Rapid isolation of non-aflatoxigenic Aspergillus flavus strains

Abstract

In the present study, a method for screening non-aflatoxigenic Aspergillus flavus in soil samples collected from major peanut-growing regions of China was developed. The single colonies were picked and cultured on Aspergillus flavus and parasiticus agar (AFPA). If the reverse side of the colony on AFPA was orange-coloured, it was considered A. flavus or Aspergillus parasiticus. After the genomic DNA of each strain was extracted, 28S rRNA and calmodulin were amplified and sequenced to determine the species. The key gene, aflR, was amplified and digested via polymerase chain reaction-restriction fragment length polymorphism. The aflatoxigenic A. flavus and the non-aflatoxigenic A. flavus and A. parasiticus were distinguished by enzyme digestion of aflR. 156 strains of A. flavus were screened, which consisted of 135 aflatoxigenic and 21 non-aflatoxigenic strains. The aflatoxin producing ability of each strain was confirmed using solid-state fermentation experiments. Using the method developed in the present study, we confirmed that the non-aflatoxigenic A. flavus strains isolated lost their capacity to produce aflatoxins. Considering there could be some alterations in other functional genes, some non-aflatoxigenic strains could be identified inaccurately as aflatoxigenic strains, although that did not occur in the present study. The growth of non-aflatoxigenic A. flavus was observed, and the most rapidly growing non-aflatoxigenic strain was selected for plate confrontation assays and toxic mixed culture experiments. The inhibition rate of non-aflatoxigenic A. flavus against aflatoxigenic A. flavus was 55.4 and 72.6% in potato dextrose agar (PDA) plate and natural soybean medium, respectively. The screened non-aflatoxigenic A. flavus strains provide a microbial resource for biological control of aflatoxin contamination.