## 3 Results and Discussion
### Effects of Sub-Lethal Concentrations of NP/Derivatives on AF Production: A. parasiticus
previously characterized the AF biosynthetic pathway gene cluster in aspergillus [61], in which at least 23 enzymatic reactions are involved. AFB\({}_{1}\)/AFG\({}_{1}\) or AFB\({}_{2}\)/AFG\({}_{2}\) are produced from the metabolic intermediates _O_-methylsterigmatocystin or dihydro-_O_-methylsterigmatocystin, respectively, by the action of oxidoreductase uncoded by _aflQ_ gene. At this moment, it is not clear why the effect of NP/derivatives in _A. parasiticus_ is enhancing mostly AFG\({}_{1}\)/AFG\({}_{2}\). Future investigation might elucidate the mechanism of action of differential AF enhancement by these compounds, and new control/intervention point(s) that can specifically inhibit AFG\({}_{1}\) and/or AFG\({}_{2}\) production. As observed in _A. flavus_, while SLD at 0.5 mM prevented the growth of _A. parasiticus_, the structural analog SA still allowed the fungal growth at the same concentration, which coincided with AFG\({}_{1}\) and AFG\({}_{2}\) enhancement in _A. parasiticus_ (structure-activity relationship). Therefore, we selected two compounds, viz., SLD (potent antifungal activity in both _A. flavus_ and _A. parasiticus_) and 4I3M (highest anti-aflatoxigenic activity), and investigated further for their utility as potential antifungal and/or anti-aflatoxigenic agents using corn seeds. Use of Salicylaldehyde (SLD) as an Antifungal Agent Targeting Corn Kernels: Petriplate Analysis (Small Scale)  
SLD is a plant-derived volatile compound classified as Generally Recognized As Safe (GRAS) [62; 63; 64]. In this study, we investigated the antifungal efficacy of SLD (0.1 to 1.0 mM) as a fumigant by targeting _A. flavus_ contaminated on corn kernels (seeds). The effect of SLD on seed germination frequency was also examined. As shown in Table 2, SLD exhibited potent antifungal activity against _A. flavus_ or seedborne fungal contaminants; the average number of _A. flavus_-contaminated corn kernels decreased from 6.8 (1-day exposure) to 3.9 (2-day exposure) and 2.9 (3-day exposure), respectively, as the exposure time increased from 1 to 3 days (see also Supplementary Figure S3). Likewise, the number of corn kernels having seedborne fungal contaminants also decreased from 3.6 (1-day exposure) to 0.6 (2-day exposure) and 0.3 (3-day exposure), respectively, as the incubation time increased. A similar trend of SLD antifungal activity was also determined in the tree nut pistachio (Supplementary Figure S4). In this testing, no _A. flavus_ was artificially inoculated onto the surface of pistachios, and therefore, antifungal testing was solely against farm-derived, seedborne fungal contaminants. We determined that the level of seedborne fungal contamination  
\begin{table}
\begin{tabular}{c c c c c c c c c c} \hline \hline  & \multicolumn{3}{c}{**1 Day**} & \multicolumn{3}{c}{**2 Day**} & \multicolumn{3}{c}{**3 Day**} \\ \hline
**SLD (mM)** & \begin{tabular}{c} **\# Seed** \\ **Geminated** \\ \end{tabular}  & \begin{tabular}{c} **\# _A/_** \\ **Contaminated** \\ \end{tabular}  & \begin{tabular}{c} **\# Seed** \\ **Contaminated** \\ \end{tabular}  & \begin{tabular}{c} **\# Seed** \\ **Geminated** \\ \end{tabular}  & \begin{tabular}{c} **\# _A/_** \\ **Contaminated** \\ \end{tabular}  & \begin{tabular}{c} **\# Seed** \\ **Contaminated** \\ \end{tabular}  & \begin{tabular}{c} **\# Seed** \\ **Geminated** \\ \end{tabular}  & \begin{tabular}{c} **\# Seed** \\ **Geminated** \\ \end{tabular}  & \ \begin{tabular}{c} **\# _A/_** \\ **Contaminated** \\ \end{tabular}  & \begin{tabular}{c} **\# Seed** \\ **Contaminated** \\ \end{tabular}  \\ \hline
0.0 (Seed only) 1 & 10 & 0 & 10 & 10 & 0 & 10 & 0 & 10 \\
0.0 (Seed only) 2 & 10 & 0 & 10 & 9 & 0 & 10 & 0 & 10 \\ \hline
0.0 1 & 9 & 10 & 0 & 9 & 10 & 0 & 6 & 10 & 0 \\
0.0 2 & 7 & 10 & 0 & 10 & 10 & 0 & 7 &  
inants on pistachios was decreased as the concentration of SLD (applied as a fumigant) increased (0.1 to 1.0 mM) (Figure S4).