CATEGORIZATION OF ASPERGILLUS FLAVUS AND ASPERGILLUS PARASITICUS ISOLATES OF STORED WHEAT GRAINS IN TO AFLATOXINOGENICS AND NON-AFLATOXINOGENICS

The objective of this study was to categorize 157 Aspergillus flavus (AF1-AF157) and 36 A. parasiticus (AP1-AP36) strains isolated from stored wheat grains from three provinces of Pakistan viz., Punjab, Sindh and NWFP, into aflatoxigenic and non-aflatoxigenic ones by cultural as well as PCR methods. None of the isolates produced aflatoxin except the AP4 isolate. The positive control in all the batches of A. flavus and A. parasiticus isolates produced maximum aflatoxin in grains at 16% moisture and 25 C temperature in a laboratory experiment of this study. The accuracy of cultural tests ELIZA assessed by percent recovery of the toxin from positive controls, spiked samples and spiked controls ranged from 98-100%. An assay based on multiplex PCR was applied for the detection of four genes located at different loci coding enzymes in the aflatoxin biosynthetic pathway of A. flavus and A. parasiticus strains. These are AflR, reported as regulatory gene and functions as a transcription activator, others are structural genes named according to their substrates Nor 1 (norsolorinic acid), Ver 1(Versicolorin) and Omt (O methylosterigmatocystin). Recovery of these four genes in the DNA template of known Aflatoxinogenic A. flavus strains in every experimental trial of present study as a positive control showed the accurate PCR experimentation. None of the A. flavus and 35 A. parasiticus (AP1-AP3 and AP5-AP36) isolates produced aflatoxin in both the flask and storage experiments. Similarly all these strains did not exhibit presence of all the four genes in the PCRs of the extracted DNA at one time. All the A. flavus isolates on the basis of their gene pattern were grouped into 11 groups and A. parasiticus isolates into 9 groups. Presence of all four genes was detected only in one aflatoxinogenic isolate of A. parasiticus ( AP4). The comparison of the cultural and PCR methods showed good agreement, as results of both the methods exclusively matched each other. All the non Aflatoxinogenic isolates of this study showed biocontrol activity against known Aflatoxinogenic A. flavus isolate during an in vitro laboratory experiment.