AFLATOXIGENIC GENE DETECTION AND THE ANTIFUNGAL ACTIVITY OF GREEN SYNTHESIZED CuO AND ZnO NANOPARTICLES AGAINST Aspergillus flavus ISOLATES FROM CHILI AND MAIZE

Aflatoxins generated by Aspergillus flavus pose a serious challenge to food safety and crop
production. The present study aimed to detect key aflatoxin biosynthetic genes (aflR, aflP, aflO,
and tub1) and to evaluate the antifungal efficacy of green-synthesized copper oxide (CuO) and
zinc oxide (ZnO) nanoparticles against A. flavus isolates collected from chili (Capsicum annuum
L.) and maize (Zea mays) grown across seven location in the Southwestern region of Bangladesh.
Polymerase chain reaction (PCR) assays were employed to confirm the presence of target genes
while nanoparticle treatments were examined for their inhibitory effects on fungal growth. Among
the four genes analyzed, aflR demonastrated the highest prevalence (97.36%) underscoring its
central regulatory role in the aflatoxin biosynthetic pathway. The tub1 and aflO genes were
detected from 94.73% and 92.10% of the isolates, respectively indicating strong genetic
conservation among the samples. In contrast, aflP exibited a lower detection frequency (63.16%)
suggesting potential genetic variability or partial gene loss within certain isolates. In chili, specific
genotypes such as AF-5 (aflR, aflO) and AF-9 (tub1, aflO, aflP) were distinct whereas other
clusters including AF-16, AF-12, AF-11, AF-7 and AF-6 showed similar gene combinations (aflR,
aflO, tub1). The largest group (AF-1, AF-2, AF-3, AF-4, AF-8, AF-10, AF-13, AF-14,AF-15, AF-
17, AF-18, AF-19 and AF-20) contained all four genes indicating close genetic relatedness.
Similarly, maize isolates demonstrated that most genotypes (AF-21 to AF-36) possessed all four
genes suggesting high aflatoxigenic potential while a few (AF-29, AF-31, AF-32, AF-33 and AF-
37) lacked aflP. Two genotypes (AF-26 and AF-27) contained only aflR and tub1.AF-38 appeared
only the aflR gene and a major cluster consisting of AF-21, AF-22, AF-23, AF-24, AF-25, AF-28,
AF-30, AF-34, AF-35 and AF-36 exibited all four genes. Additionally, zinc oxide (ZnO) and
copper oxide (CuO) nanoparticles through green synthesized methods were assessed for their
antifungal activity applying disc diffusion method. Both nanoparticles exhibited concentrationdependent inhibitory effects with CuO NPs showing comparatively higher antifungal potential
than ZnO NPs. Antifungal assays demonstrated that CuO and ZnO NPs exhibited strong,
concentration-dependent inhibitory effects against A. flavus. CuO NPs produced inhibition zones
of 21 mm, 19.33 mm, 14.33mm and 12.67 mm at 200, 150,100 and 50 ppm, respectively while
ZnO NPs produced 19.33 mm, 16.33 mm, 15.33mm and 10 mm at the same concentrations. In
comparison, the synthetic fungicide Mancozeb at 100 ppm showed smaller inhibition zones (14
mm). These results indicate that green-synthesized NPs are effective antifungal agents and could
serve as a complementary agent compare to conventional fungicides for managing A. flavus in
agricultural systems.

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