Identification and Prevention of Microbial Contaminants in Musa paradisiaca Tissue Culture


  • Nurul Izzah Hassen Faculty of Bioresources and Food Industry, Universiti Sultan Zainal Abidin, Besut Campus, 22200, Besut, Terengganu, Malaysia
  • Noor Afiza Badaluddin Faculty of Bioresources and Food Industry, Universiti Sultan Zainal Abidin, Besut Campus, 22200, Besut, Terengganu, Malaysia
  • Zakiah Mustapha Faculty of Bioresources and Food Industry, Universiti Sultan Zainal Abidin, Besut Campus, 22200, Besut, Terengganu, Malaysia
  • Dhiya Dalila Zawawi Faculty of Bioresources and Food Industry, Universiti Sultan Zainal Abidin, Besut Campus, 22200, Besut, Terengganu, Malaysia


antibiotics, antifungal, contamination, Musa paradisiaca


Banana (Musa spp.) is an economically vital fruit crop in Malaysia and therefore, micropropagation was applied to meet the market demand for disease-free seedlings. However, microbial contamination is a significant obstacle in micropropagation techniques. In this research, the microorganisms that were present in the banana culture were characterized and the efficacy of antimicrobial and antifungal agents to inhibit contaminants was evaluated. Three bacterial and two fungal isolates were isolated from the contaminated culture. Klebsiella pneumoniae, Klebsiella quasipneumoniae, and Klebsiella variicola were identified by molecular identification based on the 16S rDNA sequence. The gram-staining method confirmed all three bacteria were gram-negative. Oxidase and catalase tests showed the presence of cytochrome oxidase system and catalase enzyme in all bacteria. The bacteria can also hydrolyze starch, ferment sugars, and reduce sulfur from the amylase test. Phenotypic identification of fungi revealed the presence of conidia and hyphae, indicating the presence of Colletotrichum spp. and Aspergillus spp. In fungi characterization, Colletotrichum gloeosporioides and Aspergillus flavus were detected. Chloramphenicol was identified as an effective antibacterial agent from the disc diffusion method. Fluconazole was a potent antifungal agent by screening the sterilizing agents. The findings may potentially lead the way for the implementation of reducing the contamination rate in banana micropropagation.


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Abd. Shukor, A.R., Jamaluddin S.H., Nik Masdek, N.H., Zabedah, M. & Abdullah, H. 2000. Malaysia banana industry- prospects and challengers. In: Proceedings of the International Workshop on the Banana Fusarium Wilt Disease. University of Philippines Publisher, Laguna, Philippines. pp. 32-38.

Acsa, I., Lilly Caroline, B., Philip Njeru, N. & Lucy Wanjiru, N. 2021. Preliminary study on disinfectant susceptibility/resistance profiles of bacteria isolated from slaughtered village free-range chickens in Nairobi, Kenya. International Journal of Microbiology. 2021:1-7. DOI:

Adeniyi, A.G., Ighalo, J.O. & Onifade, D.V. 2019. Production of biochar from elephant grass (Pernisetum purpureum) using an updraft biomass gasifier with retort heating. Biofuels, 12(10):1283-1290. DOI:

Aduramigba-Modupe, A., Ekunsanmi, T.J. & Ojurongbe, T.A. 2017. Identification and elimination of bacterial contaminants from yam tissue cultures. Nigerian Journal of Ecology, 16(1): 75–85.

Agbadje, E.T., Agbidinoukoun, A., Zandjanakou-Tachin, M., Cacaï, G.T. & Ahanhanzo, C. 2021. Mass Production of bananas and plantains (Musa spp.) plantlets through in vitro tissue culture partway: A review. European Journal of Biology and Biotechnology, 2(4): 1-8. DOI:

Alam, M.S., Nahar, S., Chowdhury, M.E.K., Hasan, M.F., Islam, M.A. & Sikdar, B. 2019. Biochemical characterization of bacteria responsible for bacterial black pit disease of lime (Citrus aurantifolia) and their control system. European Journal of Biotechnology and Bioscience, 7(2): 16-22.

Alam, M.W., Malik, A., Rehman, A., Hameed, A., Tahir, U., Sarwar, M., & Shafeeq, T. 2021. First record of Colletotrichum gloeosporioides causing anthracnose of banana in Pakistan. Plant Disease, 105(7):2013. DOI:

Ali, F., Akhtar, N., Shafique, S. & Shafique, S. 2021. Isolation and identification of Aspergilli causing banana fruit rot. Open Journal of Chemistry, 4(1): 8-18. DOI:

Altan, F., Bürün, B. & Sahin, N. 2010. Fungal contaminants observed during micropropagation of Lilium candidum L. and the effect of chemotherapeutic substances applied after sterilization. African Journal of Biotechnology, 9(7): 991-995. DOI:

Anwar, A., Siddiqui, R., Hussain, M.A., Ahmed, D., Shah, M.R. & Khan, N.A. 2018. Silver nanoparticle conjugation affects anti-acanthamoebic activities of amphotericin B, nystatin, and fluconazole. Parasitology research, 117(1): 265-271. DOI:

Arsogah, S., Rathamanalan, S., Harni, S., Sam, K.K., Vivian, T., Alamudin, N.A., Geng, B.A., Kumaran, J.V. & Appalasamy, S. 2018. FSS-novel technique for in vitro propagation of Araceae and Zingiberaceae in Lojing Highlands, Kelantan. IOP Conference Series: Earth and Environmental Science, 549(1): 012036.

Aung, T.M. & Oo, P.P. 2020. Isolation and characterization of Rhizobium from root nodules of Arachis hypogaea L. (Groundnut). Journal of The Myanmar Academy of Arts and Science, 18: 197-210.

Bhuiyan, F., Campos, N.A., Swennen, R. & Carpentier, S. 2020. Characterizing fruit ripening in plantain and Cavendish bananas: A proteomics approach. Journal of Proteomics, 214: 103632. DOI:

Bohra, P., Waman, A.A., Sathyanarayana, B.N., Umesha, K., Anu, S.R., Swetha, H.G. & Gourish, R.K. 2014. Aseptic culture establishment using antibiotics with reference to their efficiency and phytotoxicity in difficult-to-establish native Ney Poovan banana (Musa, AB). Proceedings of the National Academy of Sciences, India Section B: Biological Sciences, 84(2): 257-263. DOI:

Jabatan Pertanian. 2021. Statistik Tanaman (Sub-Sektor Tanaman Makanan). Jabatan Pertanian Semenanjung Malaysia, Malaysia. 107 pp.

Brown, K.B., Hyde, K.D. & Guest, D.J. 1998. Preliminary studies on endophytic fungal communities of Musa acuminata species complex in Hong Kong and Australia. Fungal Diversity, 1: 27–51.

Chen, L.R., Hsiung, T.C., Lin, K.H., Huang, T.B., Huang, M.Y. & Wakana, A. 2017. Supplementary effect of hydrogen peroxide as a pre–disinfectant for sterilizing rhizome bud explants of Zantedeschia aethiopica L. with chlorine dioxide. Journal of the Faculty of Agriculture, Kyushu University, 62(1): 81-86. DOI:

Dangariya, M., Khandhar, D., Monpara, J., Chudasama, K. & Thaker, V. 2020. Detection and identification of microbial contaminants from plant tissue culture. Tropical Plant Research, 7(2): 388-395. DOI:

De Langhe, E., Vrydaghs, L., De Maret, P., Perrier, X. & Denham, T. 2009. Why bananas matter: An introduction to the history of banana domestication. Ethnobotany Research and Applications, 7: 165-177. DOI:

De Nardo, P., Giancola, M.L., Noto, S., Gentilotti, E., Ghirga, P., Tommasi, C. & Corpolongo, A. 2013. Left thigh phlegmon caused by Nocardia farcinica identified by 16S rRNA sequencing in a patient with leprosy: A case report. BMC Infectious Diseases, 13(1): 1-5. DOI:

De Silva, D.D., Crous, P.W., Ades, P.K., Hyde, K.D. & Taylor, P.W. 2017. Life styles of Colletotrichum species and implications for plant biosecurity. Fungal Biology Reviews, 31(3): 155-168. DOI:

Eunice, M. K., Anne, W. M., Johnstone, O. N., & Cecilia, M. M. 2019. Screening of fungal contaminants in banana tissue cultures in Jkuat, Kenya. African Journal of Microbiology Research, 13(29), 675-688.

Fang, J.Y. & Hsu, Y.R. 2012. Molecular identification and antibiotic control of endophytic bacterial contaminants from micropropagated Aglaonema cultures. Plant Cell, Tissue and Organ Culture, 110(1): 53-62. DOI:

Getnet, G. 2017. Review on in vitro propagation of sugarcane to advance the value of tissue culture. Agricultural Research and Technology, 5(4): 555670. DOI:

Hamill, S. & Rames, E. 2018. An effective indexing method for banana tissue culture provides long-term freedom from bacterial contamination. Acta Horticulturae, 1205: 741-748. DOI:

Hodkinson, T.R. & Murphy, B. 2019. Endophytes for a growing world. In: Endophytes for A Growing World T. Hodkinson, F. Doohan, M. Saunders (Eds.). Cambridge University Pres, Cambridge.pp. 3-22. DOI:

Islam, M.S., Sultana, R., Hasan, M.A., Alam, M.S., Sikdar, B., Kamaruzzaman, M. & Islam, M.A. 2020a. Characterization and biocontrol measures of Pseudomonas syringae pv. syringae associated with citrus blast disease. Vegetos, 33(3): 555-569. DOI:

Islam, R., Hossain, M.N., Alam, M.K., Uddin, M.E., Rony, M.H., Imran, M.A.S. & Alam, M.F. 2020b. Antibacterial activity of lactic acid bacteria and extraction of bacteriocin protein. Advances in Bioscience and Biotechnology, 11(2): 49-59. DOI:

Jabatan Pertanian. 2020. Statistik Tanaman Buah-Buahan, Jabatan Pertanian Semenanjung Malaysia, Malaysia. pp 180.

Kano, K., Shirai, O., Kitazumi, Y., Sakai, K. & Xia, H.Q. 2021. Enzymatic Bioelectrocatalysis. Springer, Singapore. 23–55 pp. DOI:

Kapadia, C. & Patel, N. 2021. Sequential sterilization of banana (Musa Spp.) sucker tip reducing microbial contamination with highest establishment percentage. Bangladesh Journal of Botany, 50(4): 1151-1158. DOI:

Khandaker, M.M., Nurnaeimah, N., Mat, N., Suryati Mohd, K., Badaluddin, N.A., Yusoff, N. & Sajili, M.H. 2020. The effects of hydrogen peroxide on plant growth, mineral accumulation, as well as biological and chemical properties of Ficus deltoidea. Agronomy, 10(4): 599. DOI:

Khasabulli, B.D., Musyimi, D.M., Miruka, D.M., Opande, G.T. & Jeruto, P. 2017. Isolation and characterisation of Ralstonia solanacearum strains of tomato wilt disease from Maseno, Kenya. Journal of Asian Scientific Research, 7(9): 404-420. DOI:

Kim, D.H., Gopal, J. & Sivanesan, I. 2017. Nanomaterials in plant tissue culture: The disclosed and undisclosed. RSC Advances, 7(58): 36492-36505. DOI:

Kones, C., Mwajita, M., Kariuki, L., Kiirika, L. & Kavoo, A. 2020. Isolation and characterization of rhizospheric microorganisms from bacterial wilt endemic areas in Kenya. African Journal of Microbiology Research, 14(7): 349-360.

Liang, C., Wu, R., Han, Y., Wan, T. & Cai, Y. 2019. Optimizing suitable antibiotics for bacterium control in micropropagation of cherry rootstock using a modified leaf disk diffusion method and E test. Plants, 8(3): 66. DOI:

Liu, T.H.A., Hsu, N.W. & Wu, R.Y. 2005. Control of leaf-tip necrosis of micropropagated ornamental statice by elimination of endophytic bacteria. In Vitro Cellular & Developmental Biology-Plant, 41(4): 546-549. DOI:

Liu, Y., Zhu, A., Tan, H., Cao, L. & Zhang, R. 2019. Engineering banana endosphere microbiome to improve Fusarium wilt resistance in banana. Microbiome, 7(1): 1-15. DOI:

Long, S.W., Linson, S.E., Ojeda Saavedra, M., Cantu, C., Davis, J.J., Brettin, T., & Olsen, R.J. 2017. Whole-genome sequencing of human clinical Klebsiella pneumoniae isolates reveals misidentification and misunderstandings of Klebsiella pneumoniae, Klebsiella variicola, and Klebsiella quasipneumoniae. Msphere, 2(4):e00290-17. DOI:

Mahmoud, S.N. & Al-Ani, N.K. 2016. Effect of different sterilization methods on contamination and viability of nodal segments of Cestrum nocturnum L. International Journal of Research Studies in Biosciences, 4(1): 4-9. DOI:

Maskey, C. 2018. Extraction of amylase from Bacillus subtilis isolated from soil of Dharan, Sunsari (Bachelor Degree). Central Campus of Technology, Nepal.

McEvoy, B. & Rowan, N.J. 2019. Terminal sterilization of medical devices using vaporized hydrogen peroxide: A review of current methods and emerging opportunities. Journal of Applied Microbiology, 127(5): 1403-1420. DOI:

Mizra, M.S., Ahmad, W., Latif, F., Haurat, J., Bally, R., Normand, P. & Malik, K.A. 2001. Isolation, partial characterization, and the effect of plant growth-promoting bacteria (PGPB) on micropropagated sugarcane in vitro. Plant and Soil. 237:47–54. DOI:

Msogoya, T.J., Kanyagha, H., Mutigitu, J., Kulebelwa, M., & Mamiro, D. 2012. Identification and management of microbial contaminants of banana in vitro cultures. Journal of Applied Biosciences, 55: 3987-3994.

Murashige, T. & Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiology Plant, 15: 473–497 DOI:

Nsofor, G.C. 2021. Conventional methods of controlling microbial contaminants in meristematic tissue cultures: A review. Nigeria Agricultural Journal, 52(2): 181-186.

Obuekwe, C.O. & Osagie, I.J. 1989. Morphological changes in infected wilt resistant and wilt-susceptible oil palm progenies and hydrolytic enzyme activities associated with Fursarium oxysporum f. sp. elaeidis pathogens. Oléagineux, 44(11): 8-9.

Omemu, A.M., Okafor, U.I., Obadina, A.O., Bankole, M.O. & Adeyeye, S.A.O. 2018. Microbiological assessment of maize ogi cofermented with pigeon pea. Food Science & Nutrition, 6(5): 1238-1253. DOI:

Orlikowska, T., Nowak, K. & Reed, B. 2017. Bacteria in the plant tissue culture environment. Plant Cell, Tissue and Organ Culture, 128(3): 487-508. DOI:

Prastujati, A.U., Setiadevi, S., Hilmi, M. & Khusna, A. 2019. Isolation and identification of Acetobacter sp. from pineapple (Ananas comosus L.) as nata starter. Journal of World’s Poultry Research, 9(6): 260–264. DOI:

Ray, S.S. & Ali, N. 2018. Biotic contamination and possible ways of sterilization: A review with reference to bamboo micropropagation. Brazilian Archives of Biology and Technology, 59: 1-12. DOI:

Reed, B.M., Buckley, P.M. & DeWilde, T.N. 1995. Detection and eradication of endophytic bacteria from micropropagated mint plants. In Vitro Cellular & Developmental Biology-Plant, 31(1): 53-57. DOI:

Riera, N., Ramirez-Villacis, D., Barriga-Medina, N., Alvarez-Santana, J., Herrera, K., Ruales, C. & Leon-Reyes, A. 2019. First report of banana anthracnose caused by Colletotrichum gloeosporioides in Ecuador. Plant Disease, 103(4): 763-763. DOI:

Rodrigues, C., Passet, V., Rakotondrasoa, A. & Brisse, S. 2018. Identification of Klebsiella pneumoniae, Klebsiella quasipneumoniae, Klebsiella variicola and related phylogroups by MALDI-TOF mass spectrometry. Frontiers in Microbiology, 9: 3000. DOI:

Rodriguez, R.J., White Jr, J.F., Arnold, A.E. & Redman, A.R.A. 2009. Fungal endophytes: Diversity and functional roles. New phytologist, 182(2): 314-330. DOI:

Romero-Soto, I., Dia, O., Leyva-Soto, L.A., Drogui, P., Buelna, G., Ulloa-Mercado, R.G. & Gortáres-Moroyoqui, P. 2018. Degradation of chloramphenicol in synthetic and aquaculture wastewater using electrooxidation. Journal of Environment Quality, 47(4): 805-811. DOI:

Sanderson, T.H., Wider, J.M., Lee, I., Reynolds, C.A., Liu, J., Lepore, B. & Hüttemann, M. 2018. Inhibitory modulation of cytochrome c oxidase activity with specific near-infrared light wavelengths attenuates brain ischemia/reperfusion injury. Scientific Reports, 8(1): 1-12. DOI:

Saraswathi, M.S., Uma, S., Kannan, G., Selvasumathi, M., Mustaffa, M.M., & Backiyarani, S. 2016. Cost-effective tissue culture media for large-scale propagation of three commercial banana (Musa spp.) varieties. The Journal of Horticultural Science and Biotechnology, 91(1): 23-29. DOI:

Seliem, M.K., El-Mahrouk, M.E., El-Banna, A.N., Hafez, Y.M. & Dewir, Y.H. 2021. Micropropagation of Philodendron selloum: Influence of copper sulfate on endophytic bacterial contamination, antioxidant enzyme activity, electrolyte leakage, and plant survival. South African Journal of Botany, 139: 230-240. DOI:

Sharma, V., Bhardwaj, B., Pundir, R.K. & Tiwari, S. 2017. Identification and molecular characterization of endophytic bacterial isolates from Musa rhizomes. Journal of Electronics and Communication Engineering, 12(6): 1–5.

Sharma, P. & Singh, S.P. 2021. Role of the endogenous fungal metabolites in the plant growth improvement and stress tolerance. In: Fungi Bio-Prospects in Sustainable Agriculture, Environment and Nano-Technology. V.K. Sharma, M.P. Shah, S. Parmar & A. Kumar (Eds). Academic Press, London. pp. 381-401. DOI:

Shehata, A.M., Wannarat, W., Skirvin, R.M. & Norton, M.A. 2010. The dual role of carbenicillin in shoot regeneration and somatic embryogenesis of horseradish (Armoracia rusticana) in vitro. Plant Cell, Tissue and Organ Culture, 102(3): 397-402. DOI:

Shields, P. & Cathcart, L. 2010. Oxidase Test Protocol. American Society for Microbiology, Washington, DC. 9 pp.

Suman, S. 2017. Plant tissue culture: A promising tool of quality material production with special reference to micropropagation of banana. Biochemical and Cellular Archives, 17(1): 1-26.

Tekielska, D., Peňázová, E., Kovács, T., Křižan, B., Čechová, J., & Eichmeier, A. 2019. Bacterial contamination of plant in vitro cultures in commercial production detected by high-throughput amplicon sequencing. Acta Universitatis Agriculturae et Silviculturae Mendelianae Brunensis, 67(4): 1005-1014. DOI:

Tereshchenkov, A.G., Dobosz-Bartoszek, M., Osterman, I.A., Marks, J., Sergeeva, V.A., Kasatsky, P. & Polikanov, Y.S. 2018. Binding and action of amino acid analogs of chloramphenicol upon the bacterial ribosome. Journal of Molecular Biology, 430(6): 842-852. DOI:

Tevyashova, A.N. 2021. Recent trends in synthesis of chloramphenicol new derivatives. Antibiotics, 10(4): 370. DOI:

Tripathi, N. & Sapra, A. 2022. Gram Staining [WWW Document]. StatPearls. URL (accessed 04.10.2022).

Wikström, M., Krab, K. & Sharma, V. 2018. Oxygen activation and energy conservation by cytochrome c oxidase. Chemical Reviews, 118(5): 2469-2490. DOI:

Wilber, P., Bentz, K., Fitzgerald, H., & Peterson, A. 2018. Unit 8: Oxygen utilization, streak isolation and introduction to bacterial identification [WWW Document]. Docsbay. URL (accessed 8.2.2022).

Yoo, J.H. 2018. Review of disinfection and sterilization–back to the basics. Infection & chemotherapy, 50(2): 101-109. DOI:

Zakaria, L. & Aziz, W.N.W. 2018. Molecular identification of endophytic fungi from banana leaves (Musa spp.). Tropical Life Sciences Research, 29(2): 201. DOI:

Zhou, S.N., Cao, L.X. & You, J.L. 2002. Endophytic fungi from Musa acuminata leaves and roots in South China. World Journal of Microbiology & Biotechnology, 18: 169–171.

Ziv, M. 2005. Simple bioreactors for mass propagation of plants. In: Liquid Culture Systems for in Vitro plant propagation. A.K. Hvoslef-Eide & W. Preil (Eds.). Springer, Dordrecht. pp. 79-93. DOI:



How to Cite

Hassen, N. I., Badaluddin, N. A., Mustapha, Z., & Zawawi, D. D. . (2022). Identification and Prevention of Microbial Contaminants in Musa paradisiaca Tissue Culture. Malaysian Applied Biology, 51(5), 129–143.