Endophytic Fungal Biodiversity and Antimicrobial Bioprospecting of Penicillium citrinum KIB4 from Khaya ivorensis Bark




Fungal endophytes, Khaya ivorensis, Antibiotics, Antimicrobial resistance, Penicillium citrinium KIB4


Background: The emergence of pathogens that are resistant to antibiotics has raised serious concerns about public health, forcing the hunt for alternative antibacterial substances. Endophytes, particularly those from unscreened plants, may offer new sources of antibacterial substances. Hence, this research aimed to isolate antibiotic-producing fungi from Khaya ivorensis bark, a medicinal plant widely used in Nigeria.

Methods: The fungal endophytes were isolated following conventional methods using potato dextrose agar (PDA). They were characterized by morpho-microscopical and sequencing of the internal transcribed spacer (ITS) of the fungal rDNA. The phylogenetic tree was drawn following the Neighbour-joining method and the tree was annotated using the Interactive Tree of Life (iTOL) version 6.0. The agar plug assay method was used to select putative antibiotic-producing isolate for further research. The antimicrobial activity of the fungal fermentation product was assessed by agar well diffusion while the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were estimated using the solid agar dilution technique.

Results:Atotal of 12 endophytic fungi were isolated. They were noted to be mostly Aspergillus sp. (11 isolates) while only one isolate represented Penicillium species. Only isolate KIB4 showed good inhibitory activity against several bacterial pathogens tested in the preliminary study within 10 mm to 12 mm inhibition zone diameter (IZD) range. Molecular identification proved that strain KIB4 fell into the genus Penicillium and shared the highest ITS gene sequence similarity with Penicillium citrinum D112 (98.83%), and Penicillium sp. TWJL1743 (98.65%). The fermentation extract of Penicillium citrinium KIB4 showed specific inhibition against Escherichia coli ML475, Staphylococcus aureus LN001, Klebsiella pneumoniae ML602, Bacillus subtilis ML225, and Proteus sp. UR979 with an IZD range of 11mm to 23mm.

Conclusion: We reported for the first time, the antimicrobial efficacy of fermentation extract from endophytic fungi isolated from Khaya ivorensis against several bacterial pathogens. Further fractionation of the crude extract is needed to isolate specific antimicrobial compounds. 


Alam, B., Li, J., Ge, O., Khan, M.A., Gong, J., Mehmood, S., Yuan, Y., and Gong, W. (2021). Endophytic fungi: From symbiosis to secondary metabolite communications or vise versa? Front. P l a n t S c i . 1 2 : 7 9 1 0 3 3 . https://doi.org/10.3389/fpls.2021.791033

Sridhar, K.R. (2019). Diversity, Ecology, and Significance of Fungal Endophytes. In: Jha, S. (eds) Endophytes and Secondary Metabolites. Reference Series in Phytochemistry. Springer, Cham. https://doi.org/10.1007/978.3.319.76900.4

Watts, D., Palombo, E.A., Jaimes, C. A., and Zaferanloo, B. (2023). Endophytes in Agriculture: Potential to Improve Yields and Tolerances of Agricultural Crops. Microorganisms.11(5):1276. https://doi.org/10.3390/microorganisms.110512 76

Atanasov, A.G., Zotchev, S.B., and Dirsch, V.M. (2021). Natural products in drug discovery: advances and opportunities. Nat Rev Drug Discov 20: 2 0 0 – 2 1 6 . https://doi.org/10.1038/s41573.020.00114.z

Pham, J.V., Yilma, M.A., Feliz, A., Majid, M.T., Maffetone, N., Walker, J.R., Kim, E., Cho, H.J., Reynolds, J.M., Song, M.C., Park, S.R., and Yoon, Y.J. (2019). A review of the microbial production of bioactive natural products and biologics. Front. M i c r o b i o l . 1 0 : 1 4 0 4 . https://doi.org/10.3389/fmicb.2019.01404

Newman, D.J., and Cragg, G.M. (2020). Natural Products as Sources of New Drugs over the Nearly Four Decades from 01/1981 to 09/2019. Journal of Natural P r o d u c t s . 8 3 ( 3 ) : 7 7 0 - 8 0 3 . https://doi.org/10.1021/acs.jnatprod.9b01285

Chaudhary, P., Agri, U., Chaudhary, A., Kumar, A., and Kumar, G. (2022). Endophytes and their potential in biotic stress management and crop production. Front. M i c r o b i o l . 1 3 : 9 3 3 0 1 7 . https://doi.org/10.3389/fmicb.2022.933017

Tiwari, P., and Bae, H. (2022). Endophytic Fungi: Key Insights, Emerging Prospects, and Challenges in N a t u r a l P r o d u c t D r u g D i s c o v e r y . Microorganisms.10(2):360. https://doi.org/10.3390/microorganisms.10020360

Burragoni, S.G., and Jeon, J. (2021). Applications of endophytic microbes in agriculture, biotechnology, medicine, and beyond. Microbiological research. 245:126691. https://doi.org/10.1016/j.micres.2020.126691

Abdel-Razek, A.S., El-Naggar, M.E., Allam, A., Morsy, O.M., and Othman, S.I. (2020). Microbial Natural Products in Drug Discovery. Processes. 8(4):470. https://doi.org/10.3390/pr8040470

Mushtaq, H., Ganai, B.A., and Jehangir, A. (2023). Exploring soil bacterial diversity in different microvegetational habitats of Dachigam National Park in Northw e s t e r n H i m a l a y a . S c i R e p 1 3 : 3 0 9 0 . https://doi.org/10.1038/s41598.023.30187.w

Seelbinder, B., Chen, J., and Brunke, S. (2020). Antibiotics create a shift from mutualism to competition in human gut communities with a longer- lasting impact on f u n g i t h a n b a c t e r i a . M i c r o b i o m e 8 : 1 3 3 . https://doi.org/10.1186/s40168.020.00899.6

Malarvizhi, K., Murali, T.S., and Kumaresan, V. (2023). Fungal endophytes of crop plants: diversity, stress tolerance and biocontrol potential. Egypt J Biol Pest C o n t r o l 3 3 : 6 7 . https://doi.org/10.1186/s41938.023.00711.1

M e n g i s t u , A . A . ( 2 0 2 0 ) . E n d o p h y t e s : Colonization, Behaviour, and Their Role in Defense Mechanism. Int J Microbiol. Article number: 6927219. https://doi.org/10.1155/2020.6927219

Wen, J., Okyere, S.K., Wang, S., Wang, J., Xie, L., Ran,Y., and Hu,Y. (2022). Endophytic Fungi:An Effective Alternative Source of Plant-Derived Bioactive Compounds for Pharmacological Studies. Journal of fungi (Basel, S w i t z e r l a n d ) , 8 ( 2 ) : 2 0 5 . https://doi.org/10.3390/jof8020205

Ezeobiora, C.E., Igbokwe, N.H., Amin, D.H., and Mendie, U.E. (2023). Molecular phylogenetics reveals the diversity of antagonistic fungal endophytes inhabiting medicinal plants in Nigeria. Proc Natl Acad Sci, India, Sect B Biol Sci. https://doi.org/10.1007/s40011.023.01495.y

Samapti, M.M.S., Afroz, F., Rony, S.R., Sharmin, S., Moni, F., Akhter, S., Ahmed, S.F.U., and Sohrab, M.H. (2022). Isolation and Identification of Endophytic Fungi from Syzygium cumini Linn and Investigation of Their Pharmacological Activities. Sci World J, 9529665. https://doi.org/10.1155/2022.9529665

Ezeobiora, C.E., Igbokwe, N.H., Amin, D.H., Okpalanwa, C.F., Mota'a, S.C., and Mendie, U.E. (2023). Antibacterial Potential of Endophytic Fungi from Xylopia aethiopica and Metabolites Profiling of Penicillium sp. XAFac2 and Aspergillus sp. XAFac4 by GC-MS. Trop J N a t P r o d R e s . 7 ( 7 ) : 3 5 3 8 - 3 5 4 5 . https://www.doi.org/10.26538/tjnpr.v7i7.37

Senbua, W., and Wichitwechkarn, J. (2019). Molecular identification of fungi colonizing art objects in Thailand and their growth inhibition by local plant extracts. 3 B i o t e c h . 9 ( 1 0 ) : 3 5 6 . https://doi.org/10.1007/s13205.019.1879.1

Hamzah, T., Lee, S., Hidayat, A., Terhem, R., Faridah-Hanum, I., and Mahamed, R. (2018). Diversity and characterization of endophytic fungi isolated from the Tropical Mangrove Species, Rhizophora mucronata and Identification of potential antagonists against the soil-borne fungus, Fusarium solani, Front. Microbiol. 9:1707. https//doi.org/10.3389/fmicb.2018.01707

Sathiyaseelan,A., Saravanakumar, K., Mariadoss, A.V.A., Kim, K.M., and Wang, M.H. (2021). Antibacterial activity of ethyl acetate extract of endophytic fungus (Paraconiothyrium brasiliense) through targeting dihydropteroate synthase (DHPS), Process Biochemistry, 111(2): 27-35 . https://doi.org/10.1016/j.procbio.2021.10.010

David, L. (2021). Preparation of routine media and reagents used in antimicrobial susceptibility testing. Clinical Microbiology Procedures Handbook, 5th Edition. ASM press, Washington, DC. PP. 52011-52014.

Schumacher, A., Vranken, T., Malhotra, A., Arts, J.J.C., and Habibovic, P. (2018). In vitro Antimicrobial Susceptibility Testing Methods: Agar Dilution to 3D Tissue-Engineered Models. Eur J Clin Microbiol. 37(2): 187–208. https://doi.org/10.1007/s10096.017.3089.2

Deshmukh, S.K., Gupta, M.K., Prakash, V., and Sudhakara, R.M. (2018). Mangrove-associated fungi: A novel source of potential anticancer compounds. J. Fungi. 4:101 https://doi.org/10.3390/jof4030101

Rana, K.L., Kour, D., Kaur, T., Devi, R., Negi, C., Yadav, A.N., Singh, K., and Saxena, A.K. (2020). Endophytic fungi from medicinal plants: Biodiversity and biotechnological applications. In: Kumar A., Radhakrishnan E.K., editors. Microbial Endophytes. Woodhead Publishing; Cambridge, UK: pp. 273–305.

Chimbekujwo, I.B. (2021). Potential and conservation of mahogany in Nigerian forest ecosystem. NJB. 34(2):135-162. https://doi.org/10.4314/njbot.v34i2.1

Mao, Z., Zhang, W., Wu, C., Feng, H., Peng, Y., Shahid, H., Cui, Z., Ding, P., and Shan, T. (2021). Diversity and antibacterial activity of fungal endophytes from Eucalyptus exserta. BMC microbiology; 21(1):155. https://doi.org/10.1186/s12866.021.02229.8

Tolulope, R.A., Adeyemi, A.I., Erute, M.A., Abiodun, T.S. (2015). Isolation and screening of endophytic fungi from three plants used in traditional medicine in Nigeria for antimicrobial activity. Int J Green Pharm. 9:58-62. https://doi.org/10.4103/0973.8258.150929

Amiri, N., and Tibuhwa, D.D. (2021). Antimicrobial activities of endophytic fungal crude extracts isolated from cashew tree (Anarcardium occidentale). Tanzania Journal of Sciences. 47(3):1102- 1113. https://doi.org/10.4314/tjs.v47i3.20

Ashish, V., Harshita, T., Swati, S., Priyamvada, G., Nilesh, R., Santosh, K.S., Bhim, P.S., Sombir, R., and Vibhav, G. (2023). Epigenetic manipulation for secondary metabolite activation in endophytic fungi: current progress a n d f u t u r e d i r e c t i o n s . M y c o l o g y . https://doi.org/10.1080/21501203.2023.2241486




How to Cite

Ezeobiora, C. E., Igbokwe, N. H., Adegbolagun, F. J., Okpalanwa, C. F., Mota’a, S. C., & Mendie, U. E. (2023). Endophytic Fungal Biodiversity and Antimicrobial Bioprospecting of Penicillium citrinum KIB4 from Khaya ivorensis Bark: https://doi.org/10.51412/psnnjp.2023.35. The Nigerian Journal of Pharmacy, 57(2), 747–757. Retrieved from https://psnnjp.org/index.php/home/article/view/499