony white → blue-green | Only O. sanctum |
| 7 | Macroconia fruticulosa | Dark, compact colonies; rough-walled macroconidia | Rare; single isolate in L. aspera |
| 8 | Penicillium spp. | Bluish-green; radially sulcate; terverticillate penicilli | Occasional in both hosts |
| 9 | Sterile hyaline mycelium | Unidentified coelomycete; slimy spear-shaped propagules | Single recovery in L. aspera |
Frequent multi-colony emergence from individual leaf segments suggests dense co-colonization, a pattern reported for Lamiaceae medicinal herbs.
Discussion
The observed diversity aligns with the published research showing that O. sanctum often has a richer endophyte community than related species and that L. aspera harbors several overlapping taxa.
Limitations
- Culturable bias: only plate-amenable fungi were recovered.
- Macromorphology is preliminary; taxa are putative until molecularly confirmed by labs.
- Short incubation may undercount slow-growing taxa.
Next Steps
If given more time to conduct the research, these would be the next steps I would take to advance this project.
- DNA extraction from each isolate; PCR of ITS/TEF1; Sanger sequencing.
- Crude extract preparation and initial antimicrobial assays.
- Host-specific comparison of isolate frequency with larger sample size.
Acknowledgments
I thank the University of Mysore, Department of Biotechnology, for giving me this opportunity and providing lab facilities and mentorship. I personally thank Prof. S. Umesha, for giving this opportunity, and Ravi Kumar, for his guidance throughout the project.
References
Selected works cited