Differentiation of toxigenic and atoxigenic Aspergillus flavus: Polyphasic approach, a new dimension

ABSTRACT

Contamination of food by aflatoxin of Aspergillus flavus is a major global problem affecting trade, quality, utility of food and human health. While all the members of A. flavus does not produce aflatoxin, sensitive, cost effective and reproducible methods for large scale screening and differentiation of toxigenic A. flavus from atoxigenic ones are scarce. Here, we made one such attempt using coconut milk agar (CMA), yeast extract sucrose agar (YESA), ammonium hydroxide vapour tests, enzyme linked immuno sorbent assay (ELISA) and polymerase chain reaction (PCR) for large scale screening of toxigenic strains of A. flavus. Fifty nine isolates of A. flavus obtained from major chilli growing regions of India were categorized into toxigenic and atoxigenic strains by using cultural, analytical and molecular methods. Forty two (71.18 %) isolates showed positive response in coconut milk agar (CMA), 17 (28.81%) isolates did not match while 23 (38.98 %) isolates showed red colour and 36 (61.01%) isolates did not produce red colour upon exposure to ammonia vapour in YESA. Out of 59 isolates, isolates CAF43 came out as highly toxigenic, as it produced 3128.20 μg kg-1 aflatoxin B1 followed by CAF 42 which produced 3035.10 μg kg-1. Among 59 isolates, eight A. flavus isolates were amplified with two regulatory (aflR and aflJ) and two structural (norA and ver1) genes at a region of 900, 450, 400 and 450 bp, respectively.