Antimicrobial Activity of Endophytic Fungi Isolated from Warburgia Ugandensis against Candida albicans

Abstract

Candida albicans is a dimorphic fungus which is part of normal microbial flora of an immunocompetent individual but becomes pathogenic in immunocompromised individuals causing infections such as candidiasis. Management of Candida albicans infections is by use of antifungals which are becoming increasingly resistant hence bioactive compounds from fungal endophytes can be of pharmacological value. Endophytes are microorganisms living in cells and tissues of plants without causing diseases. Both the endophyte and the host plant experience a symbiotic mode of nutrition where by the endophytes get shelter and nourishment from the host plant and in return they increase the ability of the host plant nutrients uptake and disease resistance. This study aimed at isolation of fungal endophytes from medicinal plant Warburgia ugandensis from Mt Kenya forest and determination of antimicrobial activity of their extracts against Candida albicans. Sterilized plant materials were incubated on tap water agar for six days at 27 ºC. Fungal positives were transferred to potato dextrose agar (PDA) plates followed by 4 days’ incubation at the same temperature. Colony purification gave pure cultures which were subjected to macroscopic, microscopic and sequencing methods of identification. Extraction of bioactive compounds was done by use of organic solvent ethyl acetate. Extracts were subjected to preliminary toxicity testing using brine shrimp eggs. LC50 values were calculated using regression equations from graphs of percentage mortality of brine shrimps versus log10 of the fungal extracts generated using Microsoft Excel. Preliminary phytochemical screening of the extracts was done. Antimicrobial activity of the extracts was determined using disk diffusion method according to CLSI protocols. Seventeen endophytic fungi were isolated and identified up to molecular level as; Nigrospora oryzae, Aspergillus flavus, Cladosporium spp. (two), Fusarium oxysporum, Phomopsis spp.(two), Colletotrichum acutatum, Altanaria spp. (two), Cochliobolus sativus, Bionectria ochroleuca, Phyllosticta gardeniicola, Guignardia mangiferae, Tricharina gilva, Diaporthe amygdali and Trichoderma harzianum. All the seventeen fungal endophytes had DNA base pairs ranging from 1500-2000bp. All the fungal extracts had LC50 values ˃1000 µg/ml (greater than 1000 µg/ml) when tested for toxicity with brine shrimps hence nontoxic. Colletotrichum acutatum had the lowest LC50 value (2040 µg/ml) while Phyllosticta gardeniicola had the highest LC50 value (8500 µg/ml). Average LC50 value for all the extracts was 4451.8µg/ml. Phytochemical screening of the fungal extracts showed absence of phenols and anthraquinones in all the extracts; presence of saponins, tannins, alkaloids, flavonoids, sterols and glycosides in most of the extracts. Extracts of Phomopsis mali and Alternaria alternata showed activity against Candida albicans with 2.5 mm average diameter zone of inhibition; the rest of the extracts didn’t show any activity against the test organisms. This study shows that fungal endophytes can be a potential source of metabolites which can be useful in pharmaceutical industry.