*3.7. Fungal SEM*

conidia (Figure 11D–F).

Concerning the morphological structure of *A. flavus*investigated by SEM, adjustments in conidiophore attributes were watched. *A. flavus* was refined on PDA corrected with 180 ppm of Cu-Chit/NCs hydrogel caused slight changes in mycelial structure, highlighted by hyphal twisting and by decreasing of regenerative structures, for example, conidia and conidiophores.

SEM investigation of conidiophore changes showed that fungal spores turned into glaringly extraordinary, in which mycelia and conidiophores were contracted in comparison to untreated controls (Figure 11). Alterations of hyphae structure have been determined as proven in Figure 11A–C along with lower of cytoplasmic content material and adjustments of membrane integrity. whilst, in untreated control, development of mycelium and conidiophore was regular with considerable conidia (Figure 11D–F).

**Figure 11.** Scanning electron micrographs showing shriveled conidiophores (**A**–**C**) and the healthy mycelium or conidiophores (**D**–**F**) of *A. flavus* on PDA treated with Cu-Chit/NCs hydrogel. The yellow arrows mean hyphae or conidiophore.

### **4. Discussion**

There is an enormous interest to expand effective and eco-friendly fungicides anti-toxigenic fungi with low level or zero mycotoxin residues without affecting the plant growth and crop productivity of the essential agriculture ingredients [9]. The primary target of the current examination is to evaluate the antifungal impacts of bio-polymers like chitosan cross breed with copper metals against *A. flavus* aflatoxin-creating strains. Measures of aflatoxin formed in cultural media show some alarms on the toxigenic capability of various fungal isolates to deliver a high quantity of aflatoxin in agricultural supplies [43]. In the present work, analysis of aflatoxin-producing ability by fluorescence in CA an AFPA medium showed a good correlation with the biochemical examination of aflatoxins, a cutting-edge finding in a harmony with Monda et al. [44]. However, for most purposes, we found the CA media screening technique to be simpler, faster, and much cheaper than any of the different techniques

examined [45]. Although the data indicate that the aflatoxins distinguished producer media such as AFPA is not completely persistent in differentiating between aflatoxin-producing and nontoxigenic strains of *A. flavus*, it is important that the fungal medium did not yield false-positives [46].

Our results show that 62% percent of *A. flavus* is aflatoxin-producing isolates. Fifty percent of the screened isolates of *A. flavus* collected from discolored rice grains in India can produce aflatoxin B1 [47]. These results are in agreement with Abbas et al. [48] who investigated more noteworthy producer *A. flavus* strains. Lai et al. [49] indicated that more than 35% of *A. flavus* strains secreted various quantities of aflatoxins in the rice grain. The diverse aflatoxin production capacities of the *A. flavus* isolates would be affected by the various resources of the strains and also ecological problems. The aflatoxin production pathway includes roughly 30 genes, some having unsure functions in aflatoxin biosynthesis [50].

In the present work, *A. flavus* isolates were tested for the presence of gene *alfA*, which is code for fatty acid synthases, while structural gene *aflP* is one of the main genes responsible for transforming ST into O-methylsterigmatocystin [51]. Our findings show that the two primers sets are specific for fast detection of *A. flavus*. Based on specific target genes such as *aflA* and *aflP*, the present findings confirmed the applicability of PCR assays for the detection of *A. flavus*isolated from the feeds. Researchers reported strong antifungal activity of Cu NPs and chitosan nanocomposites against *A. flavus*, for example, benzoic acid nanogel (CS-BA) [10], CuO NPs [12], nanocomposites anti-aflatoxigenic [3,5,13,14]. In the current work, Cu-Chit/NC<sup>S</sup> hydrogel showed complete inhibition of growth against *A. flavus* strains at the highest concentration (240 ppm). Furthermore, we reported that the antifungal efficacy is influenced not only by nanocomposites concentration but also by type of tested strain. The antifungal efficacy of copper oxide nanoflowers as an antifungal agent against some phytopathogenic fungi like, *A. niger, A. flavus, Penicillium notatum*, and *A. alternata* were reported [52]. In addition, CS-Cu and CSZn NCPs show strong in vitro antifungal activity against *A. alternata, Rhizoctonia solani*, and *A. flavus* and are introduced as potential materials for innovative antimicrobials in cosmetics, foodstuffs, and textiles [53]. In in vitro assays, Cu-chitosan NPs were found to be effective in inhibiting fungal growth of some plant pathogens such as *Alternaria solani* and *Fusarium oxysporum* [54]. The antifungal activity of CS NPs against two aflatoxin producers such as *A. flavus* and *A. parasiticus* was demonstrated [55] and CS NPs succeeded in reducing total aflatoxin production and inhibiting the extent of fungal growth. The main protein composition in the absence or presence of Cu-Chit/NCs gel was analyzed by SDS-PAGE in comparison with protein markers and depending on the amino acid composition. Many protein bands have not been seen in nanocomposite treatment. Subsequently, the treatment of chitosan nanocomposites generates some biological reactions, such as oxidative stress-induced metabolic changes, which in turn affect the protein synthesis rate [56]. The toxicity of nanocomposites in fungal cells is due to severe metabolic changes, in particular protein synthesis, which resulted in a maximum protein reduction, as verified by the absence of the most important protein synthesis [57].

G6PDis a housekeeping enzyme that primarily regenerates adenine dinucleotide phosphate (NADPH) nicotinamide to sustain cellular redox homeostasis. Since NADPH is necessary for NADPH oxidase (NOX), synthase of nitrogen oxides to generate reactive oxygen species, and for signaling nitrogen, several new cellular functions have been established for G6PD [58]. Lack of glucose-6-phosphate dehydrogenase isozyme can make nanoparticles more susceptible to oxidative stress [59]. Several experiments can be checked that they explain the impact of metals on G6PDH activity, also Cd++ greatly influences on G6PDH activity in bacteria, fungi, and vertebrates; Ni++ inhibits the enzyme 's kinetic properties in mammals; Zn++ has extreme effects from a variety of influences on G6PDH; Cu++ has severe effects from bacteria and animals on G6PDH [60].

G6PD stimulates xenobiotic metabolism via the Nof2 signaling pathway and impacts the xenobiotic-metabolizing expression of the enzyme [61]. The full sense is that the inhibition of *A. aculeatus* G6PD activity by zinc and many other metal nanoparticles may be reinforced by potential production or otherwise formulation of polyketide mycotoxins in toxigenic fungi, including Aspergillus [62]. Additional attempts and modes of action study are needed to examine the molecular mechanisms

on which G6PD interacts with the Nrf2 pathway. This is the first report showing G6PD isozymes activity in *A. flavus* strains treated with prepared nanocomposites to understand the antifungal mechanisms. SEM images of the treated pathogen above show that the hyphae also had a swollen appearance, damaging the plasma membrane of both fungal spores and mycelium. Similar outcomes were investigated by Rubina et al. [20], who discovered that Cu-chitosan nanocomposites deteriorate fungal mycelia of *R. solani* from cotton and also *S. rolfsii* pathogenic to onion. Gold nanoparticles may alter and disturb the fungal cell membranes of *A. flavus, F. verticillioides*, and *P. citrinumdue* [63]. Weak sporulation with shrinking spores and defects was found in all *A. versicolor* strains treated with the modified nanocomposites [64]. More omics tools such as functional genomics, transcriptomics, proteomics, and metabolomics are required for the identification of different antifungal mechanism pathways for various nanomaterials that can be used against aflatoxigenic strains of Aspergillus and also suppress their aflatoxins production.
