Fungicidal Effect of Strong Oxidative Free Radicals Against Fusarium graminearum and Their Impact on Wheat Growth and Yield

Abstract

Fusarium head blight (FHB), caused by Fusarium graminearum, is a significant fungal disease that adversely affects wheat production and food security. This study systematically evaluated the fungicidal efficacy of strong oxidative radicals (SORs) against F. graminearum and their effects on wheat growth and yield through a combination of in vitro and field experiments. In vitro experiments revealed that solutions containing different concentrations of radicals effectively suppressed the fungus. The results suggested that SOR solutions exhibited potent fungicidal activity against F. graminearum. At a concentration of 4.0 mg/L, the spore mortality rate was 96.8%, and at 5.0 mg/L, the rate reached 99.4%. The optimal concentration for the elimination of F. graminearum spores was determined to be 2.5 × 10⁵ CFU/mL. The optimal treatment duration for SORs was 10 min. Furthermore, field trials investigated the effects of SORs on wheat growth, and agronomic traits were assessed, along with their efficacy in controlling FHB in field trials, both as a standalone treatment and in combination with commercial pesticides. The results indicated that the application of SORs alone achieved an 87.9% control efficacy, demonstrating significant potential for disease control. Furthermore, SORs positively influenced wheat agronomic traits such as plant height, spike length, grain weight per plant, grain number per plant and grain yield, providing a promising new approach for the green control of FHB.

1. Introduction

Wheat, as one of the major staple crops globally, plays a crucial role in ensuring food security by maintaining the stable development of its production and quality [1,2,3]. FHB, also known as wheat head rot, rotten wheat head, or red wheat head, is a widespread disease [4]. Research into FHB dates back to the early 20th century, and as the issue of Fusarium toxin contamination emerged, the disease gained significant attention [5,6]. FHB is highly prevalent in North America, Europe, and Asia, with China’s Huang-Huai wheat region being one of the hardest-hit areas [7,8] In years with widespread outbreaks, wheat FHB in China can cause yield losses of up to 30–50%, and some severely affected areas experience total crop failure. FHB is caused by the fungus F. graminearum, which accumulates substantial quantities of toxic Fusarium toxins in infected grains, posing risks to human and animal health [9,10]. Deoxynivalenol (DON), also known as vomitoxin, is the primary mycotoxin found in wheat and its products. The widespread occurrence of FHB results in both yield losses and significant Fusarium toxin contamination, endangering food and feed safety [11,12,13]. The impact of climate change and altered agricultural practices has increased the frequency and range of FHB, making it a growing threat to global food security [14,15].

The control of FHB has long been an important challenge in agriculture. Traditional control methods include chemical treatments, breeding resistant varieties, and agronomic practices. However, each of these methods has limitations in practical application. Chemical control, primarily through fungicide application, remains the most common and direct method. These fungicides work by inhibiting pathogen growth, reproduction, or toxin synthesis. Major chemical treatments for FHB include triazoles (e.g., tebuconazole, propiconazole, fluconazole), benzimidazoles (e.g., carbendazim, thiophanate-methyl), and novel SDHI fungicides (e.g., fluopyram). Triazoles are favored for their broad-spectrum activity and systemic properties [16]. Although chemical control is effective in the short term, long-term reliance on these treatments can lead to pathogen resistance [17]. Resistance of F. graminearum to triazoles has been reported globally [18]. Furthermore, extensive pesticide use can cause environmental pollution and soil degradation, compromising the ecosystem’s health [19].

Breeding resistant varieties is considered an environmentally sustainable and cost-effective strategy for controlling wheat FHB [20,21,22]. However, environmental factors significantly impact resistance levels, and resistant varieties show varying results in different ecological zones. Breeding resistant varieties is a long-term process, and currently, no wheat varieties are entirely immune to wheat FHB [23]. Agronomic management practices also play a vital role in the integrated control of wheat FHB [24,25]. The implementation of crop rotation, precise management of sowing density and fertilization, and the prompt removal of infected plant residues post-harvest have been demonstrated to significantly mitigate the risk of pathogen reinfection in agricultural fields.

The aforementioned control strategies have mitigated the impact of wheat FHB to some degree, but the effectiveness of single measures is often limited. Therefore, modern agriculture emphasizes the Integrated Pest Management (IPM) strategy, which combines chemical treatments, resistant varieties, and agronomic management to maximize the synergistic effects of multiple methods [26]. However, in the face of climate change and pathogen variability, the effectiveness of these traditional approaches is gradually weakening, making them insufficient for meeting the needs of sustainable development. As a result, the exploration of novel green pest control technologies has become a critical research direction in wheat FHB management.

The rapid growth of the green pesticide industry has led to widespread interest in the technology of using strong oxidative radical solutions to partially replace traditional chemical pesticides, due to its significant research potential and broad market prospects. SORs, such as O²⁺, O, (O₃P), O³, and O²⁻, are generated through DBD discharge technology [27]. These radicals undergo gas–liquid mass transfer reactions to create a series of chain reactions, which, when coupled with micro-nano-gas-bubble technologies, efficiently synthesize radical solutions rich in ·OH, ·HO²⁻, and ·O³⁻ radicals. This approach has attracted considerable attention due to its advantages, including no residue, strong oxidation, spectral sterilization, and enhanced yield and profitability [28]. SORs (strong oxidizing radicals), with its extremely high redox potential, can react with a wide variety of organic molecules and has found applications in fields such as water treatment and air purification. Research has shown that SORs disrupt pathogen cell membranes through lipid peroxidation, causing the leakage of cellular contents, thus inhibiting pathogen growth. They directly target pathogen DNA, causing base oxidation and strand breaks, which impairs the pathogens’ reproductive abilities, disrupts enzyme functions, and interferes with normal metabolic processes [29,30,31].

In recent years, SORs have been widely used in various aspects of disease control, as well as in the preservation of fruits, vegetables, and seafood, due to its high-efficiency antimicrobial and environmentally friendly properties [32,33]. Ozone, whether in gaseous or aqueous form, significantly reduced the bacterial count (CFU/mL) of Staphylococcus aureus and Enterococcus faecalis at all tested concentrations and durations. The concentrations of 40 μg/mL and 60 μg/mL were notably more effective than 20 μg/mL for both bacterial strains [34]. Soaking or spraying tomatoes inoculated with Salmonella in ozonated water, with a turbidity of 2 NTU, successfully met the standards outlined by the USEPA (1997) [35]. Consequently, ozonated water is recommended as an alternative disinfectant for soaking or spraying when water turbidity is low. Studies on the detoxification of aflatoxins in peanuts using ozone revealed that under conditions of 5% (w/w) humidity and 6.0 mg/L ozone treatment for 30 min at room temperature, the detoxification rates for total aflatoxins and aflatoxin B1 (AFB1) were 65.8% and 65.9%, respectively, demonstrating the effective degradation of aflatoxins [36]. The ozonated water treatment of strawberries for 5 min delayed gray mold infection onset by 4 days and significantly reduced disease incidence during storage (approximately 17% lower than the control on day 8), thereby extending shelf life at 5 °C [37]. The antimicrobial effects of combining aqueous ozone (O₃) and peracetic acid (PAA) as a spray for whole chicken carcasses were evaluated by TetraClean Systems. The results showed that combining ozone with 500 ppm PAA significantly reduced Salmonella counts and lowered environmental PAA concentration, improving worker safety [38]. Mohammad demonstrated that low-concentration ozone (5 mg/L) significantly reduced Salmonella and Shiga toxin-producing Escherichia coli (STEC) in alfalfa seeds and sprouts, lowering food safety risks [39]. Ozonated water was also used to reduce the initial microbial content in processed foods and extend shelf life. The results showed a significant reduction in the total plate count (TPC) of semi-dried buckwheat noodles (p < 0.05), extending shelf life by 2–5 days [40]. The antibacterial effect of ozonated water disinfectant solutions on Listeria monocytogenes was assessed, and the sterilization efficiency coefficient (A) was calculated to range from 0.15 to 0.84, with lower A values indicating higher sterilization efficiency [41]. Ozone treatment on F. graminearum and DON-contaminated wheat grains demonstrated a significant effect on fungal inhibition and DON degradation, particularly under 120 min exposure and a 60 mmol/mol concentration [42]. Additionally, due to their rapid biochemical reaction rates, SORs can break down microbial cells through electrophilic addition, dehydrogenation, and electron transfer, ultimately decomposing them into H₂O, CO₂, and trace inorganic salts, with no toxic byproducts. Therefore, SORs have attracted significant attention from environmental and agricultural researchers and show great promise in the control of agricultural pathogens.

In light of the aforementioned background, the objective of this study was to examine the fungicidal mechanism of SORs against F. graminearum and their effects on wheat FHB. By optimizing the SOR sterilization parameters, this research investigated the inhibitory effects on F. graminearum, the mechanisms of action, and the practical feasibility of field application. The results of this study offer a novel approach to the green control of FHB and provide valuable references for the integrated management of other crop diseases.

2. Materials and Methods

2.1. Materials

Pathogen: F. graminearum (laboratory isolate). The F. graminearum strain Fg-01 employed in this study was obtained from infected wheat plants collected in Jiangsu Province, China. The strain was initially cultivated on PDA medium at 28 °C for 7 days and subsequently preserved on slants at 4 °C. Identification was conducted via internal transcribed spacer (ITS) sequencing, and morphological traits were verified through microscopic examination. This strain was applied in experiments assessing wheat resistance to FHB. A small amount of F. graminearum mycelium was selected and transferred to CMC liquid medium. The culture was incubated at 30 °C with shaking at 170 rpm/min under light for 3–5 days, resulting in a large amount of spore and mycelium mixture. This mixture was then filtered through sterilized gauze to obtain a pure spore suspension. A series of in vitro experiments were performed to assess the effectiveness of SORs in inactivating F. graminearum spores under different conditions. The findings offer critical insights into the potential application of SORs for managing wheat FHB in field settings.

SORs: SOR generation system (constructed by the research team). The high-ionization discharge device was designed with a discharge gap of 0.5 mm. The discharge electrode plates were fabricated from sintered silver-coated metal materials, with ceramic sheets serving as the dielectric layer. The total power of the gaseous radical generation system was 600 W, while the gas–liquid mixing unit operated at 0.75 kW. The ozone intake rate was 3.0 L/min, and the system pH was maintained at 5.2.

Wheat variety: The wheat variety “Ji Mai 23”, which is highly susceptible to wheat FHB, selected by the Crop Research Institute of Shandong Academy of Agricultural Sciences, was used as the experimental material in this study.

2.2. Effect of SOR Concentration on the Inactivation of F. graminearum

F. graminearum spores were prepared into a spore suspension using sterile water and adjusted to a concentration of 2.50 × 10⁵ CFU/mL using a Neubauer Improved hemocytometer (0650010). SORs were produced using a strong ionization discharge device, with the equipment parameters regulated according to Section 2.1. Since hydroxyl radicals and other reactive oxygen species have extremely short lifespans (typically at the nanosecond scale), direct quantification is highly challenging. Therefore, the total oxidant concentration in the solution was measured using the ATi Q45H TRO analyzer (ATI, American) as a proxy for the concentration of strongly oxidizing radicals. To systematically examine the impact of different radical concentrations on the reaction system, the SOR concentration was set at 1.00, 2.00, 3.00, 4.00, and 5.00 mg/mL [43].

In this study, a 0.5% sodium thiosulfate phosphate-buffered solution was selected as the neutralizing agent. As a reducing agent, sodium thiosulfate undergoes redox reactions with the free radical solution, thereby neutralizing it and terminating the reaction. This process eliminates the inhibitory and bactericidal effects of the free radical solution on microbial suspensions [43,44]. A 9.00 mL aliquot of each SOR solution was added to 1.00 mL of spore suspension and treated for 10 min. After treatment, 1.00 mL of the mixture was transferred into a test tube containing 9.00 mL of a neutralizing agent, mixed thoroughly, and allowed to act for another 10 min. Then, 100 µL of the mixture was evenly spread onto PDA agar plates. and incubated under 28 °C for 48 h. Following incubation, the colony count was recorded, and the sterilization rates were calculated using the following formula. All experiments were performed in triplicate, and the final results are expressed as the mean value.

Formula for sterilization rates:

$$\mathsf{\eta }={\displaystyle \frac{Nx}{No}}\times 100\%$$

η—sterilization rates (%);

N₀—colony count in the positive control group;

N_x—colony count in the experimental group.

Sterile water treatment was used as the control, eliminating microbial and non-experimental variable interference to ensure that observed experimental effects were solely attributable to the intended treatment and providing a negative control to distinguish the true effect of the experimental treatment. Through a computational analysis of the effects of different SOR concentrations on the sterilization rates of F. graminearum spores, we optimized and determined the most effective concentration for spore eradication.

2.3. Spore Suspensions at Different Concentrations

F. graminearum spores were prepared into a spore suspension using sterile water and adjusted to a concentration of 2.50 × 10⁷ CFU/mL using a hemocytometer. The suspension was then diluted in a tenfold series to concentrations of 2.50 × 10⁷ CFU/mL, 2.50 × 10⁶ CFU/mL, 2.50 × 10⁵ CFU/mL, 2.50 × 10⁴ CFU/mL, and 2.50 × 10³ CFU/mL. To each 1.00 mL of spore suspension, 9.00 mL of a 4.00 mg/L SOR solution was added, and the resulting solution was treated for 10 min. After treatment, 1.00 mL of the mixture was transferred to a test tube containing 9.00 mL of a neutralizing agent, mixed thoroughly, and allowed to react for another 10 min. Then, 100 µL of the mixture was evenly spread onto PDA agar plates. Following a 48 h incubation period, the colony counts on the agar plates for each experimental group were recorded and analyzed using the calculation formula described in Section 2.2. Based on the experimental data, the optimal spore concentration for SOR treatment of F. graminearum was determined. Sterile water treatment was used as the control. The experiment was repeated three times.

2.4. Effect of Different Treatment Durations

Graminearum spores were suspended in sterile water and adjusted to a concentration of 2.50 × 10⁵ CFU/mL using a hemocytometer. A 9.00 mL volume of a 4.00 mg/L SOR solution was added to 1.00 mL of the spore suspension and treated for 0, 3, 5, 10, 15, and 30 min, respectively. After treatment, 1.00 mL of the mixture was transferred into a test tube containing 9.00 mL of a neutralizing agent, mixed well, and allowed to react for another 10 min. Then, 100 µL of the mixture was spread evenly onto PDA agar plates. Count the number of colonies on the plates of each experimental group under different time treatments and analyzed using the calculation formula described in Section 2.2. Based on the data results, the optimal duration of SOR treatment was determined. Sterile water treatment was used as the control. The experiment was conducted in triplicate.

2.5. Methylene Blue Staining

Methylene blue staining is a widely used technique for assessing cell viability and membrane integrity. Methylene blue, as a biological stain, allows for the differentiation between living and dead cells. In viable cells, an intact membrane serves as a selective barrier that prevents methylene blue from entering the cytoplasm, leading to an absence of staining. However, in non-viable or structurally compromised cells, increased membrane permeability permits dye penetration, resulting in a pronounced blue coloration. This property makes methylene blue staining a useful tool for evaluating the antimicrobial effects of SORs on F. graminearum. The spore suspension was adjusted to a concentration of 2.50 × 10⁵ CFU/mL. To 1.00 mL of the spore suspension, 9.00 mL of a 4.00 mg/L SOR solution was added, and the resulting solution was treated for 5 min, with water serving as the control. A drop of 0.1% Löffler’s alkaline methylene blue stain was placed in the center of a glass slide, and a small amount of the spore suspension was mixed evenly with the stain under sterile conditions. A cover glass was then taken with tweezers, and one edge was placed in contact with the fungal cells. The cover glass was gently tilted and placed over the spore suspension. After approximately 3 min, the morphology and budding of the spores were observed under a low-power microscope followed by a high-power microscope. Cell states were differentiated by color.

2.6. SEM Observation of Mycelial Morphology of F. graminearum

SEM was used to observe the morphological changes induced by SOR on the mycelium at the ultrastructural level. For SEM, F. graminearum mycelium (0, 5, 20 mg/L for SOR) was pretreated with 2.5% glutaraldehyde for 24 h. The fixed cells were washed three times with a 100 mM phosphate buffer, each for 10 min. The cells were then fixed in 1% osmium tetroxide for 3 h and dehydrated using an ethanol gradient. The samples were gold-coated before being analyzed on a Hitachi SU8010 scanning electron microscope (HITACHI, Chiyoda City, Japan).

2.7. Field Trial Design

The experiment was conducted at the Jiangsu Runfruit High-Efficiency Ecological Agriculture Base in Zhenjiang, Jiangsu Province (32.14° N, 119.73° E), using the wheat variety Jimai 23. The experimental plots were selected from wheat fields under natural disease pressure in a rice–wheat rotation system. Each plot covered an area of 20 m², and treatments were arranged in a randomized complete block design (RCBD) with three replications per treatment. Tetramycin, a well-established antifungal antibiotic, was included as the positive control to evaluate and compare its efficacy with that of SORs in controlling wheat FHB. The fungicides were applied using a backpack-mounted electric sprayer (Laobaixing” LBX-16L), with formycin administered at 55 mL per 667 m² as a uniformly distributed fine mist. A real-time spraying apparatus integrated with an on-site synthesis system for a strong oxidative radical solution was applied in large-scale field trials. The working concentration of the solution was adjusted to 4.00 mg/L, with a spray volume of 240 L per 667 m², while the negative control group received only water. The operational setup is depicted in Figure 1. A total of five treatments were established in this study, based on the different fungicides and their combinations. The detailed treatment design is shown in

Table 1. Design of field experiments.

Treatment	No. 1	No. 2	No. 3	No. 4	No. 5	Application Timing
The first round of application	Tetramycin	CK	SORs	Tetramycin	SORs	BBCH 61
The second round of application	SORs	CK	Tetramycin	Tetramycin	SORs	BBCH 65

Note: BBCH61: wheat anthesis stage (emergence of the first extruded); BBCH 65: full flowering stage (50% of anthers extruded).

. The timing of fungicide application was determined using the disease warning system in conjunction with meteorological conditions. In 2021, applications were performed on 9 April (19 °C) and 16 April (22 °C), respectively.

2.8. Control Efficacy Survey

The safety of the pesticide was assessed by observing the crops at 3 and 7 days after treatment. At 7 and 14 days following the final pesticide application, field observations were made to evaluate disease incidence and wheat growth. Once FHB symptoms stabilized, the control efficacy was assessed. Following two rounds of pesticide application, five sampling sites were selected within each test field, and the incidence of the disease was recorded for 100 wheat spikes per site, resulting in a total of 500 monitored spikes per treatment group. The disease occurrence on the spikes was monitored over a period of one month, and the total number of spikes as well as the number of diseased spikes were recorded. FHB was monitored starting from the end of flowering (BBCH 69) until the medium milk stage (BBCH 75). The disease assessment and field efficacy evaluation for the management of FHB in wheat strictly adhered to the Ministry of Agriculture of the People’s Republic of China (2007) standards (NY/T 1464.15-2007: Guidelines for the Field Efficacy Trials of Pesticides, Part 15: Fungicides for Controlling Wheat Fusarium Head Blight) [45].

Level 0: no disease;

Level 1: less than 1/4 of the spikelets on a spike are diseased;

Level 3: 1/4 to 1/2 of the spikelets on a spike are diseased;

Level 5: 1/2 to 3/4 of the spikelets on a spike are diseased;

Level 7: more than 3/4 of the spikelets on a spike are diseased.

The Disease index (DI) was calculated using the following formula:

$$\mathrm{DI}={\displaystyle \frac{{\displaystyle \sum \mathrm{Disease} \mathrm{Severity} \mathrm{Level}\times \mathrm{Number} \mathrm{of} \mathrm{Diseased} \mathrm{Earsat} \mathrm{Each} \mathrm{Level}}}{\mathrm{Total} \mathrm{Number} \mathrm{of} \mathrm{Ears} \mathrm{Surveyed}}}\times {\displaystyle \frac{100}{7}}$$

The Control effect (CE) was calculated using the following formula:

$$\mathrm{CE}={\displaystyle \frac{\mathrm{Disease} \mathrm{Index} \mathrm{in} \mathrm{the} \mathrm{Control} \mathrm{Area}-\mathrm{Disease} \mathrm{Index} \mathrm{in} \mathrm{the} \mathrm{Treated} \mathrm{Area}}{\mathrm{Disese} \mathrm{Index} \mathrm{in} \mathrm{the} \mathrm{Control} \mathrm{Area}}}\times 100$$

2.9. Impact of SORs on Wheat Agronomic Traits and Yield

The effects of different treatment groups on wheat agronomic traits and yield were observed in the FHB test field. A five-point sampling method was employed in each of the five treatment groups, with 10 plants sampled at each point. The following traits were measured:

Plant height: measured from the base of the plant to the top of the spike, excluding the awn, using a tape measure.

Spike length: measured from the base to the tip of the spike, excluding the awn, using a tape measure.

Aboveground dry weight: the wheat plants were dried to a constant weight, and the aboveground dry biomass was recorded.

Grain count: the plants were threshed, and the number of grains per plant was counted.

Grain weight: the weight of grains from a single plant was measured.

Within each of the five experimental groups, three 1 × 1 m² plots were selected. The total number of spikes per square meter and the number of effective spikes (those with more than five grains) were recorded. After threshing and drying, the thousand-grain weight was measured, with three replicates for each treatment.

GPS is the abbreviation of “Grains Per Spike”; SPS is the abbreviation of “Spikes Per Square meter”, TGW is the abbreviation of thousand-grain weight.

$$\mathrm{HI}={\displaystyle \frac{\mathrm{Individual} \mathrm{GrainWeight} \left(\mathrm{g}\right)}{\mathrm{Above} \mathrm{ground} \mathrm{Dry} \mathrm{Matter} \left(\mathrm{g}\right)}}$$

$$\mathrm{Yield} (\mathrm{kg}/667{\mathrm{m}}^2)={\displaystyle \frac{\mathit{GPS}\times \mathit{SPS}\times \mathit{TGW}}{100}}$$

2.10. Data Analysis

All data were measured three times, replicates were performed, and outliers were excluded. The statistical analysis was performed using SPSS 16.0, where a one-way analysis of variance (ANOVA) was applied to the obtained data. The F-test was conducted within the same program to evaluate between-group differences, with statistical significance defined as p ≤ 0.05.

3. Results

3.1. Effect of Different SOR Concentrations on the Elimination of F. graminearum Spores

To assess the influence of SOR concentration on spore inactivation, experiments were performed under standardized conditions, with a fixed spore concentration (2.50 × 10⁵ CFU/mL) and a treatment duration of 10 min. The study examined six SOR concentrations (0, 1.00, 2.00, 3.00, 4.00, and 5.00 mg/L) to determine their effect on the inactivation rate. As depicted in Figure 2, while other experimental variables remained constant, a dose-dependent increase in the inactivation rate was observed with higher SOR concentrations.

When the SOR concentrations was 1.00–2.00 mg/L, the killing effect was relatively weak, with spore viability remaining between 20% and 40%. When the SOR concentration was increased to 3.00–4.00 mg/L, the killing effect significantly improved, with spore viability dropping to nearly 0%. This suggests that the radicals were highly efficient in reacting with the spores at that concentration range. At a concentration of 5.00 mg/L, the spore viability remained at 0%, similar to the effect observed at 4.00 mg/L. Increasing the concentration further did not significantly improve the killing effect, likely because the radicals were already sufficient to destroy all spores, and additional concentrations had no further impact. The results indicated that 4.00 mg/L was the optimal concentration of SORs for killing F. graminearum spores, achieving nearly 100% spore mortality.

3.2. Effects of SORs on Different Spore Concentrations

Under the conditions of an SOR concentration of 4.00 mg/L and a treatment duration of 10 min, this study investigated the effect of different concentrations of F. graminearum spores (2.50 × 10⁷ CFU/mL, 2.50 × 10⁶ CFU/mL, 2.50 × 10⁵ CFU/mL, 2.50 × 10⁴ CFU/mL, and 2.50 × 10³ CFU/mL) on the bactericidal efficacy. As shown in Figure 3, at high concentrations of conidia (2.50 × 10⁷ and 2.50 × 10⁶ CFU/mL), the sterilization rates of the conidia remained relatively high, and complete eradication was not achieved. This may be due to the high number of conidia, which prevents the SORs from fully covering them, or because the oxidizing agent was rapidly consumed, limiting its effectiveness. As the conidial concentration decreased (2.50 × 10⁵ CFU/mL), the killing effect became more significant, with conidial activity approaching 0%. At that concentration, the SOR concentration and the number of conidia reached an effective reaction ratio, resulting in optimal killing efficiency. In contrast, for lower concentrations of conidia (2.500 × 10⁴ CFU/mL and 2.50 × 10³ CFU/mL), the conidial mortality rate reached 100%, and the conidial activity was 0%. This indicated that at low concentrations, the SORs exerted a more thorough effect, achieving ideal sterilization results.

3.3. Effects of Different Treatment Times on Spore Inactivation

To assess the influence of treatment times on spore inactivation, experiments were performed under standardized conditions, with a fixed spore concentration (2.50 × 10⁵ CFU/mL) and an SOR concentration of 4.00 mg/L. This study investigated the effect of different treatment times (0, 3, 5, 10, 15, 30 min) on the bactericidal efficacy. As shown in Figure 4, an SOR solution (4.0 mg/L) was applied to F. graminearum conidial suspensions for 0, 3, 5, 10, 15, and 30 min. The results, based on the plate count method, showed that within 3 min, the sterilization rates of the conidia decreased from 100% to approximately 40%. This suggests that during the early stage of treatment, SORs began to damage the conidial membrane or internal structures but had not yet achieved complete eradication. After 5 min, the conidial activity dropped to nearly 0%, indicating that SORs had sufficiently acted and disrupted the majority of conidial activity. That stage represented the critical time point for effective killing. From 10 min onwards, conidial activity was fully lost, and the killing effect remained stable. Further extending the treatment time (to 15 or 30 min) did not significantly increase the killing rate, suggesting that the 5–10 min window was sufficient for complete eradication. Based on the experimental results, the optimal treatment duration is 5 min, at which point the conidial activity is almost completely lost, achieving the best killing effect. Extending the treatment to 10 min or more does not enhance the killing rate but may increase energy consumption or oxidizer usage, making it economically inefficient.

3.4. Methylene Blue Staining Experiment

In the control group with water (Figure 5a), the conidia maintained their original shape and were clearly visible. The conidia stained light blue, indicating that the cell membrane remained intact after dye adsorption. A noticeable portion of the conidia had begun to germinate, and budding was relatively common, suggesting that water treatment did not cause oxidative damage to the conidia, and they retained normal metabolic activity and viability. The SOR treatment group (Figure 5b) showed significant changes in conidial morphology, with some conidia appearing shrunken or deformed. The staining was deep blue, indicating that the cell membrane was disrupted, and the dye had penetrated the cell interior. Germination was almost absent, confirming that the conidia had lost their metabolic activity. Based on the experimental results in Section 3.1, Section 3.2 and Section 3.3, an SOR concentration of 4.0 mg/L effectively inactivates F. graminearum spores.

3.5. Observations of SEM

As red arrows depicted in Figure 6a, the control group’s mycelium maintained a smooth surface and intact structure, with no evident signs of damage or collapse. The outer wall of the mycelium maintained its normal tension, forming a continuous and tightly bound fibrous network. The mycelial morphology of F. graminearum remained unaltered in the untreated control group.

In Figure 6b red arrows showd, after treatment with 5.00 mg/L SOR, the mycelial surface exhibited some depressions and collapse, and the structure began to show irregularities. Some parts of the outer wall were likely damaged, indicating that SORs had begun to impact the mycelium. The free radicals at 5.00 mg/L disrupted the integrity of the cell wall, but some degree of continuity remained. After treatment with 20.00 mg/L SOR (Figure 6c), according to the red arrows indicited, the mycelial surface showed extensive rupture and collapse, and the fibrous structure was almost completely destroyed. Fragments of the mycelium were observed, indicating that high-concentration free radicals had caused substantial damage to the mycelial cell wall, suggesting an intense oxidative degradation of the mycelial structure.

3.6. Disease Control Efficacy

As shown in

Table 2. Statistics of field disease index and prevention efficiency index.

Treatment	The Disease Index Under Medication with 14 Days	The Control Effect Under Medication with 14 Days (%)
CK	28.29	-
SORs/SORs	3.43	87.87
Tetramycin/tetramycin	2.57	90.91
Tetramycin/SORs	1.43	94.95
SORs/tetramycin	1.71	93.94

, the disease index in the control group was the highest at 28.29, indicating significant disease severity in the untreated wheat field. The disease index in the SOR-treated group dropped to 3.43, considerably lower than in the control, showing strong disease control. The disease index in the group treated with thiabendazole alone decreased further to 2.57, outperforming the SOR-only treatment. In the combined thiabendazole and SOR treatment, the disease index reached the lowest level of 1.43, representing the most effective treatment among all groups. The disease index in the alternating SOR and thiabendazole group was 1.71, close to that of the combined treatment but slightly higher.

An analysis of the efficacy data revealed that the control group, with no treatment, showed no disease control. The SOR-only treatment exhibited an efficacy of 87.87%, demonstrating significant fungicidal effects. The efficacy of the thiabendazole-only treatment was 90.91%, slightly higher than SORs alone. The combined treatment with thiabendazole and SORs achieved the highest efficacy at 94.95%, while the alternating SOR and thiabendazole treatment had an efficacy of 93.94%, second only to the combined treatment.

3.7. Effect of SORs on Agronomic Traits and Yield of Wheat

3.7.1. Effects of Different Treatments on Wheat Plant Height, Spike Length, Aboveground Dry Matter, and Kernel Number per Plant

The asterisks (*) represent statistical significance, emphasizing differences among treatment groups. ns: p ≥ 0.05, no significant difference; *: p < 0.05, significant; **: p < 0.01, highly significant; ***: p < 0.001, very highly significant; ****: p < 0.0001, extremely significant.

As shown in Figure 7a, the average plant height (46.62 cm) in the control group was the shortest, indicating significant inhibition of wheat growth due to the disease. The plant height in the groups treated with SOR alone (59.11 cm) and thiabendazole alone (55.46 cm) showed a larger increase compared to the control group, with increases of 26.79% and 18.96%, respectively, higher than the combined treatment groups. The alternating treatments of thiabendazole and SORs (50.34 cm), as well as SORs and thiabendazole (50.84 cm), with increases of 8.00% and 9.05%, respectively, compared to the control. This suggests that combined treatments effectively promoted the healthy growth of wheat.

As shown in Figure 7b, the spike length in the control group (4.17 cm) was the shortest, indicating that the disease inhibited the reproductive growth of wheat. Compared to the control, spike length significantly increased in the SOR-only treatment group (5.35 cm) and the tetramycin-only treatment group (5.41 cm), with respective increases of 28.35% and 29.74%. The tetramycin and SOR rotation treatment (4.69 cm) showed no significant increase compared to the control, with an increase of 12.57%. The SOR and tetramycin rotation treatment (4.92 cm) exhibited a significant increase of 17.93%. Overall, all four treatment groups demonstrated a positive effect on spike length, with the single applications of SORs and tetramycin yielding the most significant improvements.

As illustrated in Figure 7c, the control group exhibited the lowest dry matter accumulation (1.70 g), indicating a significant negative impact of the disease on wheat biomass accumulation. Compared to the control, dry matter accumulation increased in the SOR-only treatment group (2.19 g) and the SOR–tetramycin rotation group (2.39 g), but these increases were not statistically significant, with respective increases of 22.78% and 40.53%. In contrast, the tetramycin-only treatment group (2.45 g) and the tetramycin–SOR rotation group (1.79 g) demonstrated significant increases of 44.00% and 5.00%, respectively. These results suggest that while all treatment groups showed improvements in dry matter accumulation, the tetramycin-only treatment was the most effective.

As shown in Figure 7d, the control group had the lowest number of grains per plant (19.63), indicating that the disease had a direct negative impact on grain development. Compared to the control, the tetramycin and SOR rotation treatment (24.22) increased the number of grains per plant by 23.37%, but this increase was not statistically significant. In contrast, the SOR-only treatment (28.91), the tetramycin-only treatment (26.75), and the SOR-tetramycin rotation treatment (25.72) all resulted in significant increases of 47.29%, 36.32%, and 31.01%, respectively. Among these treatments, the tetramycin-only treatment exhibited the most pronounced effect, suggesting that tetramycin had a strong positive influence on grain formation.

3.7.2. Effects of Different Treatments on Wheat Grain Weight per Plant and Harvest Index

As illustrated in Figure 8a, the control group exhibited the lowest grain weight per plant (0.81 g), indicating that the disease significantly reduced grain formation capacity. Among the treatments, the tetramycin-only group (1.23 g) demonstrated the most effective disease control, resulting in a significant increase in grain weight, with the highest improvement of 53.15%. The SOR-only treatment (1.08 g) and the SOR–tetramycin rotation treatment (1.07 g) followed, with respective increases of 33.83% and 33.00%. The tetramycin–SOR rotation treatment (0.95 g) showed the lowest increase (17.38%) compared to the control, and the effect was not statistically significant. These findings suggest that while all treatments improved grain weight, tetramycin alone was the most effective.

As shown in Figure 8b, the harvest index (HI) in the control group was relatively low (0.47), indicating that the disease significantly impaired the allocation of dry matter to grain formation. The SOR-only treatment (0.48) slightly improved dry matter distribution efficiency but remained inferior to the tetramycin-only treatment (0.51). The tetramycin-only treatment exhibited the highest HI, suggesting a strong positive effect on dry matter partitioning. The pesticide and SOR rotation treatment significantly increased HI compared to the control, with the largest improvement of 15.49%. In contrast, the SOR and pesticide rotation treatment (0.46) resulted in a lower HI than the control, suggesting that this rotation strategy might not effectively enhance dry matter allocation efficiency.

3.7.3. Impact of Different Treatments on Wheat Thousand-Grain Weight and Yield

As shown in Figure 9a, the control group exhibited the lowest thousand-grain weight (38.49 g), indicating that the disease significantly affected grain plumpness. Compared to the control, the SOR-only treatment (45.61 g) increased thousand-grain weight by 18.51%, showing a significant improvement in grain development. The tetramycin-only treatment (44.41 g) ranked second, with a 15.39% increase over the control. The tetramycin–SOR rotation treatment (43.18 g) and the SOR–tetramycin rotation treatment (43.05 g) resulted in slightly lower thousand-grain weights than the single SOR and tetramycin treatments but remained significantly higher than the control, suggesting that rotational treatments contributed to improving grain plumpness.

As shown in Figure 9b, the control group exhibited the lowest yield at 306.21 kg/667m², indicating that the disease had a severe impact on final yield. In contrast, the single SOR treatment group achieved the highest yield, reaching 541.25 kg/667m², a significant increase of 76.78% compared to the control group. This result suggests that SOR plays a crucial role in disease control and yield enhancement. The single tetraconazole treatment (466.10 kg/667m²), tetraconazole + SOR rotation treatment (431.38 kg/667m²), and SOR + tetraconazole rotation treatment (457.90 kg/667m²) all significantly increased yield, with improvements of 52.22%, 40.88%, and 49.54%, respectively, compared to the control. These findings indicate that all treatment strategies effectively mitigated the disease-induced yield losses, with the single SOR treatment exhibiting the most pronounced effect.

4. Discussion

This study provided a systematic evaluation of the potential of SOR in the comprehensive control of FHB by analyzing its spore-killing efficacy, disease control capacity, and impact on wheat yield across different treatment regimes.

The experimental results revealed that the optimal fungicidal effect was achieved at an SOR concentration of 4.00 mg/L, with spore activity reduced to nearly 0% and the mortality rate approaching 100%. Increasing the concentration beyond this level did not result in a significant improvement, likely due to the full destruction of spore structures by free radicals at higher concentrations. Finding this optimal concentration helps guide resource utilization and cost-effectiveness. The study also found that the highest fungicidal efficiency and oxidant utilization occurred when the initial spore concentration was 2.50 × 10⁵ CFU/mL. The most effective exposure time was determined to be 5 min, during which spore viability was nearly completely eliminated. While extending the exposure time beyond this had no substantial impact on the inactivation rate, it could lead to higher energy consumption and unnecessary resource waste.

Microbial cells react directly with dissolved ozone in the water and indirectly with potent oxidative free radicals included hydroxyl, hydrogen peroxide, and superoxide radicals generated from ozone decomposition. Due to SORs’ strong oxidative properties, ozone water exhibits rapid bactericidal action. SORs interact with cell membrane components, the cell envelope, cytoplasm, and spore coats, leading to cellular damage, metabolic disruption, and eventual microbial inactivation [46]. The oxidative stress induced by these free radicals frequently triggers oxidative reactions in pathogenic microorganisms, leading to the disruption of cell membrane integrity and the inactivation of key metabolic enzymes. This results in structural damage and physiological dysfunction, ultimately causing the cessation of microbial activity [47]. Furthermore, the findings elucidate the bactericidal effects of ozone-induced radical solutions, highlighting their potential inhibitory mechanisms against harmful microorganisms. The effectiveness of 1.5 mg/L ozone-induced radical solution and 5% imidacloprid in controlling leafy vegetable pests has been well demonstrated, reducing pest severity from level 1 to levels 3 and 4, respectively, thereby ensuring Chinese cabbage protection. The ozone-induced radical solution disrupts key physiological functions in pests, leading to neuromuscular impairment, metabolic disorders, and population decline [48]. Notably, Xu et al. reported that ozonated water treatment achieved a 76.4% control efficacy against Plutella xylostella, with a 59.3% reduction in larval population [49]. These findings indicate that ozone-based radical solutions not only provide effective pest suppression but also present a potentially sustainable alternative to synthetic chemical pesticides. Given the increasing concerns over pesticide residues and environmental safety, further investigation into the long-term ecological impact, degradation kinetics, and application feasibility of ozone-induced radical solutions is warranted to establish them as a viable component of integrated pest management strategies.

The deactivation mechanism of ozone water against E. coli and E. faecalis (representing Gram-negative and Gram-positive bacteria, respectively) was also explored [50]. Ozone water at a concentration of 1 mg/L was applied with exposure times of 30 s, 1, 5, 10, and 20 min. After 30 s, the sterilization rates of TTC dehydrogenase in E. faecalis was inactivated. However, E. coli required a longer exposure of 20 min for nearly complete inactivation. Within 30 s of treatment, both bacteria released intracellular components (DNA and proteins), with TEM images showing more severe damage to the cell wall of E. coli. The study concluded that the mechanisms of ozone water-induced inactivation differed between Gram-negative and Gram-positive bacteria, with E. faecalis relying more on the destruction of intracellular components rather than cell wall damage, while ozone inactivated E. coli by inducing lipid peroxidation and cell wall disruption.

A study by Young demonstrated that a saturated ozone water solution (25 ppm) could degrade mycotoxins into undetectable substances by ultraviolet or mass spectrometry [51]. Previous research employing saturated ozone water (ozone concentration 80 mg/L) detoxified a 15 mg/L DON standard solution within 7 min, achieving an 83% degradation rate [52]. A more recent study developed an ozone water–hydroxyl radical (-OH) treatment system, using 10 mg/L ozone water to detoxify a 15 mg/L DON solution in 7 min. The results indicated that DON was transformed into two degradation products, which were further oxidized by the OH radicals in the ozone water solution [53].

Experimental data indicate that SOR applied alone achieved 87.87% efficacy, demonstrating its oxidative fungicidal ability. However, tetramycin alone achieved a slightly higher efficacy of 90.91%, suggesting a more stable or sustained inhibitory effect against fungal pathogens. Notably, when combined, disease control efficacy was further enhanced. The tetramycin-first-then-SOR treatment achieved the highest efficacy (94.95%), followed by the SOR-first-then-tetramycin treatment (93.94%). These results suggest a potential synergistic effect, where SORs disrupt pathogen cell membranes through strong oxidative radical activity, while tetramycin inhibits fungal growth and reproduction. Additionally, the slightly superior performance of sequential application over single-agent treatments may be due to cumulative effects, optimized application timing, or enhanced pathogen suppression. Further investigation is required to elucidate the underlying molecular mechanisms of this synergistic interaction and to develop optimal application strategies. These findings provide new scientific evidence for integrated FHB control strategies and offer practical guidance for optimizing pesticide application timing and sequencing in wheat fields. Beyond disease control, such strategies may also contribute to reducing pesticide usage, minimizing environmental impact, and improving the sustainability of wheat production.

An assessment of the potential impact of SOR on wheat agronomic traits and yield revealed a positive effect. Compared with the control, both the tetramycin-first-then-SOR treatment and the SOR-first-then-tetramycin treatment led to significant improvements in plant height, spike length, single-grain weight, and thousand-grain weight. These enhancements indicate that the fungicide treatments not only effectively controlled FHB but also created a more favorable growth environment, ultimately improving grain development and weight. In terms of final yield, the tetramycin-first-then-SOR group achieved the highest yield at 457.90 kg/667m², followed by the SOR-first-then-thiophanate-methyl group, which reached 431.38 kg/667m². These findings underscore the importance of optimizing fungicide application strategies to maximize both disease control efficiency and yield potential. Further research is needed to elucidate the underlying mechanisms driving these effects, particularly in relation to fungicide interaction, application timing, and cumulative impact. These insights provide a scientific foundation for integrated disease management in wheat, contributing to enhanced field management practices and sustainable agricultural productivity.

As ozone is a biocidal agent with a broad antimicrobial spectrum, capable of killing fungi, bacteria, and viruses without leaving any residue, it is widely used in fruit and vegetable disease control. Research has shown that ozone water treatment can control diseases in grapevines, while maintaining the quality of the resulting wine. The study demonstrated that ozone-treated grapes produced wines with distinct chemical characteristics and qualities, depending on the treatment intensity [54]. Using ozone (3 mg/L) and heat treatment (70 °C hot water) alone for controlling papaya stem-end rot was effective, with a control efficiency of approximately 50%, delaying symptom onset by 3 and 4 days, respectively. When these two treatments were combined, a synergistic effect was observed, increasing the control efficiency to over 90% and delaying symptom development by 7 days. This integrated approach proved effective in controlling stem-end rot, offering a safe and sustainable alternative to the use of chemical agents in post-harvest treatment of papaya [55].

This study validated the potential of SORs in FHB control. Future research should explore its mechanism of action and verify the stability and scalability of the results through multi-site field trials, as well as optimize its application strategies for broader use in agricultural production.

5. Conclusions

This study provided comprehensive insights into the fungicidal efficacy of strong oxidative radicals (SORs) and their potential application in sustainable wheat disease management. In vitro experiments confirmed that SORs effectively eliminated F. graminearum, with a spore mortality rate exceeding 96.8% at 4.0 mg/L, and that a 10 min treatment duration was optimal for fungal inhibition.

Beyond pathogen suppression, field trials highlighted SORs’ dual role in FHB control and wheat growth promotion. The application of SORs alone resulted in an 87.9% control efficacy, significantly reducing disease severity while enhancing key agronomic traits such as plant height, spike length, and grain yield. This study underscores the potential of SORs as a green, non-chemical approach for FHB management, offering an innovative strategy for reducing fungicide dependency and promoting sustainable wheat production. Further research is warranted to optimize SOR application protocols and assess long-term effects in diverse environmental conditions.