The antifungal activity of the prepared nanoemulsions namely0_T20, 5_T20, 10_T20, 0_T80, 5_T80, and 10_T80 against P. verrucosum was determined at three concentrations (25, 50, and 100%) following the growth rate method. Briefly, P. verrucosum mycelial disks (7 mm in diameter) developed on PDA plates were carefully cut from the colony’s plate edges and deposited on plates supplemented with 1 ml of the above-mentioned concentrations of the nanoemulsions. The inoculated plates were then incubated at 25°C ± 1 for 4, 6, and 8 days. Untreated plates served as a control. Mycelial fungal radial growth was monitored after incubation in all plates, and data were reported as the percentage of inhibition (Çolak and Biçici, 2013):

Where A represents the fungal mycelial radial growth on the control (untreated) plate and a represents the fungal mycelial radial growth on the plate treated with three concentrations of each nanoemulsion individually. All laboratory experiments were repeated twice in triplicates under controlled conditions.

Maize seeds (cv. Boushy) were sterilized with an aqueous solution of sodium hypochlorite (0.25%, v/v) for 3 min. Seeds were then copiously washed with distilled water. To prime seeds, seeds were immersed in 10 ml of each of the prepared nanoemulsions (100% concentration) individually and maintained for 4 h under slow stirring. Afterward, seeds were placed on sterilized filter papers in Petri dishes (4–5 seeds/plate), supplemented with 5 ml of distilled water and 1 ml of (1 × 10⁶ spore) P. verrucosum spore suspension. The inoculated plates were then incubated in the dark (24 ± 2°C). Untreated seeds, infected seeds, and the chemical fungicide treated-seeds were used as controls. The percentages of germinated seeds and their radical length were recorded in comparison to control plates at 5 days after priming.

Seeds of maize (cv. Boushy) were sterilized as mentioned above. Seeds were then cleaned multiple times with distilled water. To prime maize seedlings, five nanoemulsions with the most appreciated concentration (100%) resulted in antifungal activity experiments at the laboratory were created. The seeds were subsequently dipped in 10 ml of these solutions for 6 h in gentle agitation for seed priming. The seeds were then placed on sterilized filter paper in Petri plates (4–5 seeds per plate) and 10 ml of distilled water was added. These samples were covered and put in a dark growing room at 22 ± 2°C. Seeds for controls were collected to prime them with distilled water. The percentage of seed germination and radical lengths were measured 4 and 5 days post-sowing (DPS), respectively. Untreated control, chemical fungicide, and infected control were used as controls.

At the third leaf stage (14 DPS), plants were collected and the physiological effects of the tested nanoemulsions on plant height, dry weight, and the level of some photosynthetic pigments (chlorophyll and carotenoid content) were evaluated in all treated seed samples. The content of photosynthetic pigments was assessed according to Venkatachalam et al. (2017). In this regard, 100 mg of maize fresh leaves were collected and grounded with 5 ml of 80% (v/v) ice-cold acetone using mortar and pestle and homogenate was aliquoted into sterile centrifuge tubes and centrifuged at 6,000 rpm for 6 min. The supernatant was then transferred into fresh centrifuge tubes and the pellet was re-extracted with 2.5 ml of 80% (v/v) ice-cold acetone, and it was repeated twice. The level of Chlorophyll a, b, and carotenoid contents was quantified using a double beam UV–Visible spectrophotometer (Spectrophotometer UV-1800, Shimadzu Tokyo, Japan) at wavelengths of 663, 645, and 470 nm, respectively. Determination of chlorophyll a, b, and carotenoid contents level was carried out using the equations proposed by Lichtenthaler (1987) and expressed as mg g¹ FW.

While anthocyanin content was determined according to the protocol of Lichtenthaler and Buschmann (2001), the anthocyanin concentration was evaluated spectrophotometrically at 535 and 650 nm.

For protein assays, a cold mortar and pestle were used to homogenize around 50 mg of leaf tissues in 5 ml of 0.1 M Tris–HCl buffer (pH 7.5). At 4°C, the extract was centrifuged for 15 min at 10,000 rpm. The protein content of the supernatant was then calculated using Bradford’s method (Bradford, 1976) using a standard of bovine serum albumin (BSA). Untreated control, chemical fungicide, and infected control were used as controls.

Acid invertase (AI) activity of leaves was assayed according to Hwang and Heitefuss (1986). Following pathogen inoculation, 0.25 g of leaf segments were collected from both the control and infected leaf tissues at the third leaf stage (14 DPS), submerged in ice-cold ethyl acetate for 20 min, and then rinsed in ice-cold distilled water. Each sample was kept in a water bath at 30°C for 60 min, during which time it was exposed to 0.1 M sodium phosphate buffer (pH 5.6), 0.5 M sucrose, and distilled water. A piece of filter paper was used to soak up any remaining moisture from the leaf surface. Twenty milliliter of 0.5 M sucrose, 2 ml of 0.1 M sodium phosphate buffer (pH. 5.6), and 6 ml of double-distilled water were placed in a 20 ml vial for each leaf sample. In a water bath with shaking, the vials were kept at 30°C for 60 min. AI activity of all samples was measured at 280 nm in a spectrophotometer (Hitachi Model: U-1100 573 × 415). Also, protease activity was assessed following the method of McDonald and Chen (1965). In this regard, about 100 mg of leaf tissue was incubated at 30°C for 1 h in 4 ml of a substrate solution (1% casein in 0.1 M sodium citrate buffer, pH 7.0). The remaining protein was precipitated by adding 5 ml of trichloroacetic acid (TCA) at a concentration of 5%. After 30 min, the tubes’ contents were filtered through filter paper to remove the precipitate (WHATMAN No. 40). After filtration, a 5-ml aliquot of the filtrate was combined with a 1-ml aliquot of an alkaline reagent mixture made from 2% sodium carbonate, 2.7% sodium potassium tartrate, and 1% copper sulfate. We then made the solution alkaline by adding 2 ml of 1 M sodium hydroxide. The Folin–Ciocalteu-phenol reagent (0.5 ml) was added after the mixture had been allowed to stand for at least 10 min. After 30 min, a spectrophotometer was used to determine the absorbance of the blue color at 660 nm in each sample. One unit of protease activity was defined as the amount of enzyme required to produce an increase in optical density at 660 nm of 0.1 h⁻¹ at 30°C at pH 7.0. Untreated control, chemical fungicide, and infected control were used as controls.

Polyphenol oxidase (PPO) activity was determined following the method of Kar and Mishra (1976) with minor modifications. Leaf tissue (1 g) collected at the third leaf stage (14 DPS) was homogenized in 2 ml of 0.1 M sodium phosphate buffer (pH 6.5) and centrifuged at 10,000 × g for 25 min at 4°C. Three milliliter reaction mixture contained 25 mM phosphate buffer (pH 6.8), 0.1 Mm pyrogallol, and 0.1 ml enzyme extract. The control reaction mixture contained no pyrogallol. The absorbance of each sample was recorded at 420 nm.

Leaf tissue (1 g) collected at the third leaf stage (14 DPS) was homogenized in 5 ml of 0.05 M phosphate buffer (pH 7.0) containing 10% polyvinylpyrrolidone (PVP-SIGMA) and 0.1 M ethylene di-amine-tetra-acetic acid (EDTA-SIGMA) and centrifuged at 14,000 rpm for 20 min at 4°C. POD activity was measured by the method of Abo-Elyousr et al. (2020). The assay mixture contained 0.1 ml enzyme extract, 1.35 ml 0.1 M mM MES buffer (pH 5.5), 0.05% H₂O₂, and 0.1% phenylenediamine. Changes in the absorbance in each sample were recorded at 485 nm for 3 min. Chitinase activity was determined by colorimetric assay following the protocol of Stangarlin and Pascholati (2000).

In this regard, the substrate CM-chitin-RBV (200 μl of 2 mg/ml) was gently mixed with 300 μl of leaf protein extract and 250 μl of 10 mM Tris–HCl, pH 7.5, containing 1% Triton X-100. The mixture was incubated at 37°C for 3–4 h. The reaction was stopped by the addition of 200 μl of 2 M HCl. Samples were then cooled on ice for 20 min followed by centrifugation at 20,000 × g for 10 min to remove the non-degraded substrate. The supernatant was collected and one unit of chitinase activity represented an increase in absorbance of 0.1 at 550 nm (Lopes et al., 2008). Untreated control, chemical fungicide, and infected control were used as controls.

Emulsifiers are required in the synthesis of nanoemulsions because they lower interfacial tension and make it easier for small droplets to form during homogenization via oil–water interface adsorption (Leão et al., 2019). The time of the ultrasonication was found to have a great impact on the droplet size of the resulting nanoemulsions formulated with either Tween 20 or Tween 80. In this study, the droplet size of 0_T20, 5_T20, and 10_T20 nanoemulsions were 248.1, 185.7, and 168.6 nm, respectively. While, the droplet size of 0_T80, 5_T80, and 10_T80nanoemulsions was 345.3, 211.6, and 188.6 nm, respectively (Figure 3). Figure 3 depicts the average size of nanoemulsions produced using the ultrasonication technique at various ultrasonication times. The results of this study showed that droplet size was greatly influenced by the time of the sonication (0, 5, and 10 min) and the longest sonication time revealing the smallest droplets for both emulsifiers (Figure 3). A longer sonication time causes more shear forces to be applied to the droplets, which leads to greater droplet deformation and fragmentation, and thence, smaller droplet sizes are expected. Results indicated that Tween 20 which has a shorter chain fatty acid moiety (lauric acid, 12 carbon atom) caused lower droplet size nanoemulsion compared to Tween 80 which has longer chain fatty acid namely oleic acid (18 carbon atom chain). The effect of the emulsifier type was also demonstrated by other researchers studies (Chong et al., 2018; Maccelli et al., 2020; Mohammed et al., 2020; Syapitri et al., 2022) who found that the type of emulsifier and homogenization time influenced droplet size and stability of prepared nanoemulsions.

It is well known that the particle size distribution index or polydispersity index (PDI) of nanoemulsion droplets is an essential factor for investigating the stability of nanoformulations in terms of physical analysis and nanoemulsion stability (Kurpiers et al., 2020). The homogeneity of nanoemulsions was assessed using the PDI, which ranged from 0.1 to 1.0. In this study, PDI values ranged from 0.181 to 0.353 (Figure 3). Nanoemulsions with lower PDI values are thought to be more homogenous. Also, an acceptable level of homogeneity is indicated by nanoemulsions with a PDI of less than 0.6 (Eid et al., 2013).

The Zeta potentials of the nanoemulsions prepared using Tween 20 and Tween 80 are displayed in Figure 4. As shown in the Figure 4, the highest zeta potential values for the synthesized nanoemulsions using Tween 20 and Tween 80 were obtained after 10 min of sonication. It was found that Zeta potential was increased by increasing the sonication time. The obtained data show that the zeta potential of the Tween 20 nanoemulsions is highest at 10 min (−46.74 mV), followed by 5 min (−39.67 mV), and 0 min (−37.34 mV). Tween 80 nanoemulsions showed the same trend with decreasing sonication time, where zeta potential of 0, 5, and 10 min recorded –27.24, −44.12, and −48.82 mV, respectively. These results may be explained by the fact that oil droplets with smaller surface areas have larger surface charges. Nanoemulsions could have a greater negative zeta potential and become more stable due to the higher surface area and smaller particle size. The obtained results were in line with the findings of Qushawy et al. (2022). The surface charge density of the produced nanodroplets, or zeta potential, is directly related to the stability of the electrostatic field and the tendency of the nanodroplets to aggregate (Ziani et al., 2011; Chung et al., 2017; Jarzębski et al., 2021). Theoretically, a high zeta potential value indicates good stability. In general, it is considered that zeta potential values above the range of –30 to +30 mV have enough repulsive force to achieve greater physical colloidal stability. Furthermore, the formation of a hydrogen bond between the water molecules and the oxyethylene group of Tweens may cause the hydroxyl ion to be selectively adsorbed at the oil/water interface, producing a negative charge (Liu et al., 2006). Visual observation of the nanoemulsions at regular intervals during 7 days indicated that 0_T20 and 0_T80 nanoemulsions showed the highest separation percentages of about 16 and 12%, respectively. While 5_T20 10_T20, 5_T80, and 10_T80 displayed physical stability against gravitational separation with slight oil separation of about 1–3% (Figure 4).

Figure 5 shows the viscosities of the nanoemulsion produced with the different emulsifiers at different sonication times. The nanoemulsion viscosity depends on the oil, water, and emulsifier components. The compositional and structural effects of interfacial packing (the ratio of tail-to-head cross-section) are also related to the viscosity change with water content. In the case of Tween 80 compared to Tween 20, the rise in nanoemulsion viscosity measurement is more noticeable (Figure 5). The lowest viscosity was found in sample 10_T20, whereas sample 0_T80 had the highest viscosity. The low viscosity may be the result of a high water content combined with a particular type of emulsifier. Tween surfactants often contain a variety of hydrophobic groups. Tween 80 has longer side chains that are responsible for more viscosity than Tween 20. Tween 20 reduced the interfacial tension and viscosity between water and oil (Taha et al., 2020). Increased sonication time resulted in a nanoemulsion with a reduced viscosity, which will increase its stability. In the same context, Gorjian et al. (2022) studied the viscosity of spearmint essential oil nanoemulsions with various surfactants (Tween 20, Tween 40, and Tween 80). An increase in hydrogen bonding causes a reduction in the molecular distances of emulsion systems and an increase in flow resistance, which means an increase in the viscosity of the system. Thus, the stability of nanoemulsions is greatly influenced by their viscosity.

Based on data recovered from the in vitro antifungal activity of the produced nanoemulsions, nanoemulsions at 100% were selected for further studies. The effect of the six produced nanoemulsions at 100% concentration on maize germination was evaluated and compared to the commercial chemical fungicide “Ridomil Gold SL (active ingredient: Mefenoxam 45.3%) at 2 g/land both control (infected and untreated) seeds (Figure 7; Table 2). Based on data presented in Table 2, all nanoemulsions had increment effects on maize seed germination by 101% in the case of 10_ T20 and 10_ T80 compared to untreated seeds (control) or the chemical fungicide treatment. The percentage of seed germination ranged between 97 and 98.7% and the radical length ranged between 2.2 and 3.9 cm for the six tested nanoemulsions. In particular, the highest germination rate was achieved by 10_T80 and 10_T20 with a rate of 98.7 and 98.2%, respectively. Also, the highest radical length was achieved by 10_T80 and 10_T20 with 3.8 and 3.9 cm, respectively. Overall, the infected seeds showed the lowest germination percentage and radical length (Table 2). In addition, a pictorial representation of the seedling growth upon nanoemulsion treatment were shown in Figure 8. The positive action of those nanoemulsions may be related to that some natural compounds have the potential to stimulate metabolic processes leading to improved seed development (Savy et al., 2017). This effect may be also attributed to a hormone-like action that Nigella Sativa-derived oils, and the phenolic compounds it contains, can exert on the early seed’s biochemical activities (R Core Team, 2019). Furthermore, natural-based compounds including essential oils can show a gibberellin-like action that results in a beneficial effect, thus influencing the seed’s hormonal status and physiological mechanisms underlying its development (Savy et al., 2017).

The prepared nanoemulsions differently affected shoot and root length in maize (Figure 9) terms of both shoot and root length, it was noticed that the applied nanoemulsions treatments “particularly (10_T20 and 10_T80) were able to more stimulate both root and shoot length as compared to the chemical fungicide treatment. Continually, the different treatment solutions on the shoot and root fresh weight in maize were monitored (Figure 9). The obtained results were also in parallel with root and shoot length data, where all nanoemulsions treatments did not passively affect root and shoot fresh weight parameters and both T0_T20 and 10_T80 nanoemulsions showed the highest values compared to other nanoemulsions. In this regard, it has been hypothesized that organic-based compounds including oil-based compounds could increase seed germination of its capacity to act directly on mitotic activity (Popa et al., 2008). Also, the efficacy of seed priming can impact the plant’s subsequent development and also inhibit/reduce the infection with P. verrucosum and consequently reduce the mycotoxins secreted by this fungal pathogen, therefore these favorable effects on the earlier growth and maturation are important and investigated. In this regard, 2 weeks after seeding, the maize seedlings were measured for root, shoot lengths, and weights for all treatments (Figure 9). The obtained data are in agreement with other studies indicating that plant-derived products are effective against different fungal pathogens infecting maize plants such as F. verticillioides, Colletotrichum graminicola, and Fusarium solani (Abo-Elyousr et al., 2009; Mandal et al., 2009; Masum et al., 2009).

The chlorophyll a and b content was ascertained in the samples exposed to the prepared nanoemulsions (Figure 10). 10_T80 and 10_T20 nanoemulsions showed the highest chlorophyll content followed by 5_T80 and 5_T20. Infected control showed the lowest chlorophyll content (Figure 10A). Concerning chlorophyll b content, 10_T80, 0_T20 and 5_T80 nanoemulsions provoked the highest increases as compared to the chemical fungicide and control samples (Figure 10B). The inductive effects of nanoemulsions on chlorophyll a and b are due to a stimulatory influence on their production. Because of their ability to promote plant nutrition, particularly nitrogen absorption, several phenolic compounds can increase the concentration of the pigments described above (Tanase et al., 2014).

Regarding carotenoids, Figure 10C showed that samples treated with 10_T80, and 10_T20 nanoemulsions demonstrated the highest increases in carotenoids content followed by 5_T80 and 5_T20 as compared to other treatments. Concerning anthocyanin, the treatments with 10_T80 and 10_T20 nanoemulsions showed the highest substantial increases in its content in comparison to other treatments. Also, 5_T80, 5_T20, and chemical fungicide treatment demonstrated no significant differences in anthocyanin content (Figure 10D). As for soluble protein, 10_T80 and 10_T20 nanoemulsions demonstrated the highest increase in soluble protein content followed by 0_T20, 5_T20, and 5_T80 with no significant difference between them (Figure 10E).

The rise in carotenoids seen can be attributed to a protective response to nanoemulsion therapy. The concentration of chlorophyll is also correlated to the number of carotenoids involved. Increases in the level of this pigment are perceived by plants as a signal, which drives carotenoid production (Zoufan et al., 2020). Based on these findings, using non-excessive dosages of nanoemulsions as a way to increase the synthesis of these beneficial compounds is recommended. In terms of anthocyanin, increases were seen in maize following various nanoemulsion treatments. Anthocyanin is a flavonoid that has important functions in plants as well as potential health benefits in humans (Bu et al., 2020). Anthocyanin can prevent and contrast lipid peroxidation by acting on ROS in the vacuole, and their content can be elevated in response to diverse environmental conditions (Dobrikova et al., 2021).

The total soluble protein content, as this is significantly backed by the ability of the plant to assimilate nutrients, can also ensure phytotoxicity (Venkatachalam et al., 2017; Alzahrani and Rady, 2019). The favorable impact of natural oils “nanoemulsions” on maize seedlings might explain this good effect. As previously stated, they can increase in treated plants the nitrogen content, which is crucial for protein biosynthesis (Ertani et al., 2011). In agreement with the above-mentioned data, different reports indicated that plant metabolites can induce systemic resistance in host plants, disease reduction, and increased different plant growth parameters in many crops (Nandakumar et al., 2001; Ramamoorthy et al., 2002). When activated by different biotic or abiotic factors, plant defense genes that are quiescent in healthy uninoculated plants can induce systemic resistance against disease (Naz et al., 2021). Furthermore, it should be noted that a good root development, as prompted by all nanoemulsion treatments, can justify the highest content of chlorophyll a and b since a better root system can allow maize to increase its capacity to take up nutrient from the growth media (Lucini et al., 2018). The amount of carotenoids is also related to the content of chlorophyll a. Indeed, plants can perceive increases in the content of this pigment as a signal which, in turn, stimulates the carotenoid biosynthesis (Bartucca et al., 2020). Based on these results, the use of non-excessive doses of nanoemulsions particularly 10_T80 and 10_T20 can be proposed as a strategy not only for disease reduction by also to raise the production of these beneficial substances.

Inoculation of P. verrucosum stimulated the activities of all the tested defense-related enzymes tested in all treatments compared to the un-inoculated control. In the pot experiment, acid invertase (AI) and protease activities in the leaves were higher in inoculated plants compared with the untreated plants (Figure 11). The nanoemulsion 5_T20seed treatment significantly increased the AI and protease activity by 68 and 64%, respectively, compared with both the un-inoculated and the infected controls. Seed treatment with 10_T80 nanoemulsion showed the highest AI and protease activity by 75 and 70%, respectively, as compared to the infected control. No significant differences were found between 0_T80, 10_T80, 0_T20, 10_T20, 5_T20, and chemical fungicide treatments for AI. Also, no significant differences were found between 10_T80, 0_T20, 5_T20, and 10_T20.

In the case of chitinase activity, results indicated that seeds treated with all nanoemulsions increased the chitinase enzyme activity, as compared to the infected control. The highest activity was recorded in seeds treated with 5_T20, 0_T20, and 10_T80 followed by 0_T80, 5_T80, and 10_T20 (Figure 11C). In the same way, inoculation of P. verrucosum induced higher PPO and POD activity in the seed-treated plants with all nanoemulsions tested than in untreated controls (Figures 11D,E). In the pot, seed treatment with both 10_T80 and 5_T20 nanoemulsion showed the maximum increase of the leaf POD and PPO activities by 60 and 52%, respectively, as compared with the infected control. Also, 10_T80, 0_T20, and 5_T20 showed the highest POD and PPO activities with no significant differences between them. The results obtained herein summarized that the prepared nanoemulsion treatments had a stimulatory effect on these enzyme activities over and above the effect of the pathogen.

These results reveal the positive effects of exogenous nanoemulsions applications to enhance resistance in maize against P. verrucosum infections, which by different reports indicated that exogenous application of specific compounds derived plants can induce resistance in the host plant via induction of higher levels of host defense enzymes and Pathogen related (PR)-proteins. This suggests that the produced L-CNPs induce systemic resistance in the host plants, resulting in a reduction in disease development (Paul and Sharma, 2002). Moreover, the onset of induced systemic resistance in plants correlates with the increased activity and expression of PR proteins, such as chitinases, β-1,3-glucanases, acid invertase, and peroxidases; consequently, such proteins are generally used as the key ingredients of systemic acquired resistance (SAR), an inducible immune response of plants that averts further infection or disease spreading to the non-infected parts of the host (Siddaiah et al., 2017; Butt et al., 2019). Different studies reported that compound-derived plants can induce systemic resistance in host plants, disease reduction, and increased plant growth in many crops (Nandakumar et al., 2001; Ramamoorthy et al., 2002).

When activated by various factors, plant defense genes that are quiescent in healthy uninoculated plants can induce systemic resistance against disease. Different reports indicated that the increment levels of chitinase, protease, PPO, POD, and AI enzyme activities (Chen et al., 2000; Nandakumar et al., 2001) as well as expression of β-1,3-glucanase and chitinase genes (Ramamoorthy et al., 2002; Saravanakumar et al., 2007) have been reported to be effective against various fungal diseases.

Penicillium verrucosum isolate grown on PDA plates showed the characteristic mycelial and conidiospores morphology, with lengthened, normal, and homogenous hyphae of constant diameter with smooth external surfaces and rounded apices (Figures 12A,D). In this regard, SEM micrographs indicated a normal growth for the fungal mycelia of the untreated control (Figures 12A,D), which consisted of conidiophores formed of smooth stipes of 200–500 μm long and ending in typically triverticillate penicillia. The length of the metulae ranged from 10 to 14 μm, while that of the tightly packed phialides was 7–12 μm, Phialide were flask-shaped and more elongated. However, significant morphological changes were noticed in fungal mycelia treated with either 10_T20 or 10_T80 nanoemulsions including alterations in conidiophores, metulae, phialides, and mature conidia characteristics were observed (Figures 12B,C,E,F, arrowed). Substantial morphological changes in the cell wall surface of the fungal mats were also observed and conidiophore improvement was abnormal (Figure 12B, arrowed), where mycelia and conidiophores were shriveled compared to the untreated control. The fungal spores were deformed and collapsed with damaged vesicles (Figure 12E), in compared to the control, and the cell walls became thinner, shriveled, crinkled, and showed a decreased cytoplasmic content and modifications of the membrane integrity, and the fungal conidiophore started to shed its spores (Figures 12C,F). The interactions between nanoemulsion molecules and the polyanionic structure of microbial cell membranes undoubtedly lead to the destabilization of the cell membranes, which is how nanoemulsions affect numerous fungal infections. This causes the fungal pathogen’s intracellular material to flow out, destroying the organism. The primary and secondary mechanisms of antimicrobial activity of nanoemulsions are likely interference with protein synthesis and membrane instability, respectively. The absorption of nanoemulsions into the cellular DNA binding site, which further inhibits RNA and protein synthesis, may also be part of the process by which such nanoemulsions work. Our findings are also in agreement with (Junior et al., 2012), indicating that chitosan nanoparticles may also cause fungal mat accumulation, and structural changes, such as excessive branching, cell wall swelling, and reduced hyphal size, which have been observed in P. expansum and Rhizopus stolonifer.