*2.7. Statistical Analysis*

All the statistical analyses were performed with the CoStat program, version 6.303, and the analysis of variance technique (CoHort software, Monterey, CA, USA). The data from the expression analysis of the aflatoxin biosynthesis genes were expressed as means standard deviation (S.D.), and the values were considered statistically significant when *p* ≤ 0.05.

### **3. Results and Discussion**

### *3.1. Identification of the Aflatoxigenic Fungal Isolate*

The aflatoxigenic isolate was found to produce AFB1 at a rate of 25.67 μg/L. The revealed sequence of the ITS region of the aflatoxigenic isolate was submitted to NCBI using a blasting tool given the high similarity with *Aspergillus flavus* fungus [60]. A coded name was given to the isolate as *A. flavus* f2 and GenBank accession no. (# MG202160).

#### *3.2. Fruit Peel Extracts Effect on A. flavus Biomass and AFB1 Production*

To evaluate the influence of different fruit peel extracts prepared with varying solvents at three different concentrations on *A. flavus* fungal biomass (dry and wet weight) and AFB1 production, the pomegranate, sugar apple, and eggplant fruit peels were extracted with diethyl ether, acetone, ethanol, and methanol at concentration percentages of 25%, 50%, and 75% (Table 2). In comparison to the control, the 75% ethanol pomegranate peel extract (0.09 and 0.03 g), 25%- methanol sugar apple peel extract (0.12 and 0.01 g), and 75%- methanol eggplant peel extract (1.89 and 0.24 g, respectively) produced the least substantial wet and dry weight values. These results agree with those previously reported for pomegranate peel extract, which has been shown to be intensely active against *A. flavus*, *A. parasiticus*, *A. fumigatus*, *Fusarium proliferatum*, and *F. verticillioides* isolates with minimal inhibitory concentration (MIC) values ranging between 1.25 and 5 mg/mL [61]. Furthermore, similar results were recorded by Oliveira and Furlong [29], according to which eggplant peel phenolic extract inhibited the growth of *A. flavus* after 72 h of incubation with 84.80%. In comparison, Basudan [62]'s findings proved that black and white eggplant peel extracts had no fungal effect against the *A.flavus* strain.


**Table 2.** *Aspergillus flavus* biomass and AFB1 inhibition ratio (AI%), as affected by applying pomegranate, sugar apple, and eggplant peel extracts.

> Furthermore, most of the solvent extracts examined were highly efficient against the formation of AFB1 at various concentrations. The AFB1 inhibition ratio (AI%) ranged from 35.52% to 91.18%. Besides, AFB1 production was less affected by the methanol extracts, with the AI% values ranging from 35.52% to 81.5%. Table 2 shows that pomegranate peel extracts, diethyl ether 25%, ethanol 25%, and acetone 25% produced promising results, with AI values of 90.87%, 90.70%, and 90.32%, respectively. Still, methanol 25% and diethyl ether 75% exhibited the lowest AI values (62.26% and 71.61%, respectively). Table 2 demonstrates that the AI% values of the sugar apple extract with different solvent concentrations differed significantly. In comparison to the AI% values of the other solvents, the highest AI%, of 82.23%, was achieved at 75% ethanol treatment. The least effective ratio was obtained with the 25%, 50%, and 75% methanol sugar apple peel extracts (38.23%, 35.52%, and 37.12%), respectively. Table 2 demonstrates that the highest levels of AFB1 inhibition were obtained with eggplant diethyl ether extract at concentrations of 50% and 75% (91.18% and 90.95%, respectively), while the 50% methanol treatment produced the lowest AI% results (35.97%).

> After 72 h of incubation, the pomegranate peel extract at the 1250 μg/mL concentration inhibited AFB1 production by 67% without affecting fungal growth [61]. According to other researchers, extracting antioxidants reduces aflatoxins by absorbing, neutralizing the free radicals, and preventing their proliferation chains, resulting in less dangerous compounds. Furthermore, the efficacy of solvents varies depending on their quantities and the components of a particular plant extract. Sugar apple and eggplant methanol and ethanol peel extracts demonstrated strong antioxidative properties against human infections, according to Bernardo and Sagum [63]; these findings endorse our results. According to Adom et al. [64] and Laddomada et al. [65], the essential antioxidants in maize bran are the phenolic acids, mainly the phenolics covalently bonded with cell wall structural components through ester bonds, which play a defensive role against plant fungal infection.

### *3.3. Maize Storage Experiment*
