A Simple Procedure to Evaluate Competitiveness of Toxigenic and Atoxigenic Isolates of Aspergillus flavus in Solid and Liquid Media

Abstract

Application of atoxigenic strains of Aspergillus flavus to reduce aflatoxin levels is the most successful strategy applied in some agricultural crops. The role of ammonium hydroxide for preliminary screening of the competitiveness of atoxigenic A. flavus isolates to interfere with aflatoxin production by highly toxigenic isolates were evaluated. Out of 270 A. flavus isolates, 17 were detected as true atoxigenic using cultural methods and confirmed by analytical assays from different pistachio agro-ecological zones during 2013. For assessment competitive ability among atoxigenic isolates of A. flavus with highly toxigenic one, rice flour, coconut agar and coconut broth medium substrates were inoculated with mixtures including combinations of toxigenic and atoxigenic isolates, simultaneously. The rice flour substrate was used to quantify the content of aflatoxin in either co-inoculations or toxigenic isolate alone on thin layer chromatography plates with a scanning densitometer. While the culture media were used to determine the intensity of color change on exposing to ammonium hydroxide vapor. The reduction rates of aflatoxin B1 in co-inoculations were varied and ranged from 2%-82%. Based on the intensity of colony color changes, the competitiveness of the isolates was classified into five groups. Atoxigenic isolates with high competitiveness have shown low color changes in culture media and high aflatoxin reduction in TLC assays with a ratio of higher than 78%. The method will facilitate preliminary screening of efficient atoxigenic isolates for mitigation of aflatoxins in food and feed as a cheap, simple and quick method.