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Morphological and Molecular Characterization of Toxigenic Aspergillus flavus from Groundnut Kernels in Kenya.

ABSTRACT: Pathogenesis of Aspergillus flavus on important agricultural products is a key concern on human health due to the synthesis and secretion of the hazardous secondary metabolite, aflatoxin. This study identified and further characterized aflatoxigenic A. flavus from groundnuts sampled from sundry shops in Kenya using integrated morphological and molecular approaches. The groundnuts were plated on potato dextrose agar for isolation and morphological observation of A. flavus based on macroscopic and microscopic features. Molecular characterization was done through amplification and comparison of the partial sequence of the ITS1-5.8S-ITS2 region. The expression analysis of aflR, aflS, aflD, aflP, and aflQ genes in the aflatoxin biosynthesis pathways was conducted to confirm the positive identification of A. flavus. The gene expression also aided to delineate toxigenic isolates of A. flavus from atoxigenic ones. Morphologically, 18 isolates suspected to be A. flavus were identified. Out of these, 14 isolates successfully amplified the 500?bp ITS region of A. flavus or Aspergillus oryzae, while 4 isolates were not amplified. All the remaining 14 isolates expressed at least one of the aflatoxigenic genes but only 5 had all the genes expressed. Partial sequencing revealed that isolates 5, 11, 12, 13, and 15 had 99.2%, 97.6%, 98.4%, 97.5%, and 100% homology, respectively, to the A. flavus isolate LUOHE, ITS-5.8S-ITS2, obtained from the NCBI database. The five isolates were accurate identification of atoxigenic A. flavus. Precise identification of toxigenic strains of A. flavus will be useful in establishing control strategies of the fungus in food products.

SUBMITTER: Okayo RO

PROVIDER: S-EPMC7492892 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Morphological and Molecular Characterization of Toxigenic Aspergillus flavus from Groundnut Kernels in Kenya.

Okayo Robert O RO Andika Darius O DO Dida Mathews M MM K'Otuto George O GO Gichimu Bernard M BM

International journal of microbiology 20200907

Pathogenesis of Aspergillus flavus on important agricultural products is a key concern on human health due to the synthesis and secretion of the hazardous secondary metabolite, aflatoxin. This study identified and further characterized aflatoxigenic A. flavus from groundnuts sampled from sundry shops in Kenya using integrated morphological and molecular approaches. The groundnuts were plated on potato dextrose agar for isolation and morphological observation of A. flavus bas ...[more]

PMID: 32963542

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Project description:Aspergillus flavus is the main producer of carcinogenic aflatoxins in agricultural commodities such as maize. This fungus occurs naturally on crops, and produces aflatoxins when environmental conditions are favorable. The aim of this study is to analyse the genetic variability among 109 A. flavus isolates previously recovered from maize sampled from a known aflatoxin-hotspot (Eastern region, Kenya) and the major maize-growing area in the Rift Valley (Kenya), and to determine their toxigenic potential. DNA analyses of internal transcribed spacer (ITS) regions of ribosomal DNA, partial β-tubulin gene (benA) and calmodulin gene (CaM) sequences were used. The strains were further analyzed for the presence of four aflatoxin-biosynthesis genes in relation to their capability to produce aflatoxins and other metabolites, targeting the regulatory gene aflR and the structural genes aflP, aflD, and aflQ. In addition, the metabolic profile of the fungal strains was unraveled using state-of-the-art LC-MS/MS instrumentation. The three gene-sequence data grouped the isolates into two major clades, A. minisclerotigenes and A. flavus. A. minisclerotigenes was most prevalent in Eastern Kenya, while A. flavus was common in both regions. A. parasiticus was represented by a single isolate collected from Rift Valley. Diversity existed within the A. flavus population, which formed several subclades. An inconsistency in identification of some isolates using the three markers was observed. The calmodulin gene sequences showed wider variation of polymorphisms. The aflatoxin production pattern was not consistent with the presence of aflatoxigenic genes, suggesting an inability of the primers to always detect the genes or presence of genetic mutations. Significant variation was observed in toxin profiles of the isolates. This is the first time that a profound metabolic profiling of A. flavus isolates was done in Kenya. Positive associations were evident for some metabolites, while for others no associations were found and for a few metabolite-pairs negative associations were seen. Additionally, the growth medium influenced the mycotoxin metabolite production. These results confirm the wide variation that exists among the group A. flavus and the need for more insight in clustering the group.

Dataset Information

Morphological and Molecular Characterization of Toxigenic Aspergillus flavus from Groundnut Kernels in Kenya.

Publications

Morphological and Molecular Characterization of Toxigenic <i>Aspergillus flavus</i> from Groundnut Kernels in Kenya.

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