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Aflatoxin B 1 (AFB 1 ) is one of the most hazardous mycotoxins for humans and livestock that mainly produced by members of the genus Aspergillus in a variety of food commodities. In this study, the effect of S. rosmarinus, T. fruticulosum, and T. caucasicum essential oils (EOs) was studied on fungal growth, AFB 1 production and aflR gene expression in toxigenic A. flavus IPI 247. The AFB 1 producer A. flavus strain was cultured in YES medium in presence of various two-fold concentrations of the plant EOs (62.5-500 μg/mL) for 4 days at 28 °C. EO composition of plants was analyzed by Gas Chromatography/Mass Spectrometry (GC/MS). The amount of fungal growth, ergosterol content of fungal mycelia and AFB 1 content of EO-treated and non-treated controls were measured. The expression of aflR gene was evaluated using Real-time PCR in the fungus exposed to minimum inhibitory concentration (MIC 50 ) of EOs. The main constituents of the oils analyzed by GC/MS analysis were elemicin (33.80 %) and 2,3-dihydro farnesol (33.19 %) in T. caucasicum, 1,8-cineole (17.87 %), trans-caryophyllene (11.14 %), α and ẞ-pinene (10.92 and 8.83 %) in S. rosmarinus, and camphor (17.65 %), bornyl acetate (15.08 %), borneol (12.48 %) and camphene (11.72 %) in T. fruticulosum. The results showed that plant EOs at the concentration of 500 μg/mL suppressed significantly the fungal growth by 35.24-71.70 %, while mycelial ergosterol content and AFB 1 production were inhibited meaningfully by 36.20-65.51 % and 20.61-89.16 %. T. caucasicum was the most effective plant, while T. fruticulosum showed the lowest effectiveness on fungal growth and AFB 1 production. The expression of aflR in T. caucasicum and S. rosmarinus -treated fungus was significantly down-regulated by 2.85 and 2.12 folds, respectively, while it did not change in T. fruticulosum-treated A. flavus compared to non-treated controls. Our findings on the inhibitory activity of T. caucasicum and S. rosmarinus EOs toward A. flavus growth and AFB 1 production could promise these plants as good candidates to control fungal contamination of agricultural crops and food commodities and subsequent contamination by AFB 1 . Down-regulation of aflR as the key regulatory gene in AF biosynthesis pathway warrants the use of these plants in AF control programs. Further studies to evaluate the inhibitory activity of studied plants EOs in food model systems are recommended., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Senna (Cassia angustifolia Vahl.) is a medicinal crop with laxative properties, and it has significant demand in the global pharmaceutical market. Senna pods are highly susceptible to aflatoxin contamination, and the successful export of pods is hindered due to the regulatory limits of importing countries. The senna pod water activity (a w ) from harvest to storage is the key factor determining AFB 1 accumulation. The temperature conditions from field to warehouse also interact with pod a w , which influences fungal growth and AFB 1 production. The determination of an ideal combination of a w and temperature led to the assessment of the critical control point for AFB 1 synthesis in senna. Hence, this study aimed to evaluate the influence of a w (0.99, 0.96, 0.93, 0.90, and 0.87 a w ) and temperature (20, 28, and 37 °C) on fungal growth, gene expression (aflR and aflS), and AFB 1 production by A. flavus in senna agar medium. The fungus showed the longest lag time (7.7 days) at 20 °C with 0.87 a w . We observed that 0.96 a w (P < 0.01) was optimum for the diametric growth rate at 28 and 37 °C. However, the peak expression of regulatory genes (aflR and aflS) and the maximum AFB 1 production were observed only at 28 °C (0.96 a w ). The highest growth rate occurred at 37 °C, which did not favor the expression of genes and AFB 1 production. However, at 28 °C, it positively correlated with gene expression and AFB 1 production. The suppressed expression of regulatory genes and a trace amount of aflatoxin B 1 were found at 20 °C with all the tested a w . In our experiments, the low a w (0.87 and 0.90 a w ) suppressed the fungal growth, gene expression, and AFB 1 production of A. flavus at all of the tested temperatures (20, 28, and 37 °C). The rapid drying of senna pods with a low water activity (≤0.87 a w ) and storage at low temperature (20 °C) are ideal conditions to avoid AFB 1 and ensure the quality of produce for export., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The regulatory gene (aflR) encodes AflR, a positive regulator of transcriptional pathway that activates aflatoxin biosynthesis. It has been demonstrated in our laboratory that L-Asp-L-Asn (DN) extracted from Bacillus megaterium inhibited the growth of Aspergillus flavus. We fused gene encoding DN with the gene encoding specific dinuclear zinc finger cluster protein of AflR, then fusion protein competed with the AflS-AflR complex for the AflR binding site and significantly improved anti-A. flavus activity (growth of A. flavus and biosynthesis of aflatoxin B 1 ) of DN. The fusion gene dn-aflR was cloned into pET32a and recombinant plasmid was introduced into Escherichia coli BL21. The highest expression was observed after 10 h induction and fusion protein was purified by affinity chromatography column. Compared with DN, the novel fusion protein DN-AflR significantly inhibited the growth of A. flavus and biosynthesis of aflatoxin B 1 (P < 0.05). This study promoted the use of competitive inhibition of fusion proteins to reduce the expression of regulatory genes in the biosynthetic pathway of aflatoxin. Moreover, it provided more supports for deep research and industrialization of such novel anti-A. flavus bio-inhibitors and biological control of microbial contamination., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The objectives of this study were to examine the efficacy of four bacterial antagonists against Aspergillus flavus using 50:50 ratio of bacterial cells/conidia for the control of aflatoxin B1 (AFB1) production on two different nutritional matrices, nutrient and maize-based media at different water availabilities (0.98, 0.94 water activity (aw) on nutrient medium; 0.995, 0.98 aw on maize meal agar medium) at 35°C. The indicators of efficacy used were the relative expression of one structural and regulatory gene in the biosynthetic pathway (aflD and aflR respectively) and the production of AFB1. These studies showed that some of the bacterial species could significantly inhibit the relative expression of the aflD and aflR genes at both 0.98 and 0.94 aw on nutrient agar. On maize-based media some of the bacterial antagonists reduced the activity of both genes at 0.94 aw and some at 0.995 aw. However, the results for AFB1 production were not consistent with the effects on gene expression. Some bacterial species stimulated AFB1 production on both nutrient and maize-based media regardless of aw. However, some bacterial treatments did inhibit AFB1 production significantly when compared to the control. Overall, this study suggests that temporal studies are required on the biosynthetic genes under different environmental and nutritional conditions to evaluate the potential of antagonists to control AFB1., (Copyright © 2015 Elsevier B.V. All rights reserved.)