Phylogenetics of aflatoxigenic moulds and prevalence of aflatoxin from in-process wheat and flour from selected major stores within northern Nigeria

Abstract

Aflatoxigenic strains that produce aflatoxins may be similar morphologically but vary genetically. Sequenced aflR-1 gene from this work was used to study relatedness of aflatoxigenic strains. Yeast extract sucrose agar (YESA) supplemented with 0.3% cyclodextrin and 0.6 % sodium Desoxycholate (YCSD) was used to characterize aflatoxigenic moulds. Total aflatoxin content of the samples was determined using Enzyme Link immunosorbent assay (ELISA). Multiplex PCR was carried out on aflatoxigenic and some non aflatoxigenic moulds using the genes; aflR-1, omt-A, ver-1 and nor-1. The aflR-1 PCR products were sequenced and used for Basic Alignment Search Tool (BLAST) and to generate dendogram. While the raw wheat samples presented highest total aflatoxin range 0.6 – 49 μg/kg, major stores presented values of 0.50 – 28 μg/kg, higher than that obtained in-process samples (0.70 – 26 μg/kg). However, there was no significant differences (p≥0.05) between the major store total aflatoxin levels and that of in-process samples. Thirty seven (37 %) and 25 % of the wheat flour samples from in-process and major stores respectively exceeded the 10 μg/kg National Agency for Food and Drug Administration and Control, Nigeria (NAFDAC) recommended limit for this product. Thirty six (36 %) and 21 % prevalence of wheat and wheat flour samples respectively analyzed from this work was contaminated with total aflatoxins. The multiplex PCR response from this work has demonstrated that there was consistency in the banding pattern of aflatoxigenic strains with respect to the major aflatoxin biosynthetic genes from this study. BLAST of the assembled AFL2T gene sequences was found to have compatibility with that of standard aflR-1 of NCBI Gene Bank that also created way of identifying the isolates. The bootstrap similarity matrix differentiated the isolates into two major clusters; sub-group and others assembled together indicating closer relatedness or general similarity. Some of the isolates varied greatly with visible mixtures of aflatoxigenic and non aflatoxigenic moulds and also random distribution of in-process and store isolates at different leaf nodes within the major clusters. Awareness and surveillance of aflatoxin levels by processors and regulatory bodies at raw material intake is advocated.

Keywords: Moulds, Aflatoxin, Gene, Dendogram