A polyphasic characterisation of Aspergillus section Flavi isolated from Malaysian and Nigerian food grains and poultry feeds by phenotypic, chemotypic, and molecular methods

Abstract

The taxonomy and toxigenicity of Aspergillus section Flavi (ASF) in various regions are still under-reported, as many potentially toxic species are continuously discovered. This study aims to determine the aflatoxigenicity and identify 74 isolates of ASF from Malaysian and Nigerian food grains and feeds using phenotypic, chemotypic, and molecular methods. Isolates were screened for sclerotium morphotype and aspergillic acid production phenotypically, while an ultra-fast reverse-phase high-performance liquid chromatography (HPLC) was optimised to quantify the cyclopiazonic acid and aflatoxins produced by the isolated ASF, based on which, they were characterised into phenotypes L-strains, S-strains, and NS-strains, and chemotypes I to VI. Chemotypic data and nucleotide sequences of β-tubulin and internal transcribed spacer (ITS) genes were utilised to identify the isolates’ species and determine their phylogenetic relationships. Aflatoxigenesis on solid media and multiplex polymerase chain reactions of four essential genes (aflR, aflM, aflD, and aflP) of aflatoxin biosynthesis were used to confirm the aflatoxigenicity of each isolate. All the six chemotypes of ASF were present among the isolated strains, with the aflatoxigenic chemotypes (55.41%) and CPA-positive isolates (68.92%) being significantly higher (p < 0.05) than the non-aflatoxigenic and CPA negative chemotypes. Aflatoxins and CPA were produced in the 0.25 – 19.58 ng/g range and 29.89 – 46.55 ng/g, respectively. Chemotypic data accurately differentiate toxigenic from atoxigenic species but only identify 90% of species names. Molecular data accurately resolved species but not toxigenicity. The results highlight the need for robust regulatory measures in the concerned regions to mitigate the risk of aflatoxicosis.