Occurrence, Molecular Characterization and Phylogenetic Relationship of Aspergillus Species Isolated from Garri Sold in Benue State, North Central, Nigeria
Characterization and identification of molds based on cultural and morphological characteristics are often not reliable and frail with limitations. The occurrence of Aspergillus species in garri on sale in markets in Benue State, Nigeria, was studied by molecular techniques. Aspergillus species were isolated and purified on Potato Dextrose Agar. DNA from the purified isolates was extracted using the ZR fungal DNA miniprep and amplified by PCR mix made up of 12.5 μL of Taq 2X Master Mix. Primer sequences for the fungi characterization were internal transcribed spacers ITS 4 and ITS 5. The phylogenetic tree was plotted between the isolated organisms and reference sequences and evolutionary analysis was conducted in MEGA X. Result revealed that one thousand, six hundred and forty-six Aspergilli were isolated comprising of 980 and 666 isolates from the white and yellow garri respectively. Aspergillusflavus, A. fumigatus, A. niger, A. aculeatinus, and A. aculeatus were identified. Twenty percent (20%) of the strains had aflatoxin D structural gene, 50% amplified AFLP and 70% of the strains expressed AFLQ genes needed for the biosynthesis of aflatoxin B1. Majority of the strains that showed the expression of these structural genes were consistent with Aspergillus flavus. Phylogenetic tree showed a close relationship among the isolates and their most identical sequence in the NCBI database.
References
[1]
Klich, M.A., Tiffany, L.H. and Knaphus, G. (1992) Ecology of the Aspegilli of Soils and Litter. In: Benneth, J.W. and Klich, M.A., Eds., Aspergillus: Biology and Industrial Applications, Butterworth Heineman, Boston, 329-354.
[2]
Klich, M.A. (2002) Biogeography of Aspergillus Species in Soil and Litter. Mycolgia, 94, 21-27. https://doi.org/10.2307/3761842
[3]
Perrone, G., Susca, A., Cozzi, G., Ehrlich, K., Varga, J., Frisvad, J.C., Meijer, M., Noonim, P., Mahakarnchanakul, W. and Samson, R.A. (2007) Biodiversity of Aspergillus Species in Some Important Agricultural Products. Studies in Mycology, 59, 53-66. https://doi.org/10.3114/sim.2007.59.07
[4]
Klich, M.A. (2009) Health Effects of Aspergillus in Food and Air. Toxicology and Industrial Health, 25, 657-667. https://doi.org/10.1177/0748233709348271
[5]
Pitt, J.I. and Hocking, A.D. (2009) Fungi and Food Spoilage. Springer, Boston.
[6]
Kullberg, B.J. and Oude-Lashof, A.M.L. (2002) Epidemiology of Opportunistic Invasive Mycoses. European Journal of Medical Research, 7, 183-191. https://doi.org/10.1007/978-0-387-92207-2
[7]
Walsh, T.J., Anaissie, E.J., Denning, D.W., Herbrecht, R., Kontoyiannis, D.P., Marr, K.A., et al. (2008) Treatment of Aspergillosis: Clinical Practice Guidelines of the Infectious Diseases Society of America. Clinical Infectious Diseases, 46, 327-360. https://doi.org/10.1086/525258
[8]
The Fungal Infection Trust (2011) How Common Are Fungal Diseases? Fungal Research Trust 20th Anniversary Meeting, London, 18 June 2011, Updated December 2012.
[9]
Eaton, D.L. and Gallagher, E.P. (1994) Mechanisms of Aflatoxin Carcinogenesis. Annual Review of Pharmacology and Toxicology, 34, 135-172. https://doi.org/10.1146/annurev.pa.34.040194.001031
[10]
Ellis, W.O., Smith, J.P., Simpson, B.K., Oldham, J.H. and Scott, P.M. (1991) Aflatoxins in Food: Occurrence, Biosynthesis, Effects on Organisms, Detection, and Methods of Control. Critical Reviews in Food Science and Nutrition, 30, 403-439. https://doi.org/10.1080/10408399109527551
[11]
Ogiehor, I.S. and Ikenebomeh, M.J. (2004) Antimicrobial Effects of Sodium Benzoate on the Growth, Survival and Aflatoxin Production Potentials of Some Species of Aspergillus in Garri during Storage. Pakistan Journal of Nutrition, 3, 300-303. https://doi.org/10.3923/pjn.2004.300.303
[12]
Abt Associates, Inc., Aflatoxin Country Assessment for Nigeria (2012) Aflatoxin Contamination and Potential Solutions for Its Control in Nigeria, A Summary of the Country and Economic Assessment. The Aflatoxin Stakeholder Workshop, Dar es Salaam, 3-4 December 2012, 1-34.
[13]
FDA (U.S. Food and Drug Administration) (2009) Compliance Policy Guides: Action Levels for Aflatoxin in Animal Feed.
[14]
Batagarawa, U.S., Dangora, D.B. and Haruna, M. (2015) Aflatoxin Contamination in Some Selected Grains, Feeds and Feed Ingredients in Katsina and Zaria Metropolis. Annals of Experimental Biology, 3, 1-7.
[15]
Roze, L.V., Hong, S.-Y. and Linz, J.E. (2013) Aflatoxin Biosynthesis: Current Frontiers. Annual Review of Food Science and Technology, 4, 293-311. https://doi.org/10.1146/annurev-food-083012-123702
[16]
Sohrabi, N. and Taghizadeh, M. (2018) Molecular Identification of Aflatoxigenic Aspergillus Species in Feedstuff Samples. Current Medical Mycology, 4, 1-6. https://doi.org/10.18502/cmm.4.2.66
[17]
Scherm, B., Palomba, O., Serra, D., Marcello, A. and Migheli, Q. (2005) Detection of Transcripts of the Aflatoxin Genes aflD, aflO, and aflP by Reverse Transcription-Polymerase Chain Reaction Allows Differentiation of Aflatoxin-Producing from Aflatoxin Non-Producing Isolates of Aspergillus flavus and Aspergillus parasiticus. International Journal of Food Microbiology, 98, 201-210. https://doi.org/10.1016/j.ijfoodmicro.2004.06.004
[18]
Rahimi, S., Sohrabi, N., Ebrahimi, M.A., Tebyanian, M., Morteza, Zadeh, T. and Rahimi, S. (2016) Application of PCR in the Detection of Aflatoxinogenic and Nonaflatoxinogenic Strains of Aspergillus flavus Group of Cattle Feed Isolated in Iran. Journal of Molecular Biology Research, 6, 121-128. https://doi.org/10.5539/jmbr.v6n1p121
[19]
Zhou, R. and Linz, J.E. (1999) Enzymatic Function of the Nor-1 Protein in Aflatoxin Biosynthesis in Aspergillus parasiticus. Applied and Environmental Microbiology, 65, 5639-5641. https://doi.org/10.1128/AEM.65.12.5639-5641.1999
[20]
Yu, J., Chang, P.-K., Ehrlich, K.C., Cary, J.W., Bhatnagar, D., Cleveland, T.E., Payne, C.A., Linz, J.E., Woloshuk, C.P. and Bennett, J.W. (2004) Clustered Pathway Genes in Aflatoxin Biosynthesis. Applied and Environmental Microbiology, 70, 1253-1262. https://doi.org/10.1128/AEM.70.3.1253-1262.2004
[21]
Ehrlich, K., Yu, J. and Cotty, P. (2005) Aflatoxin Biosynthesis Gene Clusters and Flanking Regions. Journal of Applied Microbiology, 99, 518-527. https://doi.org/10.1111/j.1365-2672.2005.02637.x
[22]
Cary, J.W., Ehrlich, K.C., Bland, J.M. and Montalbano, B.G. (2006) The Aflatoxin Biosynthesis Cluster Gene, aflX, Encodes an Oxidoreductase Involved in Conversion of Versicolorin A to Demethylsterigmatocystin. Applied and Environmental Microbiology, 72, 1096-1101. https://doi.org/10.1128/AEM.72.2.1096-1101.2006
[23]
de Hong, G., Guarro, J., Gene, J. and Figueras, M. (2000) Atlas of Clinical Fungi. 2nd Edition, Centraalbureau voor Schimmelcultures, Utrecht.
[24]
Balajee, S.A., Nickle, D., Varga, J. and Marr, K.A. (2005) Aspergillus lentulus sp. Nov., a New Sibling Species of A. fumigatus. Eukaryot Cell, 4, 625-632. https://doi.org/10.1128/EC.4.3.625-632.2005
[25]
Schoch, C.L., Seifert, K.A., Huhndorf, S., Robert, V., Spouge, J.L., Levesque, C.A., Chen, W. and Fungal Barcoding Consortium (2012) Nuclear Ribosomal Internal Transcribed Spacer (ITS) Region as a Universal DNA Barcode Marker for Fungi. Proceedings of the National Academy of Sciences of the United States of America, 109, 6241-6246. https://doi.org/10.1073/pnas.1117018109
[26]
Ernesto, M., Cardoso, A.P, Cliff, J. and Bradbury, J.H. (2000) Cyanogens in Cassava Flour and Roots and Urinary Thiocyanate Concentration in Mozambique. Journal of Food Composition and Analysis, 13, 1-12. https://doi.org/10.1006/jfca.1999.0847
[27]
Oluwole, O.B., Olatunji, O.O. and Odunfa, S.F. (2004) A Process Technology for Conversion of Dried Cassava Chips into “Garri”. Nigerian Food Journal, 22, 65-73. https://doi.org/10.4314/nifoj.v22i1.33570
[28]
Asegbeloyin, J.N. and Onyimonyi, A.E. (2007) The Effect of Different Processing Methods on the Residual Cyanides of ‘Gari’. Pakistan Journal of Nutrition, 6, 163-166. https://doi.org/10.3923/pjn.2007.163.166
[29]
Ogiehor, I.S., Ikenebomeh, M.J. and Ekundayo, A.O. (2007) The Bioload and Aflatoxin Content of Market Garri from Some Selected States in Southern Nigeria: public Health Significance. African Health Sciences, 7, 223-227.
[30]
Igbeka, J.C. (1987) Stimulation of Moisture Profile in Stored Garri. Journal of Food and Agriculture, 1, 5-9.
[31]
Ogiehor, I.S., Ekundayo, A.O. and Okwu, G.I. (2005) Shell Stability of Agidi Produced from Maize (Zea mays) and the Effects of Sodium Benzoate Treatment in Combination with Low Temperature Storage. African Journal of Biotechnology, 4, 738-743.
[32]
Ekundayo, C.A. (1984) Microbial Spoilage of Packaged Garri in Storage. Microbiology Letters, 23, 271-278.
[33]
Ogugbue, C.J., Mbakwem-Aniebo, C. and Akubuenyi, F. (2011) Assessment of Microbial Air Contamination of Post Processed Garri on Sale in Markets. African Journal of Food Science, 5, 503-512.
[34]
Amadi, J.E. and Adebola, M.O. (2008) Effect of Moisture Content and Storage Conditions on the Storability of Garri. African Journal of Biotechnology, 7, 4591-4594.
[35]
Aguoru, C.U., Onda, M.A., Omoni, V.T. and Ogbonna, I.O. (2014) Characterization of Moulds Associated with Processed Garri Stored for 40 Days at Ambient Temperature in Makurdi, Nigeria. African Journal of Biotechnology, 13, 673-677. https://doi.org/10.5897/AJB2013.12187
[36]
Lawani, E.U, Alade, T. and Pelessai, G. (2015) The Mycological Content of Ready to Eat Garri in Amassom, Bayelsa State. African Journal of Food Science, 9, 51-58. https://doi.org/10.5897/AJFS2014.1247
[37]
Ogbonna, I.O., Agbowu, B.I. and Agbo, F. (2017) Proximate Composition, Microbiological Safety and Heavy Metal Contaminations of Garri Sold in Benue, North-Central, Nigeria. African Journal of Biotechnology, 16, 1085-1091. https://doi.org/10.5897/AJB2016.15841
[38]
Thomas, B.T., Ogunkanmi, L.A., Iwalokun, B.A. and Agu, G.C. (2017) Molecular Characterization and Strain Typing of Fungal Contaminants of Processed Manihot Esculenta Crantz (“Garri”) in Ogun State, Nigeria. Annals of Health Research, 3, 112-117.
[39]
Tamura, K. and Nei, M. (1993) Estimation of the Number of Nucleotide Substitutions in the Control Region of Mitochondrial DNA in Humans and Chimpanzees. Molecular Biology and Evolution, 10, 512-526.
[40]
Kumar, S., Stecher, G., Li, M., Knyaz, C. and Tamura, K. (2018) MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Molecular Biology and Evolution, 35, 1547-1549. https://doi.org/10.1093/molbev/msy096
[41]
Mofolorunsho, C.K., Iyaji, U.S. and Agboola, K. (2016) Mycoflora and Moisture Content of Garri Sold in Anyigba, Kogi State. British Journal of Applied Science and Technology, 15, 1-5. https://doi.org/10.9734/BJAST/2016/23670
[42]
World Health Organization (WHO) (2018) Food Safety Digest: Aflatoxins. Department of Food Safety and Zoonoses, World Health Organization, Geneva.
[43]
Gautier, M., Normand, A.-C. and Ranque, S. (2016) Previously Unknown Species of Aspergillus. Clinical Microbiology and Infection, 22, 662-669. https://doi.org/10.1016/j.cmi.2016.05.013
[44]
Alastruey-Izquierdo, A., Alcazar-Fuoli, I. and Cuenca-Estrella, M. (2014) Antifungal Susceptibility Profile of Cryptic Species of Aspergillus. Mycopathologia. 178, 427-433. https://doi.org/10.1007/s11046-014-9775-z
