Monitoring the production of aflatoxin B1 in wheat by measuring the concentration of nor-1 mRNA.

A real-time reverse transcription-PCR system has been used to monitor the expression of an aflatoxin biosynthetic gene of Aspergillus flavus in wheat. Therefore, total RNA was isolated from infected wheat samples, reverse transcribed and subjected to real-time PCR. In parallel all samples were analyzed by high-pressure liquid chromatography for aflatoxin B(1) production. The primer-probe system of the real-time PCR was targeted against nor-1, a gene of the aflatoxin biosynthetic pathway. By application of this method the nor-1 transcription was quantified during the course of incubation. After 4 days of incubation nor-1 mRNA could be detected for the first time. The amount of nor-1 mRNA increased rapidly, and the maximum was achieved after 6 days. Then, starting very slowly, the mRNA was degraded until day 8, and this was followed by a very fast degradation, reaching nondetectable levels at days 9 and 10. First traces of aflatoxin B(1)could be detected between the 5th and 6th day of incubation. The aflatoxin concentration reached its maximum after 9 days of incubation and remained constant for the whole period of observation. To ensure that differences in the nor-1 mRNA concentration were due to different expression levels, the expression of the constitutively expressed beta-tubulin gene (benA56) has also been monitored. The expression of benA56 remained constant during the whole incubation time. As a parameter for fungal growth, the number of nor-1 gene copies was determined during the course of incubation. The numbers of nor-1 gene copies increased at the beginning of the incubation and reached a plateau at day 5. They correlate well with the viable counts albeit at a higher level.