Genetic variability and aflatoxigenic potential of Aspergillus flavus isolates from maize

Various isolates of Aspergillus flavus collected from infected maize seeds were tested for their ability to produce aflatoxin B1 (AFB1) in vitro. The amount of AFB1 produced by the toxigenic isolates of A. flavus was ranged from 0.57 to 82.91 ng/ml. Among the 51 isolates of A. flavus tested, the isolate AFM1 produced the highest amount of (82.91 ng/ml) AFB1. Nineteen isolates did not produce aflatoxin B1. The genetic variability among the A. flavus isolates was investigated by PCR amplification of internal transcribed spacer (ITS) including ITS1, ITS2 and the intervening 5.8S rRNA region of ribosomal DNA followed by sequencing. PCR amplification of the ITS regions of the A. flavus isolates (AFM1, AFM3 and AFM5) by using ITS1 and ITS4 primers gave products of approximately 580 base pairs in length. The nucleotide sequences were compared with ITS sequences of A. flavus from Genbank database. Analysis of the genetic coefficient matrix derived from the nucleotide sequences of the ITS regions of A. flavus isolates showed that minimum and maximum percent similarities among the tested A. flavus strains were in the range of 12 and 99%, respectively. Phylogenetic analysis of ITS sequences by the unweighted pair group method arithmetic average (UPGMA) identified three main clusters (A, B and C). Cluster A consisted of only one strain and cluster C consisted of three strains including AFM5. The sequence of AFM5 was clustered with the sequence of a clinical isolate of A. flavus (AY677676) and about 76% similarity index was observed between them at molecular level. All the remaining strains belonged to cluster B, which includes AFM1 and AFM3. The genetic variability among A. flavus isolates should be considered when A. flavus isolates are required for testing in resistance breeding programmes.