Preparation and Synergistic Effect of Composite Solid Nanodispersions for Co-Delivery of Prochloraz and Azoxystrobin

Abstract

The low efficacy of traditional single-component pesticide formulations has resulted in excessive pesticide application, the evolution of pest resistance, and a range of food safety and environmental concerns. Developing efficient composite nanopesticides represents a critical strategy for addressing the above challenges. In this study, solid nanodispersions (SNDs) co-loaded with prochloraz and azoxystrobin were constructed through a self-emulsifying carrier adsorption method. The antifungal activities of the composite SND with a 14:1 ratio of prochloraz to azoxystrobin against Fusarium graminearum and Pyricularia oryzae were 2.3-fold and 1.6-fold higher than those of commercial microemulsions (MEs) with the same proportion of active ingredients. The SND could cause severe oxidative damage to fungi, by reducing the activities of superoxide dismutase (SOD) and catalase (CAT), and break the permeability of cell membranes, resulting in fungal death. Additionally, the composite SND exhibited superior foliar wettability and biosafety with a minimal environmental cost, thereby enhancing the pesticide’s effective utilization rate. This research provides theoretical and technical support for the design and development of high-efficiency composite nano-fungicide, holding promise for sustainable disease management.

1. Introduction

Wheat (Triticum aestivum L.), rice (Oryza sativa L.), and maize (Zea mays L.) are the three most important food crops in the world. Their cereal grains directly contribute more than half of all calories consumed by human beings [1]. Nevertheless, plant diseases can lead to substantial yield reductions in agricultural crops, thereby presenting a critical challenge to global food security. Fungal diseases that frequently occur during wheat growth include rust, spot disease, Fusarium head blight, and powdery mildew [2]. Among them, Fusarium head blight, primarily caused by the Fusarium graminearum [3], is a devastating disease in wheat production. It not only results in significant declines in yield and grain quality but also allows the pathogenic fungi to secrete mycotoxins such as deoxynivalenol in the grains, posing risks to human and animal health and raising serious concerns about food safety [2]. During the growth process of rice, it is mainly affected by rice blast, sheath rot, brown spot, and bacterial leaf blight [4,5,6]. Rice blasts alone can cause a 30% drop in global rice production, and these losses are sufficient to feed 60 million people. The persistence of rice blast-infected residues in agricultural soils can lead to continuous pathogen transmission, potentially causing recurrent infections in subsequent crops and contributing to long-term environmental consequences in the agroecosystem [7]. In the prevention and control of plant diseases, the most widely adopted and effective method is the application of chemical pesticides.

Prochloraz is a broad-spectrum, highly effective, and low-toxicity synthetic imidazole fungicide. Its primary mechanism of action involves inhibiting the demethylation of lanosterol within fungi, thereby blocking its biosynthesis and disrupting the structure and function of the cell membrane, ultimately leading to fungal death. Prochloraz has excellent systemic and conductive properties and is widely used in the control of wheat Fusarium head blight [8], rice blast [9], Tomato Fusarium wilt [10], Chilli anthracnose [11], and other fungal diseases (such as Bakanae disease and Maize leaf blight) [12,13]. Azoxystrobin is a strobilurin fungicide that inhibits mitochondrial respiration by interfering with electron transfer between cytochromes, thus disrupting cellular energy supply and exerting fungicidal activity [14]. It has a remarkable control effect on fungal diseases of gramineous crops [15,16], vegetables [17], and melons [18]. However, the long-term and high-dose application of single-component pesticides readily induces pest resistance by activating bypass oxidation pathways in fungi, leading to reduced efficacy and complicating disease management efforts [19,20].

Pesticide compounding provides an economical and practical solution to address the limitations of single-component pesticide formulations. Composite formulations, consisting of two or more active ingredients, can realize synergistic effects by leveraging the complementary action mechanisms and modes of various compounds [21,22,23]. Ma et al. discovered that, compared to individual components, all combinations of prochloraz and azoxystrobin technical materials exhibited a more pronounced fungicidal effect against Fusarium graminearum, with the 4:2 ratio showing the highest efficacy [24]. Zuo et al. revealed that the integrated application of the emulsion in water (EW) containing tebuconazole and prochloraz, in conjunction with azoxystrobin water dispersible granules (WDG), demonstrated significant efficacy in controlling wheat Gibberella zeae, Rhizoctonia cerealis, and Puccinia recondite, with notable yield-increasing qualities [25]. Gu et al. prepared suspension concentrates with different proportions of prochloraz and azoxystrobin. The indoor efficacy results indicated that the composite formulations exhibited either additive or synergistic effects against Rhizoctonia cerealis, with the synergistic combination demonstrating superior field control efficacy compared to the jinggangmycin aqueous solution [26]. While the synergistic effect of prochloraz and azoxystrobin in enhancing pesticidal efficacy has been well-documented, their application remains constrained by the conventional formulation approaches, primarily limited to suspension concentrates or tank mixtures. More importantly, the underlying synergistic mechanism of prochloraz and azoxystrobin remains unclear.

In addition to pesticide compounding strategies, improving the effective utilization rate of pesticides is also a core solution to address the shortcomings of traditional formulations. Traditional formulations, which are characterized by large particle sizes, poor dispersibility, and high levels of organic solvents and emulsifying agents, experience a decrease in antifungal efficacy due to the development of resistance, leading to increased pesticide usage and heightened environmental risks. The unique properties of nanomaterials, such as small size, large specific surface area, and high permeability, provide a new avenue for the development of advanced pesticide delivery systems. In 2019, nanopesticides were rated as the top emerging technology in the field of chemistry by the International Union of Pure and Applied Chemistry (IUPAC). The term nanopesticides refers to any pesticide formulation that (a) intentionally includes entities in the nanometer size range, (b) is designated with a “nano” prefix, and/or (c) is claimed to have novel properties associated with the small size [27]. Compared to non-nanoscale pesticides, nanopesticides have demonstrated a 31.5% increase in overall efficacy against target organisms. In field trials, the efficacy has improved by 18.9%. Furthermore, the loss of active ingredients before reaching the target organisms is reduced by 41.4%, and the toxicity to non-target organisms is decreased by 43.1% [28]. The development of efficient and environmentally benign nanopesticides represents a critical advancement in integrated pest management (IPM) strategies, addressing the dual imperatives of crop protection and sustainable agricultural development [29].

A solid nanodispersion (SND) is a nanoformulation with a hydrophobic pesticide compound dispersed in a solid hydrophilic matrix. Compared to liquid formulations such as EW and suspension concentrate, SND can not only maintain the desirable solubility and redispersibility of water-based formulations but also decrease surfactant content and improve stability and safety during storage and transportation. It is particularly noteworthy that the preparation of solid nanodispersions based on self-emulsifying technology is simple, energy-efficient, and easy to scale up. In our previous research, the abamectin B₂ SND was produced by a self-emulsifying technique combined with carrier solidification. The toxicities of the nanoformulation against diamondback moths and south root-knot nematode were more than 1.7 times that of EW and WDG [30]. Nevertheless, the relevant reports on solid nanodispersions that combine multiple active ingredients remain scarce.

Therefore, in order to enhance pesticide bioavailability and overcome the shortcomings of traditional single-component formulations, this research has developed a SND co-loaded with prochloraz and azoxystrobin through a self-emulsifying carrier adsorption method. The particle size, morphology, surface tension, contact angle, and safety to non-target cells have been characterized. Furthermore, the synergistic biological effects and antifungal mechanisms of SND have been elaborated in detail. The research findings offer new insights and approaches for addressing the issue of pest resistance while providing a theoretical basis for the development of novel, high-efficiency pesticide formulations.

2. Materials and Methods

2.1. Chemicals and Microorganisms

Prochloraz technical material (TC, 97%, w/w) was supplied by Jiangxi Huihe Chemical Co., Ltd. (Jiujiang China). Azoxystrobin TC (98%, w/w) was provided by Inner Mongolia Lingsheng Crop Technology Co., Ltd. (Alxa League, China). Styryl phenol polyoxyethylene ether (emulsifier 600) was provided by Cangzhou Hongyuan Agrochemical Co., Ltd. (Cangzhou, China). Sodium benzoate was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Ethyl acetate was bought from J&K Scientific Ltd. (Beijing, China). Potato dextrose agar (PDA) and potato dextrose broth (PDB) were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). The commercial microemulsion (ME) of prochloraz and azoxystrobin (30%, prochloraz:azoxystrobin = 14:1, w/w) was provided by Hebei Bojia Agriculture Co., Ltd. (Shijiazhuang, China). Copper–zinc superoxide dismutase (CuZn-SOD, No. A001-4-1), total protein quantitative (No. A045-2-2), and catalase (CAT, No. A007-1-1) assay kits were bought from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Human hepatoma cells (HepG2) were purchased from Zhejiang Noble Biological Products Co., Ltd. (Hangzhou, China). Fusarium graminearum was provided by the Institute of Microbiology, Guangdong Academy of Sciences (Guangzhou, China). Pyricularia oryzae was provided by the China Agricultural University (Beijing, China).

2.2. Preparation of SND Co-Loaded with Prochloraz and Azoxystrobin

The SNDs containing different mass ratios of prochloraz to azoxystrobin were synthesized through a self-emulsifying carrier adsorption approach. As a representative example, the preparation process for the SND with a 14:1 (w/w) prochloraz-to-azoxystrobin ratio is described below. Initially, a solution was prepared by dissolving 4.0412 g of prochloraz, 0.2857 g of azoxystrobin, and 8.4 g of emulsifier 600 in 3 mL of ethyl acetate. This solution was subsequently incorporated into 29.2730 g of sodium benzoate. Following thorough mixing, the resulting mixture was subjected to drying in an oven at 40 °C for 12 h to yield the final solid nanodispersion product.

In the formulation optimization phase, seven distinct SND formulations were developed, featuring varying mass ratios of prochloraz to azoxystrobin (1:1, 2:1, 4:1, 5:1, 6:1, 10:1, and 14:1). The experimental work was carried out in Beijing, China, spanning the period from July 2023 to January 2025.

2.3. Particle Size and Zeta Potential Measurements

The SNDs were reconstituted as 0.1% (w/w) aqueous suspensions using deionized water for characterization. The mean particle size, polydispersity index (PDI), and zeta potential were measured at ambient temperature (25 ± 1 °C) using a Zetasizer Nano ZS 90 instrument (Malvern Panalytical Ltd., Malvern City, UK). Particle size and PDI were determined to evaluate the dispersion uniformity, while zeta potential measurements provided information about the surface charge characteristics. To ensure data reliability, all measurements were performed in three independent replicates, with the results expressed as mean values standard deviations (mean ± S.D.).

2.4. Morphological Characterization of SND

The morphological characteristics of the SNDs were examined using a JSM-7401F scanning electron microscope (JEOL, Tokyo, Japan). For sample preparation, a droplet of the aqueous dispersion was deposited onto a pristine silicon substrate, followed by air-drying and subsequent platinum coating using an ETD-800 sputter coater (Beijing Elaborate Technology Development Ltd., Beijing, China). Imaging was performed in low electron image (LEI) mode to capture the structural details.

The SEM micrographs were analyzed through particle size distribution analysis. A frequency distribution histogram was generated to illustrate the size-dependent particle population, followed by Gaussian fitting to obtain the corresponding normal distribution curve, which provided a statistical characterization of the particle size distribution.

2.5. Antifungal Activity Assay

The antifungal activities of the SND and commercial ME against Fusarium graminearum and Pyricularia oryzae were evaluated using the Mycelial growth rate method. The experimental procedure was conducted as follows. An appropriate amount of the SND or ME aqueous dispersion was added to 50 mL of a melted PDA medium and mixed thoroughly to achieve active ingredient concentrations of 0.005 mg/L, 0.01 mg/L, 0.05 mg/L, 0.1 mg/L, 0.5 mg/L, and 1 mg/L. All drug solutions were filter-sterilized (0.22 μm membrane) prior to medium incorporation to ensure aseptic conditions. The mixture was then cooled and solidified in a laminar flow cabinet for subsequent use. Sterilized disposable pipette tips with an inner diameter of 5.2 mm were used to punch holes at the edge of the colonies of Fusarium graminearum or Pyricularia oryzae to obtain the corresponding agar plugs. The mycelial side of the plugs was placed downward onto the center of the solidified pesticide-containing PDA medium, ensuring tight contact between each other. The PDA medium without any added chemicals served as the control check (CK) group. All inoculated culture media were sealed with parafilm and incubated in a biochemical incubator at 28 ± 1 °C under dark conditions. The colony diameter of mycelium was measured by the crisscross method. The mycelial growth inhibition rate was calculated using the following formula. The toxicity regression equations and the median effective concentration (EC₅₀) were calculated by probit analysis using IBM SPSS Statistics 25 statistical software (IBM Corp., Armonk, NY, USA).

$$\mathrm{G}\mathrm{r}\mathrm{o}\mathrm{w}\mathrm{t}\mathrm{h}\mathrm{r}\mathrm{e}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e}\mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{\%}={\displaystyle \frac{{\mathrm{D}}_1-{\mathrm{D}}_2}{{\mathrm{D}}_1}}\times 100\mathrm{\%}$$

(1)

where D₁ and D₂ represent the colony growth diameters in the control check group and pesticide-treated groups, respectively.

According to the calculated inhibition rate and the corresponding concentration, the Probit-log (dose) model of Probit Regression Analysis was fitted [31]. The standard virulence regression model is typically expressed as Y(probit) = a + b × log10(dose), where Y represents the probability of inhibition or mortality in probit units, a denotes the intercept, b indicates the regression coefficient (slope), and the dose corresponds to the concentration or dosage of the test compound.

2.6. Calculation of Synergistic Coefficient

The synergistic coefficient (SR) of composite formulations was calculated using the Wadley method based on the following formulas [24]:

$${\mathrm{E}\mathrm{C}}_{50}\left(\mathrm{t}\mathrm{h}\right)={\displaystyle \frac{\mathrm{a}+\mathrm{b}}{\frac{\mathrm{a}}{{\mathrm{E}\mathrm{C}\left(\mathrm{A}\right)}_{50}}+\frac{\mathrm{b}}{{\mathrm{E}\mathrm{C}\left(\mathrm{B}\right)}_{50}}}}$$

(2)

$$\mathrm{S}\mathrm{R}={\displaystyle \frac{{\mathrm{E}\mathrm{C}}_{50}\left(\mathrm{t}\mathrm{h}\right)}{{\mathrm{E}\mathrm{C}}_{50}\left(\mathrm{o}\mathrm{b}\right)}}$$

(3)

In this context, A and B represent the single-component formulations of prochloraz and azoxystrobin, respectively, while a and b refer to the mass percentages of prochloraz and azoxystrobin in the total active ingredients of the composite SNDs. EC₅₀(th) and EC₅₀(ob) are the theoretical and actual EC₅₀ values of SND, respectively.

2.7. Oxidative Stress Assay

Fusarium graminearum was inoculated at the center of the PDA plates and incubated in a biochemical incubator at 28 ± 1 °C in the dark for 5 days. Subsequently, a 5.2 mm diameter mycelial disk was excised using a puncher and transferred to 50 mL of PDB medium. The culture was incubated at 25 °C in a constant temperature shaker for 2 days. Then, the pesticide aqueous dispersion was added to the PDB medium, and the hyphae from the control and pesticide-treated groups were collected after 2 days. A certain amount of mycelium was homogenized in pre-cooled PBS using a shear machine (AD500S-H, Shanghai AngNing Instrument Co., Ltd., Shanghai, China) and an ultrasonic cell disruptor (JY92-IIN, Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China) consecutively. The homogenate was centrifuged at 12,000 rpm for 20 min at 4 °C using a refrigerated centrifuge (ST16R, Thermo Fisher Scientific, Waltham, MA, USA). The resulting supernatant was analyzed for soluble total protein content and antioxidant enzyme activities (superoxide dismutase, SOD; catalase, CAT) using commercial assay kits (CuZn-SOD, total protein quantitative, and CAT assay kits; Nanjing Jiancheng Bioengineering Institute, Nanjing, China), following the manufacturer’s protocols.

2.8. Morphological Characterization of Hyphae

Fusarium graminearum was initially cultured on PDA plates. A 5.2 mm mycelial plug excised from the colony margin was aseptically transferred into a sterile flask containing 50 mL of PDB and incubated in a constant temperature shaker at 25 °C for 48 h. To assess the antifungal efficacy of the prochloraz-azoxystrobin co-loaded SND formulation, the pesticide dispersion was introduced into the PDB culture medium. Following a two-day exposure period, hyphal samples were collected from both treated and control groups for morphological analysis. The hyphae underwent fixation in 2.5% glutaraldehyde solution at 4 °C for 12 h, followed by thorough washing with phosphate-buffered saline (PBS) to eliminate residual fixative. A sequential dehydration process was then performed using ethanol solutions at increasing concentrations (30%, 50%, 70%, 90%, and 100%). The dehydrated specimens were subsequently mounted on pristine silicon substrates and air-dried overnight at ambient temperature. Prior to scanning electron microscopy (SEM) observation, the samples were coated with a thin platinum layer using sputter coating technology.

2.9. Cell Membrane Permeability Test

Fusarium graminearum was initially cultivated on PDA plates, followed by the transfer of a 5.2 mm mycelial disk to PDB for a 48 h incubation at 25 °C. The experimental groups were then treated with either SND or ME aqueous dispersions, while maintaining identical culture conditions for an additional two days. For conductivity measurements, harvested mycelium was processed to remove the culture medium through filtration and subsequently resuspended in deionized water at a 1:50 (w/v) ratio. Electrical conductivity was monitored using a DDSJ-308F conductivity meter (Shanghai INESA Physico-Optical Instruments Co., Ltd., Shanghai, China) at predetermined intervals (1, 2, 4, 6, 8, 10, and 12 h post treatment). The relative conductivity values, serving as the evaluation metric, were calculated from the obtained measurements. To ensure data reliability, the entire experimental procedure was conducted in three independent replicates.

2.10. Contact Angle Measurement

The wettability characteristics of the formulations were assessed on both wheat and rice leaf surfaces. Contact angle measurements were performed at ambient temperature using a JC2000D contact angle analyzer (Zhongchen Digital Technic Apparatus Co., Ltd., Shanghai, China) employing the sessile drop technique. A 7-μL droplet of 0.01% (w/w) sample dispersion was carefully deposited on the leaf surface and allowed to equilibrate for 5 s. The contact angles were determined through a five-point fitting analysis, with the final reported values representing the mean of five independent measurements conducted at different positions on the leaf surface.

2.11. Surface Tension Measurement

Surface tension measurements were conducted using the du Nouy ring method with a JK998BM automatic tensiometer (Zhongchen Digital Technic Apparatus Co., Ltd., Shanghai, China). Prior to each measurement, the platinum ring underwent thorough cleaning through flame sterilization using an alcohol burner. For the measurement procedure, the ring was immersed in 0.1% (w/w) pesticide dispersions and subsequently withdrawn at a controlled rate. As the ring approached the liquid–air interface, a liquid film formed and eventually ruptured, with the maximum force being precisely recorded by the instrument. The corresponding surface tension values (γ) were automatically calculated by the integrated software system. Each example reported data represents the mean of three independent measurements, ensuring the reliability of the results.

2.12. Cytotoxicity Test

The effects of SND and ME on human hepatocellular carcinoma (HepG2) cells were tested by the methyl thiazolyl tetrazolium (MTT) method. The cells were seeded into the culture medium at a density of 1 × 10⁴ cells/well. Subsequently, 200 μL of SND or ME aqueous dispersion containing the active ingredients at concentrations ranging from 5 to 120 μM were added to the wells and incubated for 4 h. Afterwards, 20 μL of MTT solution (5 mg/mL per well) was added, and the incubation was continued in the dark for another 3 h. After removing the supernatant, 150 μL of dimethyl sulfoxide was added to dissolve the formed formazan granules. The OD value of each well was measured at 490 nm using an enzyme-linked immunosorbent assay reader (Multiskan FC, Thermo Fisher Scientific, Waltham, MA, USA) and the cell viability (%) was calculated.

2.13. Statistical Analysis

Data were analyzed by one-way analysis of variance (ANOVA) and Duncan’s multiple range test. Statistical analysis was performed with SPSS (IBM SPSS Statistics 25, International Business Machines Corporation, New York, NY, USA) and a probability less than 0.05 was deemed to be statistically significant.

3. Results

3.1. Characterization of SND Co-Loaded with Prochloraz and Azoxystrobin

The proportion of active ingredients in the composite formulations can substantially influence their biological efficacy. In this investigation, seven composite SNDs co-loaded with prochloraz and azoxystrobin along with the corresponding two single-component SNDs were all prepared by a self-emulsifying carrier adsorption technique. The preparation process is simple, energy-efficient, and easy to scale up. Additionally, the formulation composition is free of organic solvents, facilitating storage and transportation. According to previous reports, the composite formulations combining prochloraz and azoxystrobin tended to exhibit higher biological activity when the proportion of prochloraz was high [26]. Therefore, in this study, with the total active ingredient content fixed at 10% in the SNDs, the mass ratios of prochloraz to azoxystrobin were set at 1:1, 2:1, 4:1, 5:1, 6:1, 10:1, and 14:1. When the mass ratio of the two components was 1:1, the average particle size of the formed solid powder was 3162 nm measured by DLS, and it was difficult to uniformly disperse in water. In contrast, the aqueous dispersions of the SNDs with other ratios were homogeneous and transparent solutions, with a hydrated particle size of 13–16 nm and a PDI less than 0.3 (

Table 1. Particle sizes and dispersibilities of the composite SNDs with different ratios of prochloraz to azoxystrobin measured by DLS.

Ratio of Prochloraz to Azoxystrobin	Average Particle Size (nm) ± S.D.	PDI ± S.D.
Single Prochloraz	10.3 ± 0.02	0.252 ± 0.01
Single Azoxystrobin	14.7 ± 0.31	0.092 ± 0.00
2:1	15.2 ± 0.12	0.227 ± 0.01
4:1	13.9 ± 0.10	0.141 ± 0.05
5:1	13.4 ± 0.05	0.021 ± 0.01
6:1	13.4 ± 0.07	0.075 ± 0.07
10:1	16.0 ± 0.29	0.268 ± 0.01
14:1	13.3 ± 0.04	0.020 ± 0.01

).

As shown in Figure 1, the SND nanoparticles in combinations ranging from 2:1 to 14:1 were all well-dispersed spherical in shape. The statistical particle sizes based on SEM images were between 13 nm and 14 nm, consistent with the DLS results.

Interfacial charges affect the long-term stability of aqueous dispersions by influencing the electrostatic repulsion between particles. As shown in Figure 2, the zeta potentials of single prochloraz SND and azoxystrobin SND were −6.4 mV and −7.3 mV, respectively. The zeta potentials of their composite formulations also ranged from −6 mV to −7 mV.

3.2. Antifungal Activity of SND

A comparative study was conducted to evaluate the antifungal activities of the composite SNDs and the commercial composite ME of prochloraz and azoxystrobin, targeting Fusarium graminearum, the pathogen of wheat Fusarium head blight and Pyricularia oryzae, the pathogen responsible for rice blast. The fungal inhibition rates (%) of the composite SNDs and ME formulations against both pathogens, as quantitatively demonstrated in Tables S1 and S2 of the Supporting Information. As shown in

Table 2. Antifungal activities of the composite SNDs and ME on Fusarium graminearum.

Formulation	Toxicity Regression Equation *	Correlation Coefficient	EC50 (mg/L)	95% Confidence Interval	Synergistic Coefficient
Prochloraz	Y = 0.5996X + 0.7094	0.9818	0.066	0.043–0.099	—
Azoxystrobin	Y = 0.5716X + 0.3508	0.9910	0.243	0.155–0.428	—
2:1 SND	Y = 0.8003X + 0.9588	0.9975	0.067	0.046–0.087	1.384
4:1 SND	Y = 0.8382X + 1.1099	0.9950	0.047	0.035–0.064	1.644
5:1 SND	Y = 0.9525X + 1.3835	0.9935	0.035	0.013–0.078	2.146
6:1 SND	Y = 0.9625X + 1.4882	0.9945	0.028	0.012–0.057	2.631
10:1 SND	Y = 0.9392X + 1.6218	0.9808	0.019	0.007–0.038	3.720
14:1 SND	Y = 0.9625X + 1.7836	0.9757	0.014	0.005–0.027	4.955
14:1 ME	Y = 0.8282X + 1.242	0.9945	0.032	0.007–0.089	2.168

* Data were fitted by Probit-log(dose) model.

and Figure 3, the EC_50s of the prochloraz and azoxystrobin single-component SNDs against Fusarium graminearum was 0.066 mg/L and 0.243 mg/L, respectively, that is, the antifungal activity of pure prochloraz was 3.7 times that of azoxystrobin.

The EC₅₀s of the six composite SNDs with 2:1, 4:1, 5:1, 6:1, 10:1, and 14:1 ratio of prochloraz to azoxystrobin was 0.067 mg/L, 0.047 mg/L, 0.035 mg/L, 0.028 mg/L, 0.019 mg/L, and 0.014 mg/L, respectively. The antifungal activities progressively enhanced with the rising proportion of prochloraz, attributed to its stronger fungicidal efficacy. The synergistic coefficient (SR) provides a quantitative assessment of the interaction between two compounds in the composite formulation. An SR value exceeding 1.5 denotes significant synergism, whereas a value ranging between 0.5 and 1.5 suggests an additive effect. When SR was less than 0.5, it indicated an antagonistic effect. In this study, when the mass ratio of prochloraz to azoxystrobin was greater than 4:1, the SR of the composite SND was larger than 1.5, demonstrating a synergistic effect. Moreover, as the content of prochloraz increased, the degree of synergism also intensified. It is worth noting that the nano-combination of prochloraz and azoxystrobin at a ratio of 14:1 was consistent with the ratio of active ingredients in the commercial ME, yet the antifungal activity of the SND was 2.3 times higher and its synergism was also stronger than ME. The improvement in formulation performance can primarily be attributed to the small size effect and interfacial effect of nanoparticles.

The antifungal activities of the different composite SNDs and ME against Pyricularia oryzae are shown in

Table 3. Antifungal activities of the composite SNDs and ME on Pyricularia oryzae.

Formulation	Toxicity Regression Equation *	Correlation Coefficient	EC50 (mg/L)	95% Confidence Interval	Synergistic Coefficient
Prochloraz	Y = 0.7788X + 0.9461	0.9955	0.061	0.044–0.084	—
Azoxystrobin	Y = 0.5611X + 0.0928	0.9706	0.683	0.391–1.524	—
2:1 SND	Y = 0.7082X + 0.7540	0.9854	0.058	0.040–0.082	1.510
4:1 SND	Y = 0.7069X + 0.9508	0.9925	0.045	0.031–0.064	1.657
5:1 SND	Y = 0.6895X + 0.9734	0.9920	0.039	0.026–0.055	1.844
6:1 SND	Y = 0.6362X + 0.9113	0.9793	0.037	0.024–0.054	1.895
10:1 SND	Y = 0.6305X + 0.9312	0.9772	0.033	0.021–0.049	2.015
14:1 SND	Y = 0.6808X + 1.1168	0.9925	0.023	0.015–0.033	2.824
14:1 ME	Y = 0.7029X+ 1.0142	0.9884	0.036	0.024–0.051	1.804

* Data were fitted by Probit-log(dose) model.

. The control effect of the single prochloraz SND against Pyricularia oryzae was 11.2 times that of azoxystrobin SND. Similarly to the results for Fusarium graminearum, the antifungal activities of the composite SNDs against Pyricularia oryzae also enhanced as the proportion of prochloraz increased and exhibited a synergistic effect. The efficacy of the SND with a 14:1 ratio of prochloraz to azoxystrobin was 1.6 times that of ME.

3.3. Fungicidal Mechanism

The experimental findings demonstrated a concentration-dependent enhancement in fungicidal efficacy corresponding to elevated prochloraz content in the formulated preparation. Notably, the composite displayed superior inhibitory effects against Fusarium graminearum compared to other tested fungal strains. To elucidate the mechanistic basis of this differential antimicrobial performance, we conducted a systematic investigation using an optimized SND formulation (prochloraz: azoxystrobin = 14:1) with Fusarium graminearum as the target organism, employing ME as experimental controls.

3.3.1. Effect on Antioxidant Enzyme Activity

To clarify the fungicidal mechanism of the composite SND on Fusarium graminearum, SOD, CuZn-SOD, CAT, and total protein in pesticide-treated mycelia were measured to test the antioxidant defenses.

As shown in Figure 4a–c, the levels of total SOD, CuZn-SOD, and CAT in fungi all decreased after SND treatment, exhibiting a gradual decline with increasing pesticide concentration. This is attributed to the disruption of the antioxidant defense system in Fusarium graminearum by pesticide, which impairs its ability to effectively scavenge ROS and hydrogen peroxide within the cells. Consequently, unsaturated fatty acids on the cell membrane undergo oxidation, affecting the structure and function of cells, ultimately leading to death [32]. The inactive antioxidant enzymes, existing in the form of zymogens after the reaction ends, resulted in an increase in total protein content (Figure 4d).

3.3.2. Effect on Mycelial Morphology

As depicted in Figure 5a, the untreated Fusarium graminearum exhibited a smooth wall, an intact surface structure, and a three-dimensional morphology. However, the mycelia treated with the composite SND became severely shrunk and crumpled, and some hyphae even become flattened (Figure 5b). This phenomenon is similar to the previously reported impact of azoxystrobin nanosuspension on Fusarium oxysporum [33].

3.3.3. Effect on Membrane Permeability

The effects of the composite SND and ME on cell membrane permeability were studied using relative conductivity as an evaluation index. A high relative conductivity represents a strong membrane permeability and severe cell damage. As shown in Figure 6, both SND and ME resulted in an increase in relative conductivity compared to the control group, indicating that they caused different levels of damage to the cell membrane. In contrast, the treatment of composite SND, even at low concentrations, led to a substantial increase in the relative conductivity of the system compared with the ME formulation. This result further substantiates the hypothesis that SND could disrupt the structure and function of fungal cell membranes to a greater extent, more readily leading to abnormal cellular metabolism or even death, thereby enhancing antifungal activity.

3.4. Foliar Wettability

In this investigation, wheat leaves, rice leaves, and parafilm were employed as hydrophobic substrates to evaluate the wettabilities of SND and ME. As shown in Figure 7a, the contact angles of pure water on wheat leaf, rice leaf, and parafilm were 133.1°, 134.8°, and 108.7°, respectively, confirming their hydrophobic properties. Compared to water, the contact angles of the two formulations were significantly reduced due to the incorporation of surfactants, which enhanced the spreading of droplets on the surface. The contact angles of SND on parafilm were 41.9° and 4.3° smaller than those of pure water and ME, respectively, indicating improved wettability on the hydrophobic substrate with a homogeneous microstructure. Figure 7b shows the surface tensions of SND and ME. The surface tension of SND was 1.9 mN/m higher than ME. This result suggests that contact angle may be not only related to surface tension but also associated with other properties of the formulation, such as particle size effects.

3.5. Cytotoxicity to Non-Target Cells

HepG2 was used as a model cell to investigate the toxicity of the composite SND and ME towards non-target cells. As reported, prochloraz can induce oxidative stress response in cultured human cells, leading to DNA damage and cytotoxicity [34]. Azoxystrobin can inhibit the respiration rate of HepG2 cells and affect cell proliferation [35]. The cell viabilities of HepG2 after treatment with the composite SND and ME are presented in Figure 8. The commercial ME did not significantly affect HepG2 cells at lower concentrations, yet it significantly reduced cell viability at a higher concentration (95 μM), demonstrating pronounced cytotoxicity. In contrast, SND had minimal impact on cell viability within the concentration range tested, indicating favorable biosafety.

4. Discussion

Azoxystrobin and prochloraz co-loaded SNDs were fabricated using a self-emulsifying carrier adsorption technique. The composite nanoformulations were systematically characterized in terms of their physicochemical properties, antifungal efficacy, foliar wettability, and biological safety, with particular emphasis on elucidating the fungicidal mechanisms.

The DLS analysis revealed a significant increase in the particle size of the SND with a 1:1 ratio of prochloraz to azoxystrobin, which can be attributed to the limited solubility of azoxystrobin in ethyl acetate. This solubility constraint led to particle agglomeration during the carrier adsorption process, consequently increasing particle size and compromising suspension stability and dispersibility. However, the SEM results demonstrated that within an optimal ratio range (2:1 to 14:1), the proportion of prochloraz to azoxystrobin had minimal impact on the particle size and morphology. These findings suggest that the dispersibility and particle size characteristics of the composite SNDs are predominantly governed by the solubility of active ingredients in the organic solvent.

The low zeta potential of the SNDs was primarily influenced by the formulation composition, particularly the surfactant properties. Emulsifier 600, a nonionic polymer, contributes limited surface charge when adsorbed onto pesticide particles. Nevertheless, its long hydrophobic chains may induce steric hindrance against particle aggregation. It is noteworthy that the SNDs maintain a solid state before application, and their short-term stability upon aqueous dilution sufficiently meets practical spraying requirements. More importantly, as evidenced by the experimental results, the solid nanoformulation demonstrated excellent redispersibility, representing a crucial advantage for agricultural applications.

The antifungal activity results confirmed that, regardless of the type of pathogenic fungi, the biological activity of SND with a high proportion of prochloraz was superior to that of the traditional formulation. This is beneficial for reducing pesticide usage, delaying pest resistance, and minimizing environmental pollution.

The aerobic metabolism in fungal cells inevitably generates reactive oxygen species (ROS), which induce oxidative damage to cellular components including DNA, proteins, and lipids, ultimately leading to apoptosis [36]. To counteract oxidative stress, fungi have evolved comprehensive antioxidant defense systems comprising superoxide dismutases (SODs), catalases (CATs), and the glutathione system, which collectively maintain intracellular redox homeostasis through ROS scavenging [37,38]. The antioxidant mechanism involves a cascade of enzymatic reactions: SOD catalyzes the conversion of superoxide anions (O^2-) into less toxic hydrogen peroxide and molecular oxygen, while CAT subsequently decomposes hydrogen peroxide into water and oxygen, playing a crucial role in preventing intracellular hydrogen peroxide accumulation [39]. These enzymatic systems maintain the dynamic equilibrium between oxidation and antioxidation in living organisms, serving as reliable biomarkers for cellular oxidative damage [40]. In this investigation, the fungicidal mechanism of the SND co-loaded with prochloraz and azoxystrobin can be summarized as follows. SND diminishes the antioxidant defense capability of Fusarium graminearum, thereby causing severe oxidative damage due to active oxygen species and hydrogen peroxide accumulation within the cell. This subsequently alters cell membrane permeability and ultimately results in fungal death. The elucidation of fungicidal mechanisms can lay a theoretical foundation for the design and construction of highly effective fungicides.

The foliar wettability of pesticide formulations, characterized by two key parameters, contact angle and surface tension, plays a pivotal role in determining the effective utilization rate and efficacy of agricultural chemicals. Usually, a contact angle of 90° is regarded as the demarcation point of the interface hydrophilicity and hydrophobicity, with a smaller contact angle indicating superior wettability. Notably, both rice and wheat leaves exhibited typical hydrophobic surface properties, presenting challenges for effective wetting and retention of traditional formulations [41,42]. Typical approaches to improve wettability rely on increasing surfactant concentrations or incorporating organic solvents [43].

Traditional tank-mixed pesticides require the precise proportioning of different pesticides before application. In practice, variations in dosage and mixing ratios can significantly impact efficacy, often leading to overuse. Additionally, the mixing process typically necessitates the inclusion of extra additives, which complicates operations and contributes to environmental pollution. In contrast, the SND formulation can be used directly after simple dilution with water, significantly simplifying application procedures. More importantly, as demonstrated by this research work, the SND exhibited higher antifungal activity and biosafety compared to traditional formulations. Coupled with its simple and low-cost preparation process, it shows broad application prospects in disease prevention and control. In addition, the SND formulation remains in a solid state prior to application, with weak intermolecular interactions and slow molecular movement, which allows pesticides to maintain good stability. Even after dilution with water, the absence of any catalytically active ingredients in both the solution and formulation ensures the dilution stability and efficacy of SND. Nevertheless, the application of SND still faces several challenges, such as the upgrading and improvement of traditional processes and production lines, as well as limited awareness of users of new products.

5. Conclusions

The SNDs co-loaded with prochloraz and azoxystrobin were constructed through a self-emulsifying carrier adsorption method, and the most significant synergistic ratio was determined to be 14:1 (prochloraz: azoxystrobin). The average particle size of SND was 13 nm and exhibited uniformly dispersed spherical in shape. The antifungal activities of the composite SND against Fusarium graminearum and Pyricularia oryzae were 2.3-fold and 1.6-fold higher than those of ME with the same proportion of active ingredients. The fungicidal mechanism of the composite SND can be summarized as follows. The SND could cause severe oxidative damage to fungi, by reducing the activities of SOD and CAT, and break the permeability of cell membranes, resulting in fungal death. In addition, the composite SND achieved excellent foliar wettability at a small environmental cost, thus improving the effective utilization rate of pesticides. This study introduces an innovative methodology for developing efficient and compounded pesticide formulations, offering substantial potential for application in sustainable pest management.