Analysis of genes early expressed during Aspergillus flavus colonisation of hazelnut

Aflatoxins contamination by Aspergillus flavus is a matter of great concern for oil rich crops among which hazelnuts represent economically important agricultural commodities of Mediterranean countries, mainly used as mixed nuts or as ingredients in the bakery and confectionery industries. Since the biosynthetic pathway of aflatoxin biosynthesis has been elucidated in detail, expression analysis of the genes along the pathway can provide a thorough insight into the molecular mechanisms of toxin production and regulation. In the present work, we carried out a transcriptional analysis of the main genes belonging to aflatoxin biosynthetic cluster of A. flavus, namely the two regulatory genes aflR and aflS and the five structural genes aflD, aflM, aflO, aflP, and aflQ. The analysis was carried out at different stages of fungal growth on two different media: hazelnut agar medium and YES medium. The transcripts of all the genes paralleled the synthesis of aflatoxin and both were detected starting around 36h in YES medium, and 72h in hazelnut agar medium. Significantly, the amount of aflatoxin produced was about one order lower in hazelnut agar compared to YES medium. The expression of two genes encoding a lipase and a metalloprotease, potentially involved in lipid and protein catabolism, was also monitored during fungal growth. Noteworthy, the expression of the metalloprotease gene appeared to be specific for the hazelnut medium, whereas the lipase gene was expressed in both media. Finally, we verified the expression profiles of three genes encoding fatty acid dioxygenases/diol synthases involved in the biosynthesis of fungal oxylipins, namely ppoA, ppoB, ppoC. Recent findings have pointed out the importance of fungal oxylipins in fungal growth/mycotoxin production and our results indicated that all the three ppo genes are expressed during A. flavus growth on hazelnut medium. In particular, ppoB appeared to be specifically expressed in this medium. This study reports for the first time on the expression profiles of genes belonging to the biosynthetic cluster and genes potentially involved in the regulation of fungal secondary metabolism during A. flavus colonisation of hazelnuts.