**2. Mechanism of Nanoparticle Synthesis from** *Fusarium*

As discussed earlier, members of the genus *Fusarium* can synthesize metal nanoparticles both intracellularly and extracellularly. As far as the mechanism of extracellular mycosynthesis is concerned, it is proposed that metabolites such as enzymes, proteins, polysaccharides, flavonoids, alkaloids, phenolic and organic acids, etc. secreted by funguslike *Fusarium* for their survival when exposed to different environmental stresses are mostly responsible for the reduction of metals ions to metallic nanoparticles through the catalytic effect [67]. Moreover, the same metabolites act as reducing and stabilizing agents which are further responsible for the growth and stabilization of biogenic metal nanoparticles [13,22]. Figure 3 represents the schematic illustration of the general mechanism involved in the synthesis, growth, and stabilization of metal nanoparticles using fungus such as *Fusarium*.

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One of the hypothetical mechanisms proposed is that NADH-dependent nitrate reductase enzyme secreted by *F. oxysporum* is responsible for the reduction of aqueous silver ions into AgNPs [16]. A similar mechanism has been proposed by Ingle et al. [10] in case of synthesis of AgNPs from *F. acuminatum*, and they also pointed the involvement of cofactor NADH and nitrate reductase enzyme in the biosynthesis of AgNPs because they reported the presence of nitrate reductase in fungal cell-free extract using specific substrate utilizing discs for nitrate purchased from Hi-Media Pvt. Ltd. Mumbai, India.

**Figure 3.** General mechanism involved in the synthesis, growth, and stabilization of metal nanoparticles using fungus.

In addition, Duran et al. [68] and Kumar et al. [69] proposed almost similar mechanisms for the biosynthesis of AgNPs from *F. oxysporum*. In the former study, the authors reported the role of anthraquinone and the NADPH-nitrate reductase in the biosynthesis of AgNPs, and it was hypothesized that the electron required to fulfill the deficiency of aqueous silver ions (Ag<sup>+</sup> ) and convert it into Ag neutral (Ag<sup>0</sup> i.e., AgNPs) was donated by both quinone and NADPH. However, in the later study, it was demonstrated that the reduction of NADPH to NADP<sup>+</sup> and the hydroxyquinoline possibly acts as an electron shuttle transferring the electron generated during the reduction of nitrate to Ag<sup>+</sup> ions, converting them to Ag<sup>0</sup> (Figure 4).

There are reports suggesting various hydroquinones to act as electron shuttles reducing the metal ions. *F. oxysporum* f. sp. *cubense* JT1 demonstrated to have the capacity to reduce the gold ions to AuNPs [48]. Moreover, as per Ahmad et al. [16], the capacity of reducing metal ions is species-specific. The reductase specific to *F. oxysporum* and *F. moniliforme* were not able to synthesize AgNPs intracellularly and extracellularly. In addition to extracellular mechanisms, there are few mechanisms proposed for intracellular mycosynthesis of metal nanoparticles. In the case of *Fusarium*-mediated intracellular mycosynthesis, metal nanoparticles usually formed below the cell surface and this may be due to the reduction of metal ions by metabolites (i.e., enzyme) present in the cell membrane. Generally, a two-step mechanism has been proposed for intracellular mycosynthesis of nanoparticles. In the first step, aqueous metal ions are attached to the fungal cell surface by the electrostatic interaction between lysine residues and metal ions (M<sup>+</sup> ). However, in the second step, the actual mycosynthesis of nanoparticles occurs by the enzymatic reduction of metal ions (M<sup>0</sup> ), which leads to the aggregation and formation of nanoparticles [49] (Figure 5).

**Figure 4.** Hypothetical mechanism of AgNPs biosynthesis from *F. oxysporum* (Adapted from Duran et al. [67], an open-access article).

**Figure 5.** Hypothetical intracellular mechanisms for the synthesis of metal nanoparticles from *F. oxysporum* (Adapted and modified from Yadav et al. [13]; with copyright permission from Springer).

Moreover, various other studies performed on mycosynthesis proposed the role of different other enzymes and proteins. However, among all these mechanisms for extracellular mycosynthesis, the hypothetical mechanism involving the role of NADHdependent nitrate reductase enzyme has been widely accepted.
