*3.1. Antifungal Mechanism of Nanoparticles/Nanocomposites*

A promising nano-fungicide should possess an equivalent or superior activity corresponding to the bulk metal at relatively lower concentrations. Moreover, it is desirable to understand the phyto and eco-toxicity issues due to the release of metal ions. Multifarious mechanisms were involved in the antifungal activity executed by nanomaterials. The generalized antifungal activity is provided in the Figure 3. The antifungal activity of nanomaterials can be accomplished by the following events. Generally, fungal cell wall and cell membrane architecture involves chitin, lipids, phospholipids and polysaccharides with specific predominance of mannoproteins, β-1,3-D-glucan and β-1,6-D-glucan proteins [54]. Internalization of the nanomaterials occurs through three mechanisms; (i) direct internalization of nanoparticles in the cell wall, (ii) specific receptor-mediated adsorption followed by internalization, (iii) internalization of nanomaterials through ion transport proteins [55]. Post-internalization, the nanomaterials may inhibit the enzyme β-glucan synthase thereby affecting the N-acetylglucosamine [N-acetyl-D-glucose-2-amine] synthesis in the cell wall of fungi. As a consequence of enzyme inhibition, abnormalities like enhanced thickening of the cell wall, liquefaction of cell membrane, dissolution or disorganization of the cytoplasmic organelles, hyper-vacuolization, and detachment of cell wall from cytoplasmic contents indicating incipient plasmolysis might occur [56].

At the molecular level, the nanomaterials interact with various biomolecules and form complexes with different biomolecules thereby causing structural deformation in the biomolecules, inactivation of the catalytic proteins, and nucleic acid abnormalities like DNA breakage, and chromosomal aberrations [57,58]. Reactive Oxygen Species (ROS) play a critical role in antifungal activity mechanism of nano-composite materials. The metal ions trigger ROS and damage the biomolecules leading to cell death. Further, to authenticate the role of ROS in antifungal mechanism; Lipovsky et al. [59] deciphered that the increased expression of lipid peroxidation is a clear indicator of ROS generation. Meanwhile, stress enzymes like superoxide dismutase, glutathione dismutase, ascorbate peroxidase were upregulated/downregulated upon nanomaterials treatment in fungi [60].

### *3.2. Nano-Hybrid Antifungals for Control of Toxigenic Fungi and Mycotoxins Degradations*

Mycotoxins are the natural contaminants in food and feed worldwide. Changing climatic patterns have significantly affected the agricultural production due to limiting water, and land resources, temperature extremes and elevated humidity conditions [61]. The elevated humidity and temperature allowed for the proficient growth of a variety of mycotoxigenic fungal genera, *Aspergillus* spp., *Penicillium* spp., and *Fusarium* spp. which produce a variety of mycotoxins including aflatoxins (AFs), fumonisins (FBs), ochratoxins (OTs), trichothecenes (TCs), and zearalenone (ZEA). These mycotoxins cause detrimental health impacts in humans manifested as liver cancer, aflatoxicosis, malabsorption syndrome and reduction in bone strength [62].

**Figure 3.** Antifungal activity mechanisms of hybrid nanomaterials.

Conventionally, mycotoxins can be detected by chromatographic techniques like High-Pressure Liquid Chromatography (HPLC), Gas chromatography-Mass Spectrometry (GC-MS), and Liquid Chromatography-Mass Spectrometry (LC-MS) [62,63]. These techniques are robust, sensitive and specific but have high cost of analysis per sample and are therefore, expensive and time consuming. Further, to advance with innovation a simple, cheap and sensitive technique can be achieved with the aid of nanotechnology. Nano-based mycotoxin detection, and management involves specific properties such as selectivity, sensitivity, simplicity and multiple capabilities [64]. Hybrid nanomaterials are a new paradigm to counteract mycotoxin management. Generically, hybrid nanomaterials are having superior properties and multimodality (simultaneous detection, detoxification, and management abilities) when compared with polymers/metals, organic molecules when used individually in mycotoxicology [65].

Hybrid nanomaterials consisting of polymers/metals/organic molecules can synergistically interact with each other and accelerate the reaction kinetics (Figure 4). In mycotoxin management, nanohybrid materials are used for detection, detoxification and management [61]. For instance, Bhardwaj et al. [66] developed an immunosensor comprising graphene quantum dots (GOD), gold nanoparticles (AuNPs). Further, GOD-AuNPs were fabricated onto an indium tin oxide (ITO) electrode modified with an antibody (anti-AFB1) (anti-AFB1/GQDs-AuNPs/ITO) to detect Aflatoxin (AFB1). The hybrid immunosensor detected the AFB1 with high sensitivity for the presence of aflatoxin B1 even at very low concentrations (0.1 to 3.0 ng/mL) in the food sample.

Another important concern is mycotoxin detoxification. Hybrid nanomaterials are smart detoxifying agents. The hybrid nanomaterials can be incorporated in feed to sequester mycotoxin by forming a complex in the gastrointestinal tract so that the severity of the toxin gets ceased. To detoxify mycotoxin, Hamza et al. [67] devised a hybrid nanomaterial comprised of β-glucan mannan lipid particles (GMLPs) encapsulating the humic acid nanoparticles (HA-FeNPs). The specificity of this hybrid material was that the β-glucan molecules produced 3 to 4 µm hollow porous microspheres. Moreover, the addition of humic acid increased the binding affinity of Aflatoxin B1. The bare GMLPs and HA showed a moderate binding affinity for aflatoxin (10.8 µg AFB1/mg HA for GMLP HA). However, the addition of Fe increased the adsorbent capacity for GMLP HA-FeNPs AFB1 mass to

13.5 µg AFB1/mg HA and 16.8 µg AFB1/mg. This study showcased that this nanohybrid material can be used as a safe detoxification agent.

**Figure 4.** Hybrid-nanomaterial categories used in plant science: carbon nanotubes, including single-walled carbon nanotubes (SWCNTs) and multiwalled carbon nanotubes (MWCNTs), fullerenes); metallic and metal oxide NPs, silica-based nanostructures, and polymeric (dendrimers and liposomes) for the fabrication of biosensors for mycotoxin detection, detoxification of mycotoxin-contaminated food and feedstuffs through binders and management for sustainable control over fungal growth and mycotoxin contamination. (Cited from Thipe et al. with permission from Elsevier).

β β Recent findings related to nanocomposites (NCPs) based on organic polymeric and inorganic matrices or hybrid materials as effective antifungal agents against mycotoxigenic fungi and mycotoxin reduction have been summarized by Jampílek and Krá ´ lová, [68]. Spadola et al. [13] have identified an interesting alternative technique to inhibit aflatoxin production in *Aspergillus flavus.* They have formulated nanoparticles of poly-εcaprolactone polymer and loaded the generated nanoparticles with two thiosemicarbazone (benzophenone or valerophenone) compounds to curb mycotoxin production in *A. flavus.* Pirouz et al. [69] investigated the use of hybrid magnetic graphene oxides (MGOs) as an adsorbent for DON, ZEA, HT-2, and T-2 in naturally contaminated palm kernel cakes (PKC). At optimum reduction conditions at pH 6.2 for 5.2 h at 40.6 ◦C, the MGO was able to reduce the amount of DON, ZEA, HT-2. In the same direction, MGO adsorbents have been used to detoxify polluted AFB1 oils, their absorbents are made of MGO and magnetic reduced graphene oxides (MrGO) both of which are incorporated with Fe3O<sup>4</sup> nanoparticles. The MGO and MrGO were renewable, however, after seven cycles, with no major losses in the adsorption activities [70]. Copper-chitosan nanocomposite-based chitosan hydrogels (Cu-Chit/NCs hydrogel) have been prepared using metal vapor synthesis (MVS). Also, SEM measurements revealed damage to *A. flavus* cell membranes. Current findings indicate that the antifungal activity of nanocomposites in vitro can be beneficial depending on the type of fungal strain and the concentration of nanocomposites (Figure 5A). Cu-Chit/NCS hydrogel is a revolutionary nanobiopesticide developed by MVS used in food and feed to induce plant protection against mycotoxigenic fungi [71]. The fungicidal behavior of chitosan-silver nanocomposites (Ag-Chit-NCs) against *Penicillium expansum* from the feed

samples was investigated. Ag-Chit-NCs < 10 nm in size have an important antifungal inhibitory effect against *P. expansum*, the causative agent of blue mold-contaminated dairy cattle feed [72]. *P. expansum* treated with Ag-Chit *P. expansum* treated with Ag-Chit NCs was investigated by HR-SEM, alterations in conidiophores, metulae, phialides, and mature conidia characteristics had been observed to obtain information about the mode of action of Ag-Chit-NCs (Figure 5B). Therefore, nanocomposites can be utilized as viable alternatives to the already available arsenal of fungicides (Table 1).

**Figure 5.** (**A**) Antifungal activity of *Ag-Chit NCs against A. flavus* collected from feed samples. (i), control (without nanocomposite treatment), (ii), (iii), and (iv) fungal mat treated with 30, 60, and 90 milligrams of nanocomposites. All petri dishes treatment was incubated at 28 ◦C for 10 days. (**B**) Fungal mycelium of *P. expansum* treated with Ag-Chit NCs referred to the morphological changes in fungal hyphae, SEM images depicted markedly shriveled, crinkled cell walls, and flattened hyphae of the fungi (a), hyphal cell wall and vesicle damaged (b), irregular branching (a and b), and collapsed cell, formation of a layer of extruded material (d) Source (Abd-Elsalam KA. unpublished data).

To prevent fungal growth and mycotoxin production in food materials and packaging; hybrid nanofiber mats composed of cellulose acetate encapsulated with AgNPs were prepared by electrospinning. Due to close tight assembly of packaging in food materials, these nanofiber mats allowed low penetration of air permeability preventing fungal growth, and silver nanoparticles inherently inhibited the growth of yeast and molds [73]. Thus, these nanocomposite materials have shown great potential for future applications in the food packaging and preservation industry.

#### *3.3. Postharvest Management of Nanocomposite Against Pathogenic Fungi*

Perishable vegetables and fruits are spoiled due to transport, storage and growth of spoilage and opportunistic microbes. Therefore, microbial decay of fruits and vegetables is a great concern for researchers to formulate a driven strategy control measures with long-standing efficiency. Nanotechnology is an alternative solution to develop sustainable horticulture in preserving and managing post-harvest diseases of fruits and vegetables [74,75]. This technology offers various products such as packaging thin films; helping for labeling fresh products using the multiple chips (nanobiosensors), improvement of packaging appearance and prevention of the impact of gases and unsafe rays.

Conventionally, fungicides like imazalil, thiabendazole, pyrimethanil, fludioxonil and chloride-based chemicals have been used for management of post-harvest diseases of horticultural produce [61]. Though effective but prolonged use of these fungicides has led to development of among the fungal genera. Further, the active ingredients of the fungicides are toxic to humans and also to the ecosystem. Post-harvest diseases can be classified into two groups (i) diseases from field infection (ii) diseases due to post-harvest infection ([76,77]).

Citrus orange fruit, *Citrus sinensis* L. Osb., is often spoiled by *P. digitatum* during the post-harvest storage/transportation periods. To counteract against the pathogen a nanocomposite clay-chitosan nanocomposite (CCNC) was synthesized and evaluated under in-vitro conditions; at 20 µg/mL the nanocomposite completely inhibited the growth of *P. digitatum*. In *in-vivo* trials, the nanocomposite reduced the lesions by 70% and inhibited the disease in orange [47]. The CNCC coated orange were observed to be free from the disease, and exhibited high pH, chroma, peel moisture, and firmness in comparison with the control [47].

A chitosan-Titanium dioxide composite film (70 µm thickness) was used as a packaging material to extend shelf life during the postharvest storage of grapes by preventing spoilage microbial infection. The composite film enhanced the shelf life and resisted the mildew infection in stored grapes for up to 22 days [78]. Similarly, a nanocomposite of silver/gelatin/chitosan was applied as a hybrid film in grapes to improve the storage shelflife under cold conditions. The hybrid film stored grapes didn't show signs of infection, had a fresh appearance and showed no leakage of the grapes [79].

Banana (*Musa acuminata* L.) is a famed fruit consumed by all peoples and is considered to be one of the high-valued fruit in horticulture. During post-harvest, the banana fruits get deteriorated on storage and transportation periods due to its climacteric nature i.e., increased respiration rate. To overcome this and ensure delayed ripening, maintenance of fruit firmness and reduced mass loss of fruits coating of a nanocomposite containing soybean protein isolate, cinnamaldehyde, and ZnO NPs on the banana fruit was observed to be very effective [80]. In another study, a predominant disease (anthracnose) caused by *Colletotrichum musae* in banana causing a major loss to the farmers was prevented by use of metallic nanoparticles (silver, nickel, copper and magnesium) prepared from ajwain and neem leaf extract. In postharvest period, silver nanoparticles were sprayed on the banana at different concentrations ranging from 0.02 to 0.2 percent resulting in reduction in the anthracnose infection (6.67 percent disease index) on use of the least concentration of 0.2% AgNPs [81].

Apple (*Malus domestica* Bork) is a perishable fruit consumed by all ages worldwide. Since it is a climacteric fruit, it is indeed useful to pro-act for development of a sustainable methodology to prevent post-harvest losses of apple fruit. Polylactic acid incorporated with ZnO nanoparticles was applied as a thin film in a fresh-cut apple stored at 4 ◦C for 14 days. PLA-ZnO NPs exhibited effective inhibition of yeast and molds in fresh-cut apples. This showed the possibility that PLA nanocomposite can be used as a packaging material in apples during the storage period [82].

Mango (*Mangifera Indica* L.) is also supposed to be highly prone to infections by post-harvest pathogen(s) causing anthracnose diseases. A chitosan-silver NPs composite was prepared and evaluated for the antifungal effect against conidial germination of *C. gloeosporioides*. The prepared chitosan-silver nanocomposite (at 100 µg mL−<sup>1</sup> ) completely suppressed the spore germination. Under in vivo conditions, mango fruits were inoculated with the fungal spores. The infected mango fruit were then coated with the aforementioned nanocomposite and evaluated for the disease incidence. The nanocomposite prominently lowered the disease incidence with 45.7% and 71.3% at 0.5 and 1.0% of nanocomposite respectively [83]. Likewise, a nanocomposite containing aloe vera gel, ZnOPs and glycerol was coated on an edible mango and stored for 9 days at room temperature. After the storage days, the edible mango didn't show any sign of infection/diseases [84]. Similarly, nanoemulsion containing chitosan was effectively inhibited *C. musae* and *C. gloeosporioides*. However, the chitosan nanoemulsion showed better prospective results than chitosan nanocomposite in banana, papaya and dragon fruits [85]. New biopolymers composite oligochitosan (OCS) and OCS/nanosilica (OCS/nSiO2) hybrid materials with impressive synergistic action are likely to be considered potential protections for plants infected with

*Colletotrichum* sp. Conjugated nanomaterials may be considered possible biotic elicitors that not only avoid anthracnose disease but also efficiently enhance plant growth [86]. The same team found that silica and hybrid material had good antifungal properties against *P. infestans*, the causal organism for late blight in tomato and potato, but the antifungal properties of hybrid materials, due to their synergistic effect, had better antifungal capacities than that of each individual component. Interestingly, the inhibition zone diameters of OCS/nSiO<sup>2</sup> were approximately 4–5 mm and 7–9 mm larger than those of OCS and nSiO2, respectively [87]. Bio-synthetized MgO nanoparticles are produced using the native bacterial strain like *Bacillus* sp. The RNT3 strain was used to render CS-Mg nanocomposite. CS-Mg nanocomposite has demonstrated impressive antimicrobial activity against *Acidovorax oryzae* and *R. solani* and substantially inhibited development compared to the non-treated control [88]. In vivo assays with two plant hosts including tomatoes and peppers affected by *Fusarium* wilt and root rot diseases in which traditional chemical fungicides were used for comparative purposes displayed better antifungal activity of rGO-CuO NPs and a long-lasting impact at a very low concentration of 1 mg/mL. Interestingly, as CuO is a plant nutrient, the study of treated plants showed a positive impact on flowering, plant height and dry weight, as well as the aggregation of photosynthetic pigments [89].


**Table 1.** Nano-composite formulation and their application for curbing the plant fungal pathogens.


**Table1.***Cont.*
