Genetic and chemical methods of ascertaining aflatoxigenicity give discordant results

A Case of Aspergillus species from Eastern Kenya


  • Micah Lagat Egerton University


aflatoxin; non-aflatoxigenic; aflatoxigenicty; Aspergillus section Flavi; Dichlorvos-ammonia


Aflatoxins (AFs) are poisonous compounds produced by species of fungi belonging to the genus Aspergillus mainly A. flavus and A. parasiticus. However, there are some members of these species that do not produce toxins and have since become of interest for use in Biological Control programs. The species that produce AFs are believed to have clusters of aflatoxin-associated genes that confer aflatoxigenicity. However, recent findings suggest that the presence of aflatoxin genes may not imply aflatoxin production capability. We therefore examined the extent to which the presence or absence of aflatoxin-associated genes (genetic) infers aflatoxigenicity using the Dichlorvos-Ammonia (chemical) method as reference.  We report a dissonance between the genetic and chemical methods of ascertaining aflatoxigenicity. Fungi (n = 314) that were morphologically identified as A. flavus and A. parasiticus were isolated from maize kernels and soil from Eastern Kenya. The fungi were further characterized to confirm their identities using a PCRsequence analysis of the internal transcribed spacer (ITS) region of rDNA which confirmed them to be Aspergillus species. We selected and induced 16 isolates in YES media amended with Dichlorvos to produce AFs. Seven of the sixteen isolates were DM-AM positive (aflatoxigenic) but lacked at least one of the key aflatoxin-associated genes. Even more confounding, A. flavus isolate (1EM2606) had all aflatoxin-associated genes but was non-aflatoxigenic (DVAM negative) while A. flavus (1EM1901) having all genes was aflatoxigenic (DV-AM positive). The genetic abundance of the AF-asscociated genes among the isolates was as follows: aflQ (100%), aflD (62.5%) followed by aflR (37.5%) and aflP (25%).   We report that either method alone is limited in ascertaining aflatoxigenicity among Aspergillus section Flavi. The discordance between presence of aflatoxinassociated genes and aflatoxigenicity may be due to epigenetic factors that affect secondary metabolism, or even other signaling processes that alter toxin production that still remain poorly understood.


Varga J, Frisvad JC, Samson R: Two new aflatoxin producing species, and an overview of Aspergillus section Flavi. Studies in Mycology 2011, 69:57-80.

Salano EN, Obonyo MA, Toroitich FJ, Odhiambo B, Omondi , Aman B, Omondi: Diversity of putatively toxigenic Aspergillus species in maize and soil samples in an aflatoxicosis hotspot in Eastern Kenya. African Journal of Microbiology Research 2016, 10(6):172-184.

Afzal H, Shazad S, Qamar S, Nisa S: Morphological identification of Aspergillus species from the soil of Larkana District (Sindh, Pakistan). Asian J Agric Sci 2013, 1(105):e17.

Okoth S, De Boevre M, Vidal A, Diana Di Mavungu J, Landschoot S, Kyallo M, Njuguna J, Harvey J, De Saeger S: Genetic and Toxigenic Variability within Aspergillus flavus Population Isolated from Maize in Two Diverse Environments in Kenya. Frontiers in Microbiology 2018, 9(57).

Cotty PJ, Antilla L, Wakelyn PJ: Competitive exclusion of aflatoxin producers: farmer driven research and development. Biological control: a global perspective 2007:241-253.

Lagat MK, Toroitich FJ, Obonyo MA: Development of an ELISA-based method for testing aflatoxigenicity and aflatoxigenic variability among Aspergillus species in culture. Scientific African 2020, 7:e00266.

Lagat MK, Toroitich FJ, Obonyo AM, Makonde HM: Modulation of Aflatoxin Production by Interaction of Aspergillus Species from Eastern Kenya. Journal of Environmental Science, Toxicology and Food Technology 2019.

Dawlatana M, Shahida S, Rahim M, Hassan MT: Investigation on the occurrence of ochratoxin A in maize in Bangladesh. Bangladesh Journal of Scientific and Industrial Research 2008, 43(4):495-500.

Fakruddin M, Chowdhury A, Hossain MN, Ahmed MM: Characterization of aflatoxin producing Aspergillus flavus from food and feed samples. SpringerPlus 2015, 4(1):159.

Gallo A, Stea G, Battilani P, Logrieco AF, Perrone G: Molecular characterization of an Aspergillus flavus population isolated from maize during the first outbreak of aflatoxin contamination in Italy. Phytopathologia Mediterranea 2012, 51(1):198-206.

Oloo RD, Okoth S, Wachira P, Mutiga S, Ochieng P, Kago L, Nganga F, Domelevo Entfellner J-B, Ghimire S: Genetic profiling of aspergillus isolates with varying aflatoxin production potential from different maize-growing regions of kenya. Toxins 2019, 11(8):467.

Jamali M, Karimipour M, Shams-Ghahfarokhi M, Amani A, Razzaghi-Abyaneh M: Expression of aflatoxin genes aflO (omtB) and aflQ (ordA) differentiates levels of aflatoxin production by Aspergillus flavus strains from soils of pistachio orchards. Research in microbiology 2013, 164(4):293-299.

Bhatnagar D, Cary JW, Ehrlich K, Yu J, Cleveland TE: Understanding the genetics of regulation of aflatoxin production and Aspergillus flavus development. Mycopathologia 2006, 162(3):155-166.

Chiou C-H, Miller M, Wilson DL, Trail F, Linz JE: Chromosomal location plays a role in regulation of aflatoxin gene expression in Aspergillus parasiticus. Applied and environmental microbiology 2002, 68(1):306-315.

Chang P-K, Horn BW, Dorner JW: Sequence breakpoints in the aflatoxin biosynthesis gene cluster and flanking regions in nonaflatoxigenic Aspergillus flavus isolates. Fungal Genetics and Biology 2005, 42(11):914-923.

Chang P-K, Horn BW, Dorner JW: Clustered genes involved in cyclopiazonic acid production are next to the aflatoxin biosynthesis gene cluster in Aspergillus flavus. Fungal Genetics and Biology 2009, 46(2):176-182.

Yu J, Chang P-K, Bhatnagar D, Cleveland TE: Cloning of a sugar utilization gene cluster in Aspergillus parasiticus. Biochimica et Biophysica Acta (BBA)-Gene Structure and Expression 2000, 1493(1-2):211-214.

Yu J, Chang P-K, Ehrlich KC, Cary JW, Montalbano B, Dyer JM, Bhatnagar D, Cleveland TE: Characterization of the Critical Amino Acids of anAspergillus parasiticus Cytochrome P-450 Monooxygenase Encoded by ordA That Is Involved in the Biosynthesis of Aflatoxins B1, G1, B2, and G2. Applied and environmental microbiology 1998, 64(12):4834-4841.

Trail F, Chang P-K, Cary J, Linz JE: Structural and functional analysis of the nor-1 gene involved in the biosynthesis of aflatoxins by Aspergillus parasiticus. Applied and environmental microbiology 1994, 60(11):4078-4085.

Yu J, Chang P-K, Payne GA, Cary JW, Bhatnagar D, Cleveland TE: Comparison of the omtA genes encoding O-methyltransferases involved in aflatoxin biosynthesis from Aspergillus parasiticus and A. flavus. Gene 1995, 163(1):121-125.

Price MS, Yu J, Nierman WC, Kim HS, Pritchard B, Jacobus CA, Bhatnagar D, Cleveland TE, Payne GA: The aflatoxin pathway regulator AflR induces gene transcription inside and outside of the aflatoxin biosynthetic cluster. FEMS Microbiology Letters 2006, 255(2):275-279.

Rodrigues P, Venâncio A, Kozakiewicz Z, Lima N: A polyphasic approach to the identification of aflatoxigenic and non-aflatoxigenic strains of Aspergillus section Flavi isolated from Portuguese almonds. International journal of food microbiology 2009, 129(2):187-193.

Sweeney MJ, Pàmies P, Dobson AD: The use of reverse transcription-polymerase chain reaction (RT-PCR) for monitoring aflatoxin production in Aspergillus parasiticus 439. International Journal of Food Microbiology 2000, 56(1):97-103.

Huang C, Jha A, Sweany R, DeRobertis C, Damann KE: Intraspecific aflatoxin inhibition in Aspergillus flavus is thigmoregulated, independent of vegetative compatibility group and is strain dependent. PLoS One 2011, 6(8).

Kushiro M, Hatabayashi H, Yabe K, Loladze A: Detection of Aflatoxigenic and Atoxigenic Mexican Aspergillus Strains by the Dichlorvos–Ammonia (DV–AM) Method. Toxins 2018, 10(7):263.

Mahmoud MA: Detection of Aspergillus flavus in stored peanuts using real-time PCR and the expression of aflatoxin genes in toxigenic and atoxigenic A. flavus isolates. Foodborne pathogens and disease 2015, 12(4):289-296.

Adhikari BN, Bandyopadhyay R, Cotty PJ: Degeneration of aflatoxin gene clusters in Aspergillus flavus from Africa and North America. AMB Express 2016, 6(1):62.

Levin RE: PCR detection of aflatoxin producing fungi and its limitations. International Journal of Food Microbiology 2012, 156(1):1-6.

Obonyo MA, Salano EN: Perennial and seasonal contamination of maize by aflatoxins in eastern Kenya. International Journal of Food Contamination 2018, 5(1).

Klich MA: Identification of common Aspergillus species. Centraalbureau voor Schimmelcultures 2002.



How to Cite

Lagat, M. (2022) “Genetic and chemical methods of ascertaining aflatoxigenicity give discordant results: A Case of Aspergillus species from Eastern Kenya”, Egerton University International Conference. Available at: (Accessed: 4 February 2023).



Health Systems, Science and Technology