Chitosan-Loaded Inorganic Oxide Nanocomposites (SiO2, ZnO, CuO) for Effective Control of Postharvest Fungal Diseases and Maintaining Apple Fruit Quality

TL;DR

Scientists created chitosan-based nanocomposites that effectively fight fungal diseases in apples, offering a sustainable alternative to traditional fungicides.

Contribution

The study introduces scalable, eco-friendly chitosan-inorganic oxide nanocomposites with dual antifungal and host-defense-enhancing properties.

Findings

CS-CuO nanocomposites showed over 80% growth inhibition against Botrytis cinerea and Alternaria alternata.

In vivo trials on apples demonstrated an 81% reduction in lesion area with CS-CuO treatment.

CS-CuO treatment increased total phenolic content and antioxidant activity in infected apples.

Abstract

Phytopathogenic fungi pose a critical threat to global food security through substantial pre- and post-harvest crop losses, intensified by climate change and fungicide resistance. To address this, we synthesized low-concentration chitosan–inorganic oxide nanocomposites (CS-SiO2, CS-ZnO, CS-CuO) via ionic gelation, a green and scalable method. Comprehensive characterization (DLS, UV-Vis, FTIR, XRD, SEM) confirmed nanocomposite formation, CS-SiO2 exhibited uniform particle sizes (200–250 nm), while CS-CuO showed slightly larger particles, all with excellent dispersity. Zeta potential analysis confirmed strong colloidal stability, with pure chitosan nanoparticles (CSNPs) displaying a surface charge of +12.9 mV, while all nanocomposites retained positive charges, enhancing adhesion to negatively charged fungal membranes. In vitro antifungal assays against Alternaria alternata, Botrytis cinerea, Colletotrichum graminicola, and Fusarium graminearum demonstrated hierarchical efficacy: CS-CuO > CS-ZnO > CS-SiO2, with CS-CuO achieving >80% growth inhibition against B. cinerea and A. alternata. SEM revealed severe hyphal damage and spore collapse in CS-CuO-treated fungi, attributed to synergistic reactive oxygen species (ROS) generation and chitosan-mediated membrane disruption. In vivo trials on B. cinerea-infected apples showed CS-CuO reduced lesion area by 81% and elevated host defense markers, including a 1.5-fold increase in total phenolic content and higher DPPH radical scavenging activity compared to controls. These nanocomposites, particularly CS-CuO, offer a sustainable, dual-action solution direct antifungal activity and enhanced host resilience while minimizing environmental impact. By integrating scalable synthesis, eco-compatibility, and efficacy, this work advances chitosan–inorganic oxide nanocomposites as viable alternatives to conventional fungicides, with immediate potential for agricultural and postharvest applications.

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1. Introduction

Phytopathogenic fungi, notably Botrytis cinerea and Alternaria alternata, represent a persistent and escalating threat to global food security, causing devastating pre- and post-harvest crop losses estimated at ~40% of global agricultural waste and incurring annual economic damages exceeding $200 billion [1,2]. The widespread emergence of fungicide-resistant fungal strains further compounds this challenge, drastically diminishing the efficacy of conventional chemical controls [3]. This is particularly true for B. cinerea, where resistance has compromised approximately 80% of commercial fungicides [4]. Exacerbated by climate change, this escalating crisis necessitates the urgent development of effective yet sustainable alternatives to traditional fungicides. Among these, chitosan (CS) stands out as a particularly promising candidate. Its inherent biodegradability, intrinsic antifungal properties, reduced environmental toxicity, and polycationic nature, which enhances nanoparticle adhesion to fungal cell walls, position it as a multifaceted solution to address fungal resistance while minimizing ecological harm [5,6].

Chitosan (CS) is a polycationic linear polysaccharide derived from the deacetylation of chitin, primarily sourced from crustacean exoskeletons and fungal cell walls [7]. It is composed of β-(1→4)-linked D-glucosamine and N-acetyl-D-glucosamine units. CS is soluble in dilute acidic aqueous solutions due to the protonation of its amino groups, and it exhibits a compelling combination of properties for diverse applications in biomedicine and agriculture. These include low toxicity, biodegradability, stability across a wide pH range, and inherent antioxidant, antifungal, and antibacterial activities [8]. Chitosan nanoparticles (CSNPs), often formed via ionic cross-linking leveraging the interaction of CS’s positively charged amino groups with negatively charged polymers or ions like tripolyphosphate (TPP) [9,10], provide a versatile platform for enhancing these bioactivities. In particular, nanotechnology, specifically metal oxide nanoparticles (MONPs), offers a promising avenue for augmenting fungal disease control [11]. MONPs, such as ZnO and CuO, are recognized for their potent antifungal mechanisms that are less prone to inducing resistance. Strategically combining MONPs with CS leverages synergistic effects, enhancing both stability and bioefficacy while maintaining an eco-friendly profile that contrasts sharply with conventional fungicides [5,12]. CS’s inherent antimicrobial properties and biocompatibility further establish it as an excellent matrix for developing advanced antifungal nanocomposites [6,13].

MONPs, including ZnO and CuO, further amplify antifungal efficacy through mechanisms such as reactive oxygen species (ROS) generation, while SiO_2_ can enhance the nanocomposite’s mechanical stability [14,15]. The strategic combination of CS with MONPs capitalizes on synergistic mechanisms: chitosan facilitates nanoparticle adhesion to fungal cells, while inorganic oxides induce oxidative stress and disrupt membrane integrity [16]. Therefore, this study addresses this critical gap by developing and evaluating low-concentration chitosan–inorganic oxide nanocomposites (0.1% w/v) synthesized via facile ionic gelation [17,18]. We comprehensively assess their synergistic antifungal efficacy against key postharvest pathogens, elucidating their mechanisms of action and exploring their capacity to enhance fruit defense responses [18]. This research aims to provide a scalable, cost-effective, and eco-friendly solution to mitigate postharvest losses, offering a viable alternative to conventional fungicides for sustainable food preservation.

We strategically selected ZnO, CuO, and SiO_2_ inorganic oxides based on their distinct and complementary antifungal mechanisms. While chitosan composites with individual inorganic oxides have been explored, a systematic comparative study evaluating their efficacy when embedded within an identical chitosan matrix via a unified green synthesis route remains less common. Such a direct comparison is crucial for establishing a clear hierarchy of antifungal performance. Therefore, by integrating CS with MONPs via ionic gelation, this study aims to: (i) synthesize low-concentration chitosan–inorganic oxide nanocomposites; (ii) rigorously evaluate their synergistic antifungal efficacy against key postharvest pathogens (A. alternata, B. cinerea, C. graminicola, and F. graminearum); and (iii) elucidate their role in enhancing biochemical defense mechanisms in treated apple fruit. Furthermore, advancing beyond direct toxicity, we investigate the potential of these nanocomposites to simultaneously elicit host plant defense responses, presenting a promising dual-mode action strategy for sustainable disease management. This study focuses on a low-concentration (0.1% w/v) formulation from the outset, prioritizing environmental compatibility while seeking potent efficacy. Ultimately, our findings seek to establish a sustainable and effective alternative to conventional fungicides, contributing to both enhanced food security and reduced environmental impact in postharvest disease management.

2. Materials and Methods

2.1. Materials

2.1.1. Chemical Reagent

Chemicals used in this study were obtained from the following suppliers: Chitosan (80–95% deacetylation, (C_6_H_11_NO_4_)n F.W.), Zinc acetate (Zn(CH_3_CO_2_)2·2H_2_O) and ammonium hydroxide solution (NH_4_OH) (25%) from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China); Rose Bengal Agar and Potato Dextrose Agar from Beijing Land Bridge Technology Co., Ltd. (Beijing, China); CuCl_2_·2H_2_O from Xilong Scientific Co., Ltd. (Shantou, China); NaOH from Guangdong Guanghua Sci-Tech Co., Ltd. (Shantou, China); Ethanol, Isopropyl alcohol, acetic acid and HCl from Tianjin Kemiou Chemical Reagent Co., Ltd. (Tianjin, China); tetraethyl orthosilicate (TEOS) from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China); tripolyphosphate (TPP) from Macklin Co., Ltd. (Shanghai, China); and KBr from ChengDu Chron Chemicals Co., Ltd. (Chengdu, China).

2.1.2. Fungal Strains

Alternaria spp. and Fusarium spp. were isolated from infected sweet cherry fruits and ginger, respectively. These isolates underwent molecular identification (Section 2.1.3). Initial isolations were performed on Rose Bengal Agar (RBA) medium supplemented with chloramphenicol (50 mg/L) to inhibit bacterial growth. Pure cultures were established through single-spore isolation on a Potato Dextrose Agar (PDA) medium. Botrytis cinerea (GenBank accession number PQ569562) obtained from the department of plant pathology Al-Azhar university, Cairo, Egypt, and Colletotrichum graminicola, obtained from plant pathology department, Huazhong Agriculture University, Wuhan, China, which used as reference strains.

2.1.3. Morphological and Molecular Characterization

Fungal identification was performed using morphological and molecular techniques for Alternaria spp. and Fusarium spp. Morphological characterization involved examining colony characteristics, spore morphology, and mycelial features using light microscopy. Species confirmation was achieved by sequencing the Internal Transcribed Spacer (ITS) region. Amplification products were sequenced using an ABI3730 Sanger sequencer (Applied Biosystems, Foster City, CA, USA). Fungal cultures were maintained by two conservation methods: short-term storage on PDA slants at 4 °C and long-term storage in 50% glycerol at −80 °C.

2.2. Synthesis of Nanoparticles

2.2.1. Synthesis of Inorganic Oxide Nanoparticles

Synthesis of CuO Nanoparticles

Copper Oxide Nanoparticles (CuO NPs): CuO NPs were synthesized using a wet chemical method. Copper chloride dihydrate (CuCl_2_·2H_2_O, 9.0 g) was dissolved in 200 mL of ethanol. Separately, sodium hydroxide (NaOH, 5.4 g) was dissolved in 50 mL of ethanol. The NaOH solution was added dropwise to the copper chloride solution under constant stirring at room temperature until the color changed from green to black. The resulting precipitate was collected by centrifugation (10,000 rpm, 10 min), washed sequentially with ethanol 50% followed by deionized water, dried at 65 °C overnight, and then annealed at 500 °C for 4 h. The annealed product was ground to obtain a powdered form of CuO NPs [19].

Synthesis of ZnO Nanoparticles

Zinc Oxide Nanoparticles (ZnO NPs): ZnO NPs were prepared by dissolving zinc acetate dihydrate (Zn (CH_3_CO_2_)2·2H_2_O, 8.78 g) in 840 mL of isopropyl alcohol at 50 °C. A solution of sodium hydroxide (NaOH, 3.2 g in 160 mL of 7:1 isopropyl alcohol:water) was added dropwise under ice-cold conditions, followed by heating at 65 °C for 2 h and aging for 7 days. The resulting product was centrifuged, dried at 65 °C overnight, and calcined at 400 °C for 2 h [20].

Synthesis of SiO2 Nanoparticles

Silicon Dioxide Nanoparticles (SiO_2_ NPs): SiO_2_ NPs were synthesized using tetraethyl orthosilicate (TEOS, 45 mL) dissolved in 50% ethanol solution (55 mL). The pH was adjusted to 11 using ammonium hydroxide solution (25%), and the mixture was stirred at 70 °C overnight. The product was collected by centrifugation, washed with deionized water until neutral pH, dried at 65 °C, and annealed at 550 °C for 4 h [14].

2.2.2. Preparation of Chitosan–Inorganic Oxide Nanocomposites

A 0.1% (w/v) CS solution was prepared by dissolving CS in 1% acetic acid with magnetic stirring overnight at room temperature until a clear solution was obtained. The pH of the CS solution was adjusted to 4.8 using 5 M NaOH and then stored at 4 °C for 4 h. MONPs (0.1% w/v) were dispersed in 1% acetic acid by sonication, stirred for 1 h, and then added dropwise to the CS solution under constant stirring for 5 h. A 0.05% (v/v) tripolyphosphate (TPP) solution was prepared, and the pH was adjusted to 5 using 1 M HCl, and then added dropwise to the CS-inorganic oxide mixture under constant stirring. CSNPs were prepared by adding TPP to the CS solution under the same conditions. The working concentration of 0.1% (w/v) for nanocomposites was selected based on preliminary assays with chitosan nanoparticles, which indicated this concentration provided a sub-maximal inhibitory effect suitable for evaluating the enhancement conferred by inorganic oxide incorporation. The resulting nanocomposites (CS, CS-CuO, CS-ZnO, CS-SiO_2_) were purified by centrifugation (10,000 rpm, 20 min), washed three times with deionized water to remove residual acetic acid, and then freeze-dried for storage [14]. For biological assays, a 10% (w/v) concentration of the rehydrated pellet was prepared in deionized water.

2.3. Characterization of Nanoparticles

The synthesized MONPs (CuO, ZnO, and SiO_2_) and their chitosan composites (CS, CS-CuO, CS-ZnO, and CS-SiO_2_) were characterized using multiple techniques to determine their size, surface properties, chemical structure, and crystalline nature. Particle size distribution and zeta potential were measured using dynamic light scattering (DLS) with a Zetasizer Nano ZS (ZEN3600, Malvern Instruments Ltd., Malvern, UK). Samples were diluted to 0.01% (w/v) in deionized water. Samples were briefly sonicated (10 min) prior to analysis to ensure complete dispersion. Measurements were then performed at 25 °C with a scattering angle of 173°, with each sample measured in triplicate [14]. UV-Vis absorption spectra were recorded using a UV-2600i spectrophotometer (Shimadzu, Kyoto, Japan) in the 200–800 nm range. The Samples were diluted to 0.01% (w/v) in deionized water for UV-Vis analysis. Fourier transform infrared (FTIR) spectra were obtained using a Bruker Vertex 70v Spectrometer (Bruker, Ettlingen, Germany) within the 4000–400 cm^−1^ range with a resolution of 4 cm^−1^, using the KBr pellet method and averaging 64 scans for each spectrum. X-ray diffraction (XRD) patterns were collected using a D8 Advance diffractometer (Bruker, Karlsruhe, Germany) with Cu Kα radiation (λ = 1.5406 Å) at 40 kV and 40 mA, with scans performed in the 2θ range of 10–80° with a step size of 0.02° and a scan rate of 2°/min. The morphology and size of the nanoparticles were examined using a field emission scanning electron microscope (Hitachi SU 8600 SEM, Hitachi High-Technologies, Tokyo, Japan) operated at a voltage of 5 kV, after deposition on a silicon wafer and sputter-coating with a thin layer of gold.

2.4. In Vitro Antifungal Activity Assessment

2.4.1. Fungal Culture and Preparation of Treatment Media

The antifungal efficacy of the synthesized nanoparticles was evaluated against four phytopathogenic fungi: A. alternata, B. cinerea, C. graminicola, and F. graminearum. Fungi were cultured on potato dextrose agar (PDA) medium. B. cinerea was incubated at 22 °C, while the remaining fungi were incubated at 28 °C for 7 days. Nanoparticle suspensions (CSNPs, SiO_2_NPs, ZnONPs, CuONPs, CS-SiO_2_NPs, CS-ZnONPs, and CS-CuONPs) were sonicated for 10 min to ensure uniform dispersion. Nanocomposites were tested at 0.1% (w/v) in PDA, with fungal plugs standardized to 7 mm diameter to ensure uniform exposure.

2.4.2. Antifungal Assay

Mycelial plugs (7 mm diameter) were excised from the periphery of 7-day-old fungal cultures using a sterile cork borer and placed at the center of the treatment plates. Control plates contained only PDA medium. All treatments were performed in triplicate and incubated at their respective optimal temperatures for 7 days [21]. Antifungal activity was quantified by measuring the radial growth of fungal colonies. The inhibition rate was calculated using the following formula:

$$[eqn]$$

where A represents the colony diameter in control plates, and B represents the colony diameter in treated plates [22].

2.4.3. SEM Analysis of Fungal Morphological Changes

Morphological changes in fungal structures following nanoparticle treatments were examined using scanning electron microscopy. Small segments (2–3 mm) were carefully excised from the growing edge of the fungal colonies cultured on PDA media containing different nanoparticle treatments after 4 days. These samples were fixed in a 2.5% glutaraldehyde solution overnight at 4 °C, followed by a sequential dehydration in a graded series of ethanol (30%, 50%, 70%, 90%, and 100%) for 15 min at each concentration and then left at 100% until drying. Dehydrated specimens were dried using a critical point dryer (EM CPD300 Critical Point Dryer; Leica, Wetzlar, Germany), mounted on aluminum stubs, and sputter-coated with gold for 30 s. SEM imaging was performed using a field emission scanning electron microscope operated at 10 kV, focusing on hyphal morphology and spore structural modifications [23].

2.5. In Vivo Antifungal Activity Assessment on Apple Fruits

The protective effects of chitosan-based nanocomposites against B. cinerea were evaluated using apple fruits. Healthy, uniform-sized apples were selected and randomly assigned to five treatment groups (n = 3 per group). Fruits were surface sterilized by immersion in a 1% sodium hypochlorite solution for 2 min, followed by a thorough rinse with deionized water for 5 min, and a surface treatment with 75% ethanol. Three equidistant holes (3.5 mm × 3 mm, width × depth) were created on the equator of each apple using a sterile tool. 30 µL of nanoparticle suspension was applied per wound. Control fruits received an equivalent volume of deionized water. After 30 min, B. cinerea mycelial plugs (7 mm diameter) from 10-day-old cultures were placed in the treated holes [24]. Treated fruits were incubated in glass desiccators at 25 ± 2 °C with 80–90% relative humidity. Disease progression was monitored daily, and lesion measurements were recorded on day 5. The antifungal effects of nanocomposites were determined as described in Section 2.4.2.

2.6. Biochemical Analysis of Apple Fruits

2.6.1. Sample Extraction

Apple tissue samples (20 g) were homogenized using a laboratory blender (mixer) for a brief period (approximately 1–2 min) to disrupt the tissue structure. The homogenate was then immediately mixed with 100 mL of 80% ethanol and stirred continuously for 4 h at room temperature. This ratio of tissue to solvent and extraction time was selected based on previously established optimal extraction conditions for phenolic compounds in apple tissue [25]. The extract was then filtered through Whatman No. 1 filter paper and centrifuged at 8000 rpm at 4 °C for 15 min to remove any particulate matter. The supernatant was collected and stored in darkness at −20 °C until analysis.

2.6.2. Total Phenolic Content (TPC) Analysis

The total phenolic content was quantified using the Folin–Ciocalteu method, with some modifications. A gallic acid calibration curve was prepared using standard solutions ranging from 50 to 600 µmol/L. The reaction mixture contained: 0.2 mL of sample extract, 1.0 mL of Folin–Ciocalteu reagent, 0.8 mL of sodium carbonate solution (75 g/L). After 30 min of incubation at room temperature in darkness, the absorbance was measured at 765 nm using a UV-visible spectrophotometer. Results were expressed as μmol/L of gallic acid equivalents (GAE) [26].

2.6.3. DPPH Radical Scavenging Activity

DPPH radical scavenging activity was measured in apple tissue extracts to assess antioxidant capacity induced by nanocomposite treatments. A 0.1 mM DPPH solution was prepared in ethanol. The reaction mixture contained 3.5 mL of DPPH solution and 0.5 mL of apple tissue extract samples. The mixture was vigorously shaken and incubated in darkness at room temperature for 30 min. The decrease in absorbance was measured at 517 nm [27]. The radical scavenging activity was calculated using the following formula:

$$[eqn]$$

where A0 the absorbance of the control, and A1 the absorbance of the sample. The experiment was performed in triplicate for each treatment group.

2.7. Data Analysis

Statistical analyses were conducted using a one-way ANOVA followed by Duncan’s multiple range test, with a significance threshold of p < 0.05. Data were analyzed using SPSS, Version 27.

3. Results and Discussion

3.1. Fungal Identification

Molecular identification of fungal isolates using ITS1 and ITS4 primers successfully amplified the ITS region, yielding 550 bp bands for both isolates. Sequencing analysis identified the isolates as Alternaria alternata (GenBank: PQ047825) and Fusarium graminearum (GenBank: PQ049134). These identifications were further validated through morphological characterization, aligning with previous studies that characterize these species as important plant pathogens. The amplification using ITS1 and ITS4 primers generated fragments of approximately 550–570 bp (Figure 1A), consistent with the expected sizes for both Alternaria and Fusarium species [28]. The successful amplification and subsequent sequencing confirm the reliability of these primers for fungal identification.

3.2. Characterization of Nanoparticles

The synthesized MONPs and their chitosan composites were characterized to understand their size, surface charge, optical properties, chemical structure, and crystallinity, which are crucial for their performance. Dynamic light scattering (DLS) analysis (Figure 1B) revealed that CSNPs exhibited a narrow size distribution with a mean hydrodynamic diameter of approximately 210 nm (range: 174 to 898 nm), consistent with previous reports [29]. This size range is suitable for effective interaction with microbial cells. Upon incorporation of inorganic oxides, the nanocomposites showed increased but well-defined sizes: CS-SiO_2_ had a mean diameter of 635 ± 15 nm, CS-ZnO measured 650 ± 25 nm, and CS-CuO displayed slightly larger particles averaging 720 ± 30 nm, all with good dispersity. The surface charge characteristics, as determined by zeta potential measurements (Figure 1C), provided critical insight into composite formation and stability. While the bare inorganic oxides exhibited negative surface charges (SiO_2_: −23.0 mV; ZnO: −11.8 mV; CuO: −20.6 mV), all chitosan-based composites showed positive zeta potentials (CS: +12.9 mV; CS-SiO_2_: +14.1 mV; CS-ZnO: +7.6 mV; CS-CuO: +5.4 mV). This distinct shift from negative to positive values confirms the successful coating of inorganic oxide surfaces by cationic chitosan through ionic gelation [12,30], which enhances colloidal stability and promotes electrostatic interaction with negatively charged fungal cell membranes, a key factor underlying their antimicrobial efficacy.

The UV-Vis absorption spectra of chitosan–inorganic oxide nanocomposites (CS-SiO_2_, CS-ZnO, and CS-CuO) are presented in Figure 1D. Pure chitosan (CS) exhibited negligible absorption across the 200–600 nm range, consistent with its non-semiconducting properties [31]. In contrast, CuO nanoparticles displayed a broad absorption band spanning 215–275 nm, characteristic of charge-transfer transitions in monoclinic CuO, which aligns with its semiconducting behavior [32]. ZnO nanoparticles showed a distinct absorption peak at 370 nm, corresponding to the intrinsic bandgap transition (~3.3 eV) of hexagonal wurtzite ZnO [33]. SiO_2_ nanoparticles, as expected for wide-bandgap oxides, exhibited no absorption in the visible range [34]. In the composite spectra, CS-CuO retained the CuO absorption band but exhibited a red shift to 280 nm, while CS-ZnO showed a slightly broadened peak at 375 nm compared to pure ZnO. CS-SiO_2_ displayed a weak absorption edge near 300 nm, absent in pure CS or SiO_2_, likely due to interfacial interactions between chitosan and silica. The enhanced absorbance intensity in all composites, particularly CS-CuO suggests the successful encapsulation of metal oxides within the chitosan matrix, which modifies light scattering and electronic transitions at the polymer-metal oxide interface [35,36]. These spectral shifts and intensity changes confirm the formation of nanocomposites.

The functional groups of the synthesized nanoparticles were analyzed via FTIR spectroscopy (Figure 1E). Pure chitosan (CS) exhibited two prominent absorption bands: a broad peak at 3401 cm^−1^, attributed to overlapping O–H and N–H stretching vibrations, and a sharp peak at 1607 cm^−1^, corresponding to the amide I (C=O stretching/N–H bending) of its acetylated units [31]. Upon formation of the nanocomposites, distinct alterations in the amide I band were observed, confirming successful interaction between chitosan and the inorganic oxides. In the CS-CuO spectrum, the amide I band underwent a blue shift to 1599 cm^−1^, accompanied by a change in intensity, indicating coordination between chitosan’s functional groups and CuO. For the CS-ZnO and CS-SiO_2_ composites, the amide I band either significantly decreased in intensity or became indistinguishable, which similarly suggests interaction through electrostatic or hydrogen bonding between chitosan and the inorganic oxide surfaces. These spectral modifications shifts, attenuation, and disappearance of the amide I band are consistent with reported studies on chitosan–inorganic oxide nanocomposites and serve as clear evidence of successful composite formation [31]. For SiO_2_ nanoparticles, characteristic vibrational modes were observed, including a dominant asymmetric Si–O–Si stretching band at 1095 cm^−1^, symmetric Si-O-Si stretching at 830 cm^−1^, and bending vibrations at 463 cm^−1^, consistent with amorphous silica networks [37]. The ZnO spectrum displayed a broad absorption band spanning 480–570 cm^−1^, assigned to Zn-O stretching in the hexagonal wurtzite phase [38], while CuO nanoparticles showed a distinct Cu-O stretching vibration at 529 cm^−1^ [39].

In the composites, spectral shifts and intensity modifications confirmed interactions between chitosan and inorganic oxides. For CS-SiO_2_, the O–H/N–H band at 3401 cm^−1^ broadened and shifted to 3420 cm^−1^, indicating enhanced hydrogen bonding between chitosan’s hydroxyl groups and silica’s surface silanols [40]. In CS-ZnO, the Zn-O band narrowed and shifted to 515 cm^−1^, suggesting coordination between ZnO and chitosan’s amino groups, while the amide I peak at 1607 cm^−1^ attenuated, reflecting electrostatic interactions [30]. Similarly, CS-CuO exhibited a redshifted Cu–O vibration (529 → 535 cm^−1^) and intensified chitosan-related bands, confirming nanocomposite formation via ionic gelation [12]. These results collectively validate the successful integration of inorganic oxides into the chitosan matrix, with spectral alterations underscoring interfacial bonding mechanisms critical to antifungal efficacy.

XRD patterns (Figure 1F) indicated that SiO_2_ was amorphous, while ZnO had a hexagonal wurtzite structure, and CuO had a monoclinic structure [41,42]. The broad peak observed for chitosan at 2θ = 20.091° was consistent with the findings reported [12]. XRD patterns of ZnO and CuO could well match with the corresponding standard reference patterns, indicating the successful preparation of ZnO and CuO. The chitosan–inorganic oxide composites showed modified patterns, indicating successful incorporation of inorganic oxides while maintaining the structural integrity of both components. Crystallite sizes ranged from 10 to 55 nm, which is optimal for enhanced surface interactions and biological activity [42]. These XRD results collectively confirmed the successful formation of the crystalline nanocomposites while preserving the characteristic features of both CS and the inorganic oxide components.

Scanning electron microscopy (SEM) revealed distinct morphological features of the synthesized nanoparticles and their CS composites, as shown in Figure 2. Pure CS (Figure 2A) exhibited a relatively smooth surface with some irregular formations. In contrast, SiO_2_ nanoparticles (Figure 2B) displayed a well-dispersed, flake-like morphology, with evidence of porosity and minimal aggregation, consistent with the observations of [43]. These flake-like structures suggest a high surface area that can benefit interactions. ZnO nanoparticles (Figure 2D) exhibited a more uniform spherical morphology with some agglomeration [42]. CuO nanoparticles (Figure 2F) showed a globular morphology with evidence of crystalline characteristics, consistent with findings from [44]. The SEM micrographs clearly demonstrate the impact of incorporating inorganic oxides into the CS matrix. The CS-SiO_2_ composite (Figure 2C) showed enhanced surface roughness compared to pure CS and SiO_2_. CS-ZnO composites (Figure 2E) exhibited a broadened particle distribution and a modified surface topology, unlike the more uniform ZnO nanoparticles. This also suggests good interaction between the CS and ZnO. Likewise, CS-CuO composites (Figure 2G) displayed altered CuO arrangements, and an increased presence of chitosan-related surface features compared to pure CuO, which indicates interaction [45]. These findings support that the ionic gelation method effectively incorporated the MONPs into the CS matrix, altering the composite’s morphology [46]. These observed changes in surface characteristics, particularly the increased surface area and structural modifications, are hypothesized to contribute to the enhanced antifungal activity of the composites. The morphological changes potentially lead to greater surface contact with fungal cells, facilitate the release of metal ions, and disrupt the fungal cell wall.

3.3. In Vitro Antifungal Activity

The in vitro antifungal efficacy of different nanoparticle formulations (CS, SiO_2_, ZnO, CuO, CS-SiO_2_, CS-ZnO, and CS-CuO) was assessed against four phytopathogenic fungi: A. alternata, B. cinerea, C. graminicola, and F. graminearum (Figure 3A). The radial growth assay quantified fungal growth inhibition after a 7-day incubation at their respective optimal temperatures. The chitosan–inorganic oxide nanocomposites generally exhibited superior antifungal activity compared to their individual components, suggesting a synergistic interaction between CS and inorganic oxides (Figure 3B,C). Among the tested nanocomposites, CS-CuO consistently showed the highest inhibitory effects across all four fungal species.

Specifically, CS-CuO showed the greatest impact on B. cinerea (85.88% growth inhibition) and A. alternata (81.71% growth inhibition), followed by C. graminicola (75% growth inhibition) and F. graminearum (64.70% growth inhibition). These results are consistent with prior studies demonstrating the antifungal properties of copper-based nanomaterials [47] and enhanced efficacy when combined with CS [48]. CS-ZnO and CS-SiO_2_ exhibited more variable inhibitory effects across the tested fungi, which may be attributed to the diverse mechanisms of action and physicochemical properties of the inorganic oxides, as well as their specific interactions with fungal cell walls [49,50]. The chitosan–inorganic oxide nanocomposites frequently exhibited enhanced antifungal activity compared to their individual components against most fungi, suggesting potential synergistic or additive interactions in many cases. However, the degree of enhancement was pathogen-dependent, with CS-CuO showing the most consistent and pronounced improvement (Figure 3B,C). These effects are likely due to a combination of chitosan’s membrane-disrupting abilities [51,52] and the capacity of metal oxides to generate reactive oxygen species (ROS) [53]. Variation in efficacy against different fungal species may be attributed to differences in fungal cell wall composition [54]. Higher concentrations of (0.1% w/v) showed maximum inhibitory effects [55]. The high performance of CS-CuO nanocomposites was attributed to their optimal particle size and positive surface charge, which facilitates better interaction with fungal cell membranes [56]. Furthermore, incorporating inorganic oxides into the CS matrix improved stability and enhanced the surface area for fungal contact [57].

The consistent hierarchical efficacy observed (CS-CuO > CS-ZnO > CS-SiO_2_ > CS) indicates that the combined effects of CuO and CS contribute significantly to the observed antifungal activity. This highlights the potential of chitosan–inorganic oxide nanocomposites, particularly CS-CuO, as effective broad-spectrum antifungal agents. A. alternata, B. cinerea, C. graminicola, and F. graminearum.

3.4. SEM Analysis of Fungal Morphological Changes

SEM analysis showed altered hyphal morphology and spore collapse in fungi treated with nanocomposites, particularly CS-CuO (Figure 4). Untreated control groups displayed typical, well-defined hyphae with smooth surfaces and intact spores. Treatment with CS alone showed minimal alterations. However, CS-SiO_2_-treated fungi exhibited visible distortions in hyphal morphology and a partial reduction in spore density. More pronounced damage was observed in fungi treated with CS-ZnO, including hyphal swelling, alteration in diameter, and a reduction in spore germination. Fungi treated with CS-CuO showed the most severe damage, with significant swelling, complete collapse of hyphal structures, and spore damage, which indicates that CS-CuO had the greatest effect on fungal structural integrity. The structural damage observed in SEM (Figure 4) aligns with prior studies where CuO-generated ROS disrupts fungal membranes, while chitosan enhances nanoparticle adhesion. These results are consistent with previous studies showing that MONPs induce oxidative stress and cellular damage in fungal cells. Copper oxide, in particular, is known to disrupt cell membrane integrity by generating reactive oxygen species (ROS) that can damage fungal cell components [53]. Chitosan’s interaction with cell walls and membranes alters permeability, which further contributes to the antifungal effect [51,52]. The observed variation in damage between CS-ZnO and CS-CuO may be attributed to differences in ROS generation potential and their interactions with fungal cells [50]. The SEM data emphasize the potential of chitosan–inorganic oxide nanocomposites, particularly CS-CuO, as potent antifungal agents because of the extensive damage they induce on fungal structures.

3.5. In Vivo Antifungal Efficacy of Chitosan–Inorganic Oxide Nanocomposites on Apple Fruit

The in vivo antifungal efficacy of chitosan-based nanocomposites (CS, CS-SiO_2_, CS-ZnO, and CS-CuO) was evaluated on apple fruits infected with B. cinerea (Figure 5). The results demonstrated a significant reduction in lesion size in apples treated with nanocomposites, compared to controls. CS-CuO exhibited the most pronounced inhibitory effect, with statistically significantly smaller lesion areas compared to all other treatments (Figure 5B). Graphic documentation also clearly illustrates the reduced disease progression in CS-CuO-treated apples, compared to other treatments and controls (Figure 5A). Lesion areas in CS-ZnO and CS-SiO_2_ treated samples were lower than in controls, but the differences were not statistically significant. The inhibition efficiency calculated relative to the control samples revealed that CS-CuO had the highest inhibition efficiency, which confirms its strong in vivo antifungal effect (Figure 5C). The in vivo performance of CS-CuO is consistent with the in vitro findings, which further supports the synergistic effects of CS and CuO [58]. The lower activity of CS-ZnO and CS-SiO_2_ in vivo compared with in vitro may be attributed to differences in environmental conditions, matrix composition, and interactions with the apple tissue, which could alter the formulations’ behavior. These results suggest that CS-CuO nanocomposites hold promise for sustainable and eco-friendly post-harvest disease treatment of apples.

3.6. Biochemical Defense Response of Apple Fruit to Chitosan–Inorganic Oxide Nanocomposite Treatments

The biochemical defense response of apple fruit to B. cinerea infection and nanocomposite treatment was assessed by quantifying total phenolic content (TPC) and DPPH radical scavenging activity (Figure 6). All nanocomposite treatments elevated TPC compared to the infected control, with the CS-CuO treatment triggering the most significant increase (Figure 6A). Similarly, DPPH radical scavenging activity was significantly enhanced in all treatment groups, with CS-CuO again demonstrating the highest antioxidant capacity (Figure 6B). This marked upregulation of phenolic compounds and associated antioxidant activity indicates that the nanocomposites, especially CS-CuO, elicit a systemic host defense response [59,60]. By boosting the fruit’s innate ability to neutralize reactive oxygen species (ROS) generated during fungal attack [61], these treatments enhance tissue resilience, contributing to the observed reduction in disease severity. This dual mechanism direct antifungal action coupled with host defense priming underscores the multifaceted potential of chitosan–inorganic oxide nanocomposites for sustainable postharvest protection.

3.7. Cytotoxicity Assessment of Chitosan–Inorganic Oxide Nanocomposites

To evaluate biosafety for potential applications, the cytotoxicity of the nanocomposites was assessed against RAW 264.7 murine macrophage cells, a relevant model for mammalian immune response. All formulations exhibited high cell viability at concentrations up to 300 µg mL^−1^), with CS-CuO maintaining high viability even at 600 µg mL^−1^ (see Supplementary Figure S1). This favorable profile is notable given the established cytotoxicity of unencapsulated CuO nanoparticles, which induce cell death through Cu^2+^ ion release and ROS generation [13,53]. The significant mitigation of toxicity in our CS-CuO composite is attributed to the effective encapsulation within the chitosan matrix, which acts as a biocompatible scaffold and controls ion dissolution kinetics [13,62,63]. Crucially, the effective in vivo antifungal concentration (~300 µg mL^−1^) is well within this biocompatible range, establishing a wide therapeutic window. This clear separation between the effective dose and any toxic dose is a fundamental requirement for practical application and strongly supports the biosafe use potential of these nanocomposites, particularly CS-CuO, in agriculture.

From a translational perspective, the effective in vivo concentration of the lead CS-CuO nanocomposite (0.1% w/v, equating to ~190 µg/mL of elemental copper) is notably lower than the application concentrations of many conventional copper-based fungicides, which often range between 1000 and 2000 µg/mL of copper. This lower effective dose, combined with the reduced cytotoxicity of the chitosan-encapsulated formulation, addresses initial concerns regarding environmental load and non-target toxicity. However, the practical deployment of such nanocomposites requires a consideration of existing regulatory frameworks for inorganics in agriculture. Future work must therefore prioritize large-scale field trials to evaluate residue dynamics, long-term environmental fate, and integration with standard horticultural practices. This study serves as a compelling proof-of-concept, demonstrating that low-concentration chitosan–inorganic oxide nanocomposites can achieve significant disease control through a dual mechanism of direct antifungal action and host defense priming, while presenting a potentially improved safety profile.

The present work advances the field of bio-nanocomposites for plant protection by providing a direct, controlled comparison of three chitosan–inorganic oxide systems synthesized under identical conditions. This approach unequivocally established the superior and broad-spectrum performance of the CS-CuO nanocomposite. Beyond direct antifungal activity, our results demonstrate a novel dual-functional role for these materials: they act as both a direct antimicrobial agent and a primer of host systemic resistance, as evidenced by the significant upregulation of phenolic compounds and antioxidant activity in treated fruit. Importantly, achieving this significant disease reduction at a low application concentration (0.1%) coupled with the mitigated cytotoxicity of CS-encapsulated CuO addresses critical concerns regarding dosage, environmental load, and non-target toxicity. These findings position CS-CuO as a promising, scalable alternative to synthetic fungicides for both pre- and post-harvest applications. The formulation’s low concentration, combined with chitosan’s biodegradability and the encapsulation of inorganic oxides within its matrix, is designed to minimize environmental and health risks by reducing nanoparticle leaching and toxicity. Future studies should prioritize translational formulations, such as nanoparticle-infused spray coatings for field applications or biofilm-integrated packaging materials for extended storage protection. Additionally, large-scale trials assessing residue thresholds, environmental impact, and compatibility with existing agricultural practices remain critical steps toward commercial viability and sustainable adoption.

3.8. Regulatory Considerations and Translational Pathway

While zinc oxide and silicon dioxide nanomaterials are recognized as Generally Recognized as Safe (GRAS) by the U.S. Food and Drug Administration for certain food-contact applications, copper oxide nanoparticles currently lack such regulatory approval for direct application on edible plant tissues. This study does not claim that CS-CuO nanocomposites are immediately deployable; rather, it establishes a proof-of-concept demonstrating that chitosan encapsulation significantly reduces CuO-associated cytotoxicity while preserving potent antifungal and host-defense-priming activities at low concentrations.

The in vivo wound inoculation model employed here is a standard, controlled method for evaluating direct antifungal efficacy under severe infection pressure and is not intended to simulate commercial application. In practical scenarios, whether in the field, during postharvest handling, or in packaging, nanocomposite formulations would be applied as sprays, dips, or edible coatings without wounding the fruit. Moreover, routine postharvest washing procedures commonly used in fruit packinghouses can substantially reduce surface-bound nanomaterials; however, quantitative assessment of residue removal and potential migration into fruit tissues requires dedicated future study.

4. Conclusions

This study successfully synthesized low-concentration chitosan–inorganic oxide nanocomposites, utilizing a facile ionic gelation method, and rigorously evaluated their antifungal efficacy against key postharvest phytopathogens. Among the tested formulations, the CS-CuO nanocomposite consistently exhibited higher antifungal activity, both in vitro and in vivo, effectively suppressing fungal growth and significantly reducing Botrytis cinerea lesion development on apple fruits. This enhanced efficacy is attributed to a synergistic mechanism, where chitosan’s cell membrane-disrupting properties complement copper oxide’s capacity to generate reactive oxygen species, leading to enhanced fungal cell damage. Moreover, CS-CuO treatment resulted in a notable increase in total phenolic content and DPPH radical scavenging activity in treated apple tissue, which is indicative of a robust activation of the host defense system. These findings underscore the considerable potential of low-concentration CS-CuO nanocomposites as a sustainable, multi-faceted approach for managing postharvest diseases, and they warrant further investigation in the development of effective bio-based strategies. Future studies should explore translational applications, such as sprayable nanocomposite coatings for field use or biofilm-integrated packaging for extended postharvest protection, ensuring compatibility with existing agricultural practices.