Discrimination of Aspergillus flavus group fungi using phylogenetic tree analysis and multiplex PCR

: We describe a simple method for discrimination of Aspergillus flavus group fungi, including aflatoxin (AF) producers, by means of molecular-biological analysis of 45 strains of A. flavus. First, 20 strains of A. flavus were compared using phylogenetic tree analysis based on the nucleotide sequences of ITS 1-5.8S-ITS 2 (ITS-1/2) and aflR-aflJ intergenic spacer (aflR/J-IGS). In this analysis, the tested strains were discriminated into 4 groups at the aflR/J-IGS region. Although ITS-1/2 region analysis could not discriminate between A. flavus (AF producers) and A. oryzae/A. flavus (AF nonproducers), aflR/J-IGS region analysis could discriminate between these groups. Moreover, 45 strains of A. flavus were compared by means of both phylogenetic tree analysis based on the aflR/J-IGS region and the conventional aflatoxin production test (culture method). The phylogenetic tree analysis of the tested strains was consistent with the findings of the culture method. In addition, 49 strains of A. flavus and related species (Aspergillus spp.) were tested by multiplex PCR with primers designed on the basis of the phylogenetic tree analysis. These results were consistent with phylogenetic tree analysis based on the aflR/J-IGS region for 41 strains.