## 4 Discussion  
Agricultural foods and feedstuffs need to be safe until the customer can consume them properly, and thus, fungal infection and mycotoxin contamination during the storage and/or transport should be periodically monitored [18]. Additionally, effective control strategies for fungal infection and toxin production should be organized for the right place and time [19]. Chemical treatment has been seriously considered as an effective method to reduce fungal infection and toxin production in agricultural foods and feedstuffs [9; 15; 20]. Recently, [PERSON] et al. [21] reported that five food additives might be used to protect foods and feedstuffs from aflatoxin contamination. These authors demonstrated that benzoic acid up to 0.1% exhibited potent antifungal and antiaflatoxigenic activities against _A. flavus_ at concentration levels below the legal limit for standard use; it can be used as an alternative food additive to propionic acids at those levels, because propionic acid, the currently used disinfectant, cannot be utilized due to the limitations of its standard use. On the other hand, naturally occurring bioactive compounds, including plant essential oils and their major constituents, have been introduced, due to their strong antifungal activities [7; 8; 11; 22]. Among them, essential oil from _Mentha longifolia_, which contained 2.2% of 1,8-cineole, showed strong  
Figure 5: Expression of genes (_afIC_, _afID_, _afIE_, _afIK_, _afIL_, _afIO_, _afIP_, _afIR_, _afIS_, and _yap_) involved in aflatoxin biosynthesis in _Aspergillus flavus_ treated with \(t\)-cinnamaldehyde. An ergosterol biosynthetic gene, _erg28_, was comparatively measured after treatment with \(t\)-cinnamaldehyde. Different letters above the same column indicate significant differences from the control group (\(p<0.05\)). antifungal and antiaflatoxigenic activities towards _A. flavus_[22]. 1,8-Cineole showed moderate antifungal activity against _A. flavus_ (Figure 1A) at a treatment concentration of up to 250 ppm. However, _t_-cinnamaldehyde exhibited strong antifungal activity when compared to 1,8-cineole (Figure 1B). In addition to the antifungal activity, the antiaflatoxigenic activity of _t_-cinnamaldehyde on aflatoxin production was higher than that of 1,8-cineole (Figures 2 and 3). AFB1 and AFB2 decreased with increasing concentrations in the chemical-treated growth medium. In this study, 1,8-cineole showed antifungal and antiaflatoxigenic activities towards _A. flavus_ growth and AFB1 production at a concentration of 250 ppm. Additionally, a recent report examined 1,8-cineole and showed that its minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) were lower than those of the currently used fungicide Nystatin [13]. They used 1,8-cineole at a concentration of 0.918 mg/mL (about 918 ppm) in the growth medium. Therefore, 1,8-cineole completely inhibited _A. flavus_ growth, at least at a concentration of 918 ppm. With this antifungal activity, 1,8-cineole strongly inhibited AFB1 production without non-phytotoxic effects on chickpea seed germination [13]. However, in the report, they did not explain how 1,8-cineole possessed antiaflatoxigenic activity. In our study, 1,8-cineole strongly inhibited _afIE_ and _afIL_ gene expression in _A. flavus_ (Figure 4). These two genes are responsible for the expression of nonsolorinic acid reductase and cytochrome P450 monooxygenase/desaturase. Norsolorinic acid (NOR) reductase is responsible for the conversion of NOR to averantin [23]. This enzyme functions in aflatoxin biosynthesis after polyketide synthase [23], which is at a very early part of the biosynthetic pathway. Therefore, 1,8-cineole inhibits the expression of the first enzyme to suppress aflatoxin production. Moreover, 1,8-cineole downregulated the _afIL_ gene, which encodes cytochrome P450 monooxygenase/desaturase and it presumably participates in the conversion of versicolorin B (VERB) to VERA [23]. This enzyme plays a key role in the separation of the AFB1 subtype from the AFB2 subtype.