Report on the identification of genes directly related to the regulation of AFB biosynthesis in Aspergillus flavus infecting almond under stress conditions

This deliverable describes the results obtained from the expression analysis of some genes involved in the biosynthesis of aflatoxin B1 (AFB1) in Aspergillus flavus grown on almond medium under physical stress (aw and T°). In particular, the aim was to study the effect of varying (various?) water activity (aw) conditions on the activation of the two regulatory genes, aflR and aflS, located in the biosynthesis cluster, during the growth of the fungus, in relation to aflatoxin production. In addition, the relation to the expression of some structural genes (aflD, aflM, aflO, aflP, and aflQ) of the biosynthesis pathway during the time course experiment was assessed. The analyses were performed on fungal mycelium harvested in early days of incubation (seven time points, every 24 hours, for all the conditions tested) and then after 10 days and 15 days of growth. Fungal growth, AFB1 production and relative quantification of the expression for each gene during the growth for each aw value were evaluated. Additionally, at days 5 and 7 among the different aw, relative expression of each gene and the expression levels of all the genes considered were compared. Mycelial growth and AFB1 production resulted delayed with the lowering of aw values. During the early stages of incubation, they were especially reduced at 0.90 aw. At this condition, all the tested biosynthesis genes exhibited a reduced level of transcription, with aflS showing a lower expression level than aflR. At 0.99 and 0.96 aw, aflR and aflS were transcribed from the beginning, whereas at 0.93 aw their expression started later, and reached the maximum just in the proximity of the peak of AFB1 accumulation, at 15 days of growth. Finally, the comparison of the relative expression of all the genes at different aw showed the highest transcription levels at 5 days for aw 0.99, and at 7 days for 0.96 aw.