Ethnobotanical Survey and Characterization of Antimicrobial Producing Fungal Endophytes from Selected Medicinal Plants Found in Cherangani Forest, Elgeyo-Marakwet County, Kenya
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Infectious diseases remain a global health challenge, and contemporary medicine has been used for a long time to treat them. Apart from contemporary medicine, medicinal plants have been used repeatedly to treat some of these diseases. Due to these plants' overuse, the existence of these plants are threatened hence a need for conservation. Medicinal plants are host to fungal endophytes, which produce bioactive metabolites with antimicrobial activity. Therefore, fungal endophytes in these medicinal plants can be utilized since the intimate association with the plants may allow them to have similar antimicrobial properties. Owing to the importance of fungal endophytes, this study aimed to document medicinal plants in Cherangani forest, isolate and characterize antimicrobial fungal endophytes, and identify their bioactive metabolites. Structured questionnaires were used to gather information on uses of medicinal plants in Cherangani forest. It is from the questionnaires that two medicinal plants, namely; Zehneria scabra and Croton macrostachyrus were selected for fungal endophytes isolation. The leaves, roots and bark of the two plants were randomly collected and placed in polythene bags before transporting to Kenyatta University microbiology laboratories. Each plant's leaves, roots, barks, and stems were surface sterilized and plated on PDA at 25°C for five days. The fungal endophytes were selected based on morphology and fermented in PDA broth culture. The isolates' crude extracts were screened for antimicrobial activity against the test organisms; Bacillus subtilis ATCC 23857, Staphylococcus aureus ATCC 977, E. coli ATCC 35218 and Candida albicans ATCC 14053 using well and disk diffusion methods. Isolates with antimicrobial activity were identified based on morphology and sequencing the ITS region using ITS1F and ITS4R primers. To identify the bioactive compounds in the fungal extracts, ethyl acetate was used to extract the active compound from the isolate extracts with broad-spectrum antimicrobial activity. GC-MS analysis was used to profile the compounds present in active isolate extracts. A total of 26 medicinal plants were documented in Cherangani forest. A total of 172 isolates were isolated from the two plants and placed into 30 morphological groups. Out of the 30 groups, 18 had antimicrobial activity. These isolates were from genera; Fusarium, Chaetomium, Clonostachys, Meyerozyma, Aporospora, Lasiodiplodia, Acremonium, Penicillium, Aspergillus, Leptosphaeria, Phyllosticta and Ampelomyces. Antimicrobial bioassays showed 94.44% of the isolates showed activity against Bacillus subtilis, 63.1% against Staphylococcus aureus and 31.6% against Escherichia coli in disk diffusion method. However, none of the isolates had activity against Candida albicans. Antimicrobial bioassays from well diffusion method showed 83.33% of the isolates had activity against Bacillus subtilis, 61.11% activity against Staphylococcus aureus, and 38.89% had activity against Escherichia coli. The most active isolates in disk diffusion were; MR26, MR28 and MR29, with mean of inhibition ranging from 10mm to 18mm while in well diffusion isolates MR22, MR26, MR28, and MR29 were the most active isolates with mean zones of inhibition ranging from 9mm to 19mm. Analysis of variance showed a significant difference between the test organisms at p = 0.0001 in disk and well diffusion methods. There was a significant difference between the fungal extracts at p = 0.0001 in disk and well diffusion methods. In addition, there was a significant difference in interaction between the fungal extracts and the test organisms at p = 0.0001 in well and disk diffusion methods. Generally, the highest antibacterial activity was from isolate MR26 from Croton macrostachyrus bark, MR29 and MR22 from Zehneria scabra root, and MR28 from Zehneria scabra stem. The GC-MS analysis of the extracts revealed bioactive metabolites in chemical classes, monoterpenoids, carboxylic acid and long-chain fatty acids. This study has shown that medicinal plants are reservoir to fungal endophytes, which could be exploited as a source of natural products of pharmaceutical importance.