Assessment of Toxigenic Potential and Genetic Relatedness of Aspergillus Flavus Populations From Different Maize Growing Regions in Kenya

Abstract

Maize (Zea mays L.) is a staple food in Kenya, being consumed at 98 Kg/person/year. Aflatoxin, a metabolite of Aspergillus flavus is a common maize contaminant, and Aflatoxin B1 (AFB1) is the most carcinogenic natural compound known. Highly toxigenic strains of Aspergillus flavus have been reported to frequently contaminate maize, causing fatal aflatoxin poisoning in Kenya. This study was carried out to assess genetic relatedness and toxigenic potential of A. flavus isolates (n=72) recovered from maize grains (n=120) sampled from Eastern, Coastal and Western regions of Kenya. Analyses of genetic sequences of internal transcribed spacer (ITS) regions of ribosomal DNA and partial calmodulin were used in assessing genetic relatedness. The isolates were further screened through PCR detection for presence of five aflatoxin structural genes (aflD, aflM, aflO, aflP and aflQ) and two regulatory genes (aflR and aflS). Profiling of aflatoxigenicity was performed through HPLC-FLD quantification of aflatoxin produced in growth medium. Levels of AFB1 contamination of the maize samples were analyzed using ELISA and UPLC-FLD. The ITS gene sequence grouped all isolates as A. flavus whereas Calmodulin gene sequence data placed them in two clades; A. flavus and A. minisclerotigenes. A. flavus was widespread across all regions whereas A. minisclerotigenes was mostly found in Eastern region. The sequences of calmodulin gene exhibited wider genetic variation than those of ITS. All A. minisclerotigenes were highly toxigenic. Aspergillus flavus isolates varied widely in toxigenicity. Aspergillus minisclerotigenes isolates from Eastern region produced more of AFB1 while those from Coastal and Western regions produced more of AFG1. Aspergillus minisclerotigenes isolates produced more total aflatoxins than A. flavus isolates, with an average total aflatoxin production of 41883.9μgkg-1 and 54.4μgkg-1, respectively. Of seven aflatoxin genes tested, only two genes (aflD and aflS) were corelated to aflatoxin production. Maize samples from coastal region were most contaminated with AFB1. Contamination levels of maize in Western and Eastern Kenya were not significantly different. This study lays foundation in formulating aflatoxin mitigation measures in Kenya and contributes to the understanding on molecular basis for the toxigenicity in Aspergillus species.