**3. Nanocomposites and Their Mode of Action on the Fungal Phytopathogens**

Nanocomposite materials include multi-phase components. These materials may be comprised of components with variable phase domains with atleast one continuous phase while another having nano-scale dimensions [23]. These hybrid nanomaterials can be generated through co-synthesis/impregnation of diverse inorganic and organic components [24]. Nanocomposites have been extensively studied due to the properties of inorganic and organic materials that enact concurrently to perform the desired activity [25]. Generally, nanocomposites are derived by the addition of nano-particulate materials in long-chain or short-chain polymeric matrices. The derived nanocomposites exhibit improved properties not observed for any of the individual components. Most likely, the combination of polymers with nanoparticles is anticipated to increase the properties of the polymer significantly [26]. Such kind of nanocomposites are now widely being used in food processing, pest detection and management, food health screening, water treatment, disease detection, drug-delivery systems, and improvement of sustainable agriculture [27–30]. Likewise, the polymer composites act as fertilizers which increase the nutrients uptake, decrease soil toxicity [31,32]. Moreover, nanocomposites are well being used to increase the shelf life of food materials by acting as antimicrobial dispositions and as sensors [33].

Plant disease management using hybrid polymer nanocomposites is focused on making mulch films to control weeds; as nano pesticides and as a biostatic agent [34]. Min et al. [35] demonstrated that silver nanoparticles can effectively inhibit the phytopathogens such as *Rhizoctonia solani*, *Sclerotinia sclerotiorum*, and *S. minor*. Further, silver nanoparticles can cause extensive damage by breaking the hyphal wall membrane followed by internal damage of the hyphae. Sepiolite, a magnesium silicate, was blended with MgO to form a (SE-MgO) nanocomposite that exhibited excellent antifungal activity against rice pathogen *Fusarium verticillioides*, *Bipolaris oryzae*, and *Fusarium fujikuroi* with ED90 > 249 µg/mL compared with MgONPs [36].

Carbon nanomaterials have shown to possess strong antifungal activity against pathogens *Fusarium graminearum* and *Fusarium poae* [37]. Six carbon nanomaterials (CNMs), single-walled carbon nanotubes (SWCNTs), multi-walled carbon nanotubes (MWCNTs), graphene oxide (GO), reduced graphene oxide (rGO), fullerene (C60), and activated carbon (AC) were evaluated against the phytopathogens. The outcome imposes that SWCNTs, MWCNTs, GO and rGO at varying concentrations decreased the biomass, mycelia growth and inhibited spore germination of the fungal pathogens. However, the C<sup>60</sup> and AC didn't exert any activity against the tested pathogens.

In another study, GO-AgNPs nanocomposite was synthesized through interfacial self-assembly and was evaluated for its antifungal activity against the pathogen *Fusarium graminearum* under in vitro and in vivo conditions [38]. The fungicidal activity of GO-AgNPs against the *F. graminearum* spores was 4.68 µg/mL; the nanocomposite also constrained the germination of spores and hyphae. Moreover, the authors demonstrated response to GO-AgNPs through the SEM analysis of the events of spore germination of *F. graminearum*. The images portray that *F. graminearum* conidia were crumpled, widened, and damaged heavily as seen in Figure 2. The fungicidal activity of nanocomposite is due to the adsorption of nanoparticles to the fungal cell wall membranes and oxygen groups of GO form hydrogen bond with lipopolysaccharide subunits of the cell membrane which contains sugars, phosphates, and lipids.

**Figure 2.** SEM images of *F. graminearum* spores incubated with sterile water (**C**) control and treated with GO-AgNPs nanocomposite (**T**). Images obtained with permission from Chen et al. [38]. (Cited from Chen et al., with permission from ACS).

Downy mildew is a disease caused by *Plasmopara viticola* in grapes plants that leads to extremely heavy yield loss. To combat the disease, a nanocomposite construct consisting of Graphene oxide (GO) and Iron oxide (Fe3O4) as GO-Fe3O<sup>4</sup> nanocomposite was developed [39]. The study demonstrated that the pretreatment of the leaf discs with nanocomposite followed by inoculation with *P. viticola* sporangium suspension strongly inhibited the spore germination. This effect may be attributed to the blockage of the water channels of sporangia by surface adsorption of the nanocomposites. From the images, it is

evident that bare GO and Fe3O<sup>4</sup> possessed moderate spore germination inhibition activity while the nanocomposites triggered stupendous activity [39].

Silver-Titanate nanotubes (AgTNTs) nanocomposite was synthesized through a onepot chemical method and functionalization with AgNPs. These nanocomposites were further evaluated against the phytopathogenic fungi *Botrytis cinerea* by the photoinactivation method. The nanocomposite stimulated Reactive Oxygen Species (ROS) cascades and damaged the conidia which eventually led to cell death [40]. In a microwave-assisted method, a magnetically separable Fe3O4/ZnO/AgBr nanocomposite was synthesized. These synthesized nanocomposites inactivated the *Fusarium graminearum* and *Fusarium oxysporum* within a short period of 120 and 60 min. Thus, the efficacy of nanocomposites can be identified as the combined aggregation of the inorganic metal complexes [41].

The leaf extract of *Adhatoda vasica* was utilized as a reducing agent to synthesize Copper oxide nanoparticles/Carbon nanocomposite through the green chemistry approach [42]. These nanocomposites exhibited effective growth inhibitory activity against *Aspergillus niger*. Thus, from these reports, it is evident that inorganic metals possessing inherent properties when combined as nanocomposites as a result of synergistic effects result in the fabrication of nanocomposites which exhibit activities on comparison with bare metals. However, the behavior of metal alloys during nanocomposite synthesis will differ according to the synthesis methods; elemental composition, and the applications.

Nanocomposites can be manufactured from any combination of materials like polymers, metals, and ceramics [43]. Among the materials, polymers and inorganic/organic materials will have a high aspect ratio and surface properties which enable them to be widely used in a different range of industries. In agriculture, polymers play an indispensable role in the release of chemical moieties such as fungicides, insecticides, growth stimulants and germicides [44]. The promising advantage of polymers is to control the release rate and rate of biodegradability of the embedded or encapsulated compound. These features make polymers widely used as a delivery agent in medicine and agrochemicals [45,46]. Polymers such as cellulose acetate phthalate, gelatin, chitosan, gum Arabic, polylactic acid, poly-butadiene, poly-lactic-glycolic acid, polyhydroxyalkanoates, polyvinyl alcohol (PVA), polyacrylamide, and polystyrene are widely used as delivery agents for drugs and agrochemicals [13,23].

In agriculture, the usage of polymer nanocomposites is to be critically chosen according to the application. Further, natural polymers are preferably chosen for the agriapplications owing to the nature of degradability and controlled release behavior. Chitosan polymer is gaining a new avenue in the plant protection field due to its outstanding properties. Nanochitosan exhibits anti-microbial potentials against bacteria and fungi at varying levels of concentrations. Combined formulation comprised of metal or metal oxide nanomaterials encapsulated or embedded in chitosan exhibit improved antimicrobial potentials. For instance, nanocomposite Ag-chitosan prominently exhibited antibacterial activity to a higher extent. Likewise, the fungicidal activity of clay chitosan nanocomposite was evaluated against *Penicillium digitatum* under in vivo and in vitro conditions [47]. The results were promising in terms of the superior activity exhibited by the nanocomposite formulation. Likewise, copper nanoparticles (Cu NPs), zinc oxide nanoparticles (ZnNPs), and chitosan, zinc oxide, and copper nanocomposites (CS–Zn-Cu NCs) were chemically fabricated and evaluated against plant pathogenic fungi *A. alternata*, *R. solani*, and *B. cinerea* [48]. The results intrinsically acclaimed that nanocomposite displayed higher activity at a concentration of 90 µg mL−<sup>1</sup> . Antifungal activities of Bimetallic blends and Zn-Chitosan, and Cu-Chitosan at concentrations of 30, 60, 90 µg mL−<sup>1</sup> effectively inhibited the growth of *Rhizoctonia solani*. Further, it indicated effective control of cotton seedling damping-off under greenhouse conditions [49]. Silver/chitosan nanocomposite portrayed incremental growth-inhibitory effect against phytopathogens isolated from chickpea seeds [50]. Moreover, the individual metal and polymer components exhibited inhibitory activity lower than the Silver/Chitosan nanocomposite against the test pathogen, *Aspergillus niger*. A combination of chitosan/silica nanocomposite was evaluated against *Botrytis cinerea* under

in vitro and in vivo (natural and artificial infections) conditions [51]. The in vitro study revealed complete reduction of the fungal growth by the nanocomposite compared to 72% and 76% inhibition potential of chitosan and silica nanoparticles respectively. Moreover, under natural conditions, the Chitosan/silica nanocomposite effectively hampered the gray mold disease in Italian grapes by 59% and in Benitaka grapes by 83% without affecting the grape quality.

The chitosan conjugated Ag nanoparticles functionalized with 4(*E*)-2-(3-hydroxynaphthalene-2-yl) diazenyl-1-benzoic acid were prepared which demonstrated improved effectiveness against *A. flavus* and *A. niger* forming larger inhibition zone of 20.2 mm and 27.0 mm respectively [52]. Likewise, chitosan hydrogel with cinnamic acid encapsulating *Mentha piperita* essential oil markedly inhibits the growth of mycelia of *A. flavus* at a concentration of 800 mg/mL [53]. Thus, with the above appropriate scientific evidence, it can be inferred that organic/inorganic-metal-polymer hybrid nanocomposites exhibit exceptionally superior anti-fungal activities under in vitro and in vivo conditions.
