Compound preparation for preventing and treating tobacco mosaic disease and application thereof

Abstract

The invention relates to a compound preparation for preventing and treating tobacco mosaic disease and application thereof. Aiming at the problems that chemical control easily causes pesticide residues, biological control takes effect slowly and is unstable, the threshold of a gene editing technology is high and the like in the existing tobacco mosaic disease control technology, the invention provides a medicament compounded by azelaic acid and beta-aminobutyric acid according to a molar ratio of 1: (2-10). According to the agent, antioxidant enzyme systems such as SOD (superoxide dismutase), CAT (catalase) and POD (peroxidase) in tobacco plants are synergistically activated, and meanwhile, SAR (synthetic aperture radar) and SA / ABA double-signal channels are triggered, so that the self-defense capability of the plants is Experiments show that the SOD activity is improved by more than 40% after transplanting for 30 days after treatment by the medicament, and the CAT activity is improved by more than 125% after 60 days, so that the morbidity of the tobacco mosaic disease is obviously reduced, the disease index in 30 days is lower than 1.0, and the morbidity in 60 days is reduced by more than 10% compared with that of a control group. The method is easy and convenient to operate, environmentally friendly and free of residual risk, achieves the dual effects of early-stage rapid inhibition and long-term continuous control, and is efficient and safe in tobacco virus disease prevention and treatment.

Description

Technical Field

[0001] This invention relates to the field of plant disease control technology, and in particular to a compound preparation for controlling tobacco mosaic disease and its application. Background Technology

[0002] Tobacco mosaic virus (TMV) is a systemic infectious disease caused by Tobacco Common Mosaic Virus (TMV) and Cucumber Mosaic Virus (CMV), and is one of the major diseases affecting tobacco production. This disease can occur throughout the entire growth period of tobacco. In the early stages, the lateral veins and branch veins of young leaves become translucent. As the virus further proliferates, leaf mesophyll cells become malformed, forming a yellow-green mosaic pattern. As the disease progresses, large areas of brown necrotic spots appear on the leaves. In severe cases, leaf deformities, wrinkling, and twisting occur, and even blistering and inward curling of leaf margins may form. Early-infected tobacco plants experience stunted growth, shortened internodes, and significant dwarfing. Flowering is also abnormal and prone to shedding, resulting in a significant decrease in seed yield and quality, severely impacting the yield, quality, and economic benefits of tobacco.

[0003] Statistics show that among the 16 major tobacco-producing provinces in China, 14 provinces have suffered from tobacco mosaic virus infection, resulting in yield losses of 40% to 60%, causing huge economic losses to the tobacco industry. Existing control technologies for this disease mainly include the following three categories, but all have significant limitations: While chemical control methods offer advantages such as rapid effectiveness and ease of operation, long-term use can lead to increased pesticide residues in tobacco leaves. This not only poses a safety hazard of poisoning to humans and animals but also promotes drug resistance in pathogens and can even cause recurring outbreaks, threatening sustainable agricultural development. Furthermore, the widespread use of chemical agents may disrupt the ecological balance in tobacco fields and negatively impact the survival environment of beneficial microorganisms.

[0004] Biological control methods, such as the use of plant immune activators like amino oligosaccharides and chitosan, enhance disease resistance by inducing plants to produce disease-resistant factors. These methods offer advantages such as being environmentally friendly, pollution-free, and not inducing drug resistance. However, a significant drawback of these methods is their slow onset of action and the susceptibility of their effectiveness to environmental factors (such as temperature, humidity, and soil conditions). This makes it difficult to achieve timely and effective control during periods of high disease incidence, leading to inconsistent field application results.

[0005] Gene editing technology works by knocking out or modifying key genes in tobacco (such as...). NtSPS1 While cultivating disease-resistant varieties has the advantage of being highly targeted, it also faces challenges such as high technical barriers, long research and development cycles, and expensive application costs. Furthermore, it may encounter practical obstacles such as low market acceptance of genetically modified products and strict regulatory approvals, making it difficult to achieve large-scale promotion in the short term.

[0006] In summary, the current field of tobacco mosaic disease control still lacks a comprehensive control solution that is highly efficient, safe, stable, economical, and easy to operate, making it difficult to simultaneously meet the dual requirements of rapid early suppression and long-term continuous control. Therefore, developing a novel control technology that can effectively activate the plant's own defense system, reduce dependence on chemical agents, and is not significantly affected by environmental factors has become a key breakthrough for improving tobacco yield and quality.

[0007] The information disclosed in this background section is intended only to enhance the understanding of the background technology of this disclosure and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0008] This invention addresses the technical problem of limited efficacy of single-agent pesticides in the control of tobacco mosaic virus, which cannot simultaneously achieve rapid early suppression and long-term sustained control. It employs a foliar spray of a mixture of azelaic acid (AzA) and β-aminobutyric acid (BABA) at specific concentration ratios (e.g., 0.5mM:2.5mM and 1mM:5mM). This treatment synergistically activates antioxidant enzyme systems such as SOD, CAT, and POD within the plant, forming a multi-layered defense network. This significantly reduces the incidence and disease index of tobacco diseases, achieving the dual technical effects of rapid early suppression (disease index below 1 after 30 days) and effective long-term disease control (incidence rate reduced by 10 percentage points compared to the control after 60 days).

[0009] According to one aspect of this disclosure, a compound formulation is provided, comprising an active ingredient consisting of azelaic acid (AzA) and β-aminobutyric acid (BABA) in a molar ratio of 1:(2~10), wherein the concentration of the active ingredient when the compound formulation is applied is 1~4 mM.

[0010] In some embodiments of this disclosure, the molar ratio of azelaic acid to β-aminobutyric acid is 1:(4~6), and the concentration of the active ingredient when the compound preparation is applied is 1.5~3 mM.

[0011] In some embodiments of this disclosure, the molar ratio of azelaic acid to β-aminobutyric acid is 1:5.

[0012] According to another aspect of this disclosure, the compound preparation is used in at least one of the following (1) to (4): (1) To prevent and control tobacco mosaic virus or to prepare products for preventing and controlling tobacco mosaic virus; (2) Inhibit tobacco common mosaic virus and / or cucumber mosaic virus, or prepare products that inhibit tobacco common mosaic virus and / or cucumber mosaic virus; (3) To improve the activity of antioxidant enzymes in plants or to prepare products that improve the activity of antioxidant enzymes in plants; wherein the antioxidant enzyme is at least one of SOD, CAT, and POD; (4) Improve plant resistance to TMV and / or CMV, or prepare products that improve plant resistance to TMV and / or CMV.

[0013] In some embodiments of this disclosure, the plant includes tobacco.

[0014] According to another aspect of this disclosure, a method for preventing and controlling tobacco mosaic virus is provided, comprising: The compound reagent was sprayed onto the tobacco seedlings 1-2 days before transplanting and 28-32 days after transplanting.

[0015] This invention addresses the aforementioned technical deficiencies in existing tobacco mosaic disease control technologies by providing a novel control scheme based on azelaic acid (AzA) and β-aminobutyric acid (BABA). By synergistically activating the plant's antioxidant enzyme system, it achieves highly efficient control of viral tobacco diseases, specifically yielding the following technical effects: 1. Overcoming the limitations of single-agent action and achieving synergistic effects: This invention is the first to combine azelaic acid and β-aminobutyric acid in a specific ratio, overcoming the limitations of single-component treatments. Experimental data show that the combined agent, by simultaneously activating the system, acquires both the resistance (SAR) and SA / ABA dual signaling pathways, significantly improving the overall resistance of tobacco to tobacco mosaic virus. The incidence rate 60 days after transplanting was reduced by more than 10 percentage points compared to the control group, and the disease index was reduced by more than 50%, effectively solving the technical problem that traditional single-agent agents cannot simultaneously achieve early rapid inhibition and long-term continuous control.

[0016] 2. Highly efficient induction of plant immune mechanisms and enhanced disease resistance stability: Azelaic acid promotes G3P synthesis and accumulation by upregulating the expression of GLY1 and GK genes, activating the SAR resistance pathway; β-aminobutyric acid (GABA) induces the expression of defense enzymes through the SA pathway, while simultaneously promoting ABA accumulation, enhancing resilience to adverse conditions. The synergistic effect of these two substances forms a multi-layered defense barrier, enabling plants to establish an effective defense system before pathogen infection. This not only significantly shortens the time to effectiveness of biological control methods but also greatly improves the stability of disease resistance, overcoming the shortcomings of traditional biological control methods that are heavily influenced by environmental factors.

[0017] 3. Strengthening the antioxidant defense system and reducing oxidative damage: This invention, through the treatment of tobacco plants with compound agents, significantly enhances the activity of key antioxidant enzymes such as POD, SOD, and CAT. Experimental results show that 30 days after transplanting, the activity of superoxide dismutase (SOD) increased by more than 40% compared to the control group; 60 days after transplanting, the activity of catalase (CAT) increased by more than 125% compared to the control group. This multi-enzyme synergistic activation mechanism effectively reduces oxidative damage caused by pathogen infection, maintains cellular homeostasis, provides continuous redox balance protection for plants, and fundamentally enhances the plant's resistance to viral diseases.

[0018] 4. Environmentally friendly and without residue risks, meeting the needs of green agriculture: Compared with chemical control, the compound agent used in this invention is a plant-derived active ingredient. Its mechanism of action is to activate the plant's own defense system rather than directly killing pathogens. Therefore, it will not produce pesticide residues, avoid the safety hazards of human and animal poisoning, and also eliminate the problem of pathogen resistance. It meets the requirements of green agriculture and sustainable development, and solves the environmental and safety risks brought about by chemical control methods.

[0019] 5. Simple operation and broad applicability, easy to scale up: This invention can induce resistance through exogenous spraying, without the need for complex gene manipulation or special equipment, making it suitable for large-scale field application. The dual signaling pathway synergistic mechanism has broad-spectrum control potential against a variety of diseases (including bacterial wilt, TMV, etc.), providing an efficient and environmentally friendly comprehensive solution for tobacco disease control, overcoming the shortcomings of high threshold and high cost of gene editing technology.

[0020] In summary, this invention, through the scientific combination of azelaic acid and β-aminobutyric acid, not only effectively activates multiple defense mechanisms in plants, achieving efficient multi-target control of tobacco mosaic virus, but also resolves the contradictions between "speed of effectiveness" and "long-term effect," and between "safety and environmental protection" and "control efficiency" in existing technologies. It provides an innovative technical solution for the control of viral diseases in tobacco, with significant economic benefits and ecological value. Attached Figure Description

[0021] Figure 1 Bar chart comparing SOD activity in different treatment groups at 30 and 60 days post-transplantation. The T3 group showed the highest activity at 30 days, demonstrating its advantage in early stress response.

[0022] Figure 2 The chart shows the trend of CAT activity over time in different treatment groups. The T4 group showed the highest activity at 60 days, demonstrating its tolerance to long-term oxidative stress.

[0023] Figure 3 The graph shows the results of peroxidase (POD) activity assays in different treatment groups at 30 and 60 days after transplanting.

[0024] Figure 4 The following is a statistical chart showing the incidence rates of each treatment group. The CK group had the highest incidence rate, while the T4 group had the lowest incidence rate at 60 days, indicating that the compound medication had a significant disease-suppressing effect.

[0025] Figure 5 The graph shows the changes in the disease index. The disease index was lowest in group T3 at 30 days (<1), while it was lowest in group T4 at 60 days (≈7.2), demonstrating the optimal performance of different concentrations of the compound at different time points.

[0026] Note: In all charts, light gray bars represent data 30 days after transplanting, and dark gray bars represent data 60 days after transplanting. The legend is located in the upper right corner. The horizontal axis represents the treatment group (CK, T1, T2, T3, T4), and the vertical axis represents the corresponding indicator value. Detailed Implementation

[0027] To facilitate those skilled in the art to understand and implement the technical solutions proposed in this application, the preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings and specific experimental data.

[0028] In this embodiment, unless otherwise specified, all instruments and equipment are conventional laboratory or field test equipment; all reagent preparation, sample processing and detection methods adopt the standard operating procedures recognized in the art.

[0029] Examples and field control trials I. Reagent Preparation AzA (azelaic acid): Analytical grade azelaic acid produced by Comio was used. An appropriate amount was weighed and dissolved in distilled water under stirring in a 60°C water bath to prepare stock solutions with concentrations of 0.5 mM and 1.0 mM. These solutions were stored at 4°C protected from light for later use.

[0030] BABA (β-aminobutyric acid): Purchased from Sigma-Aldrich, prepared as 1.0 mM and 5.0 mM aqueous solutions, and stored at 4°C. Before use, mix equal volumes according to the experimental design to obtain the desired compound.

[0031] II. Field Experiment 1. Experimental Design and Field Management This experiment was conducted in the tobacco experimental field at the Xuchang campus of Henan Agricultural University. The field had been planted with tobacco for more than three consecutive years, and the disease occurred naturally year-round, providing a favorable background for disease occurrence. The soil type was yellow loam, with flat terrain and moderate fertility. The fertilization scheme was: nitrogen application rate of 37.8 kg / hm². 2The ratio of N:P₂O₅:K₂O is 1:2:5. Tobacco seedlings were transplanted on May 1st, with a row spacing of 110 cm × 50 cm. Field management was carried out in accordance with local high-quality tobacco cultivation technical regulations.

[0032] The tested variety was NC55. Each treatment had 200 tobacco seedlings, and the experiment was repeated three times. Protective rows were set up around the experimental area to reduce edge effects.

[0033] The processing group is configured as follows: CK: Sprayed with an equal volume of distilled water (control group); T1: Spray with 1 mM AZA solution; T2: Spray with 5 mM BABA solution; T3: Spray with a compound solution of equal volumes of 0.5 mM AzA and 2.5 mM BABA; T4: Spray with a compound solution of equal volumes of 1mM AzA and 5mM BABA.

[0034] Spraying time and method: The first spray should be carried out 2 days before transplanting, and spray evenly until the leaves are dripping with water. The second spraying was carried out 30 days after transplanting.

[0035] Leaf samples were collected 30 and 60 days after transplanting to determine antioxidant enzyme activity, and the incidence and disease index were investigated simultaneously. Fresh samples were immediately stored at -80℃ for testing.

[0036] 2. Assay for antioxidant enzyme activity (1) Assay of superoxide dismutase (SOD) activity Methods: The WST-8 micro-detection kit (catalog number BC5165) from Beijing Solarbio Science & Technology Co., Ltd. was used. The operation steps are as follows: Preheat the microplate reader for ≥30 min and set the wavelength to 450 nm; prepare the reaction system according to the instructions, add crude enzyme solution and working solution to a 96-well plate, gently shake to mix, and let stand at room temperature for 30 min; read the absorbance value A at 450 nm. Calculation formula: Inhibition percentage = (Control A - Assay A) / Control A × 100% Superoxide dismutase (SOD) activity (U / g fresh weight) = 20 × inhibition percentage / (1 - inhibition percentage) / W.

[0037] The results show (e.g.) Figure 1As shown in the figure, the SOD activity of group T3 (0.5 mM AzA + 2.5 mM BABA) reached a peak of 810 U / g FW 30 days after transplanting, significantly higher than other treatment groups and the control group. Notably, the SOD activity of group T2 (5 mM BABA single agent) dropped sharply to 270 U / g FW at 60 days, only 42.2% of that at 30 days, indicating that BABA treatment alone cannot maintain long-term antioxidant capacity. Group T3, however, showed stable high activity at both time points, indicating that the low-concentration compound formulation can effectively activate and maintain the SOD system in plants, which is positively correlated with the later disease suppression effect (see...). Figure 4 , Figure 5 This demonstrates that the stable increase in SOD activity is one of the key mechanisms by which compound drugs exert their anti-disease effects.

[0038] (2) Assay of catalase (CAT) activity Assay method: Ammonium molybdate colorimetric method (micro-method). The reagent kit is also a product of Solarbio. The operation steps are as follows: (1) Preheat the microplate reader for more than 30 minutes and adjust the wavelength to 405 nm; (2) Prepare the reaction system according to the instructions, mix well, and add 200 µL to a 96-well plate; (3) Measure the absorbance at 405 nm. Calculate using the following formula: ΔA = A 对照 -A 测定 ; Catalase (CAT) activity (µmol / min / g fresh weight) = 8.9 × (ΔA - 0.0013) / W.

[0039] Figure 2 The results clearly show that the CAT activity in group T4 (1 mM AzA + 5 mM BABA) reached 450 U / g FW 60 days post-transplantation, 2.25 times that of the control group (200 U / g FW). Particularly noteworthy is that group T1 (1 mM AzA single agent) showed the highest CAT activity (290 U / g FW) at day 30, but plummeted to 50 U / g FW at day 60, indicating that while AzA treatment alone can rapidly induce CAT activity, it lacks sustainability. In contrast, group T4 exhibited a significant "delayed effect," with its CAT activity peaking at day 60, which is consistent with... Figure 4 The results showed that the incidence rate in the T4 group (approximately 48%) was significantly lower than that in the T1 group (approximately 50%) at 60 days, which is consistent with the results indicating that the continuous improvement of CAT activity plays a key role in long-term disease control.

[0040] (3) Peroxidase (POD) activity assay Method: The o-methoxyphenol method was used.

[0041] ① Take 0.25 g of leaf tissue, add 3 mL of 50 mmol / L pH 7.0 phosphate buffer, homogenize in an ice bath, centrifuge at 12000 rpm for 20 min at 4℃, and store the supernatant at -20℃. ② Dilute the crude enzyme solution 10 times, take 1 mL of the diluted solution + 1 mL of buffer solution + 1 mL of 1% guaiacol, and incubate in a water bath at 30℃ for 5 min; ③ Add 1 mL of 3% H2O2 to start the reaction, and immediately measure the change in OD value at 470 nm; ④ Calculation formula: POD activity (△OD•g) -1 •min -1 ) = (ΔOD 470 ×VT) / (FW×t×V1) In the formula, t is the reaction time (min); VT is the total volume of the sample solution (ml); V1 is the amount of sample used during the determination (ml); and FW is the fresh weight of the sample (g).

[0042] Figure 3 The results showed that group T1 (1 mM AzA single agent) induced POD activity to peak at 9800 U / g FW 30 days after transplanting, but significantly declined at 60 days. In contrast, group T4 (high-concentration compound), although showing no significant increase in activity at 30 days, reached 7700 U / g FW at 60 days and maintained a high level. Interestingly, group T2 (5 mM BABA single agent) showed significantly lower POD activity than the CK group (approximately 35%) at both time points, which explains why group T2's disease control effect was not as good as other treatment groups (see...). Figure 4 The POD activity in both the T3 and T4 combination groups was higher than that in the CK group at 60 days, indicating that the combination of AzA and BABA can synergistically promote the long-term activation of the POD system, which is consistent with... Figure 5 The results of lower disease index at 60 days were highly consistent between the T3 and T4 groups.

[0043] 3. Investigation of disease occurrence (1) Survey methods and evaluation criteria Survey time: Surveys were conducted 30 days and 60 days after transplanting, with the 60-day data used as the final basis for evaluating the control efficacy.

[0044] Investigation methods: The occurrence of tobacco mosaic disease was observed and recorded at fixed times daily, and classified according to GB / T 23222-2008 "Classification and Investigation Methods for Tobacco Diseases and Pests". Grade 0: The entire plant is disease-free; Grade 1: The veins of the heart leaves are clear or there is slight mosaic pattern; the diseased plants are not obviously stunted. Grade 3: 1 / 3 of the leaves are mosaic but not deformed, or the diseased plant is stunted to more than 3 / 4 of its normal height; Level 5: 1 / 3-1 / 2 of the leaves are mosaic, or a few leaves are deformed, or the main vein turns black, or the diseased plant is stunted to 2 / 3-3 / 4 of the normal plant height; Level 7: 1 / 2-2 / 3 of the leaves are mosaic, deformed, or have necrosis of the main and lateral veins, or the diseased plant is stunted to 1 / 2-2 / 3 of the normal plant height; Level 9: The entire plant has mosaic patterns on its leaves, is severely deformed or necrotic, or the diseased plant is stunted to more than half the normal height.

[0045] Calculation formula: Disease index = ∑ (number of diseased plants × representative value of the disease level) / (total number of plants surveyed × highest representative value). Relative efficacy = (Control disease index - Treatment disease index) / Control disease index × 100%.

[0046] (2) Interpretation of the results of incidence and disease index from Figure 4 Observations showed that all treatment groups significantly reduced the incidence of tobacco mosaic virus, especially in the early stage (30 days). The T1 group (1 mM AzA) showed an incidence rate of only 2% at 30 days, demonstrating excellent early control, but this rose to 50% at 60 days, indicating that single-drug treatment could not maintain long-term protection. The T3 and T4 groups showed significantly lower incidence rates at both time points than the T1 and T2 groups, with the T4 group showing a 10 percentage point reduction in incidence rate (48%) at 60 days compared to the CK group (58%). Particularly noteworthy is that the rate of increase in incidence rate (30-60 days) in the T3 and T4 groups was significantly smaller than in other treatment groups, indicating that the combined treatment effectively slowed the disease progression. This is consistent with... Figure 1-3 The stable increase in antioxidant enzyme activity in the compound formulation showed a corresponding relationship, further confirming that the compound formulation achieves sustained disease resistance by enhancing the plant's own defense system.

[0047] Figure 5 The study showed dynamic changes in the disease severity index (DMI). The T3 group (0.5 mM AzA + 2.5 mM BABA) had a DMI of only 0.8 at 30 days, close to a disease-free state (grade 0), significantly better than other treatment groups. While the T4 group performed slightly worse than the T3 group at 30 days, its DMI at 60 days was 7.2, slightly lower than the CK group's 7.5, indicating better long-term control. The T1 group (AzA single dose) had a DMI of only 2.3 at 60 days, but combined with… Figure 4It is evident that the actual incidence rate was as high as 50%, indicating that while the T1 group could alleviate the severity of disease on individual plants, it could not effectively control the spread of the disease. The T3 group had a significantly lower disease index (3.0) at 60 days compared to other groups, demonstrating that the low-concentration compound formulation had a unique advantage in controlling the severity of the disease, which is related to its significant increase in SOD activity (…). Figure 1 It is closely related to the disease index of the T2 group (BABA single dose) at 60 days, which is close to that of the CK group, further confirming that the effect of BABA alone is limited, while the effect is significantly improved when combined with AzA (T3, T4).

[0048] 4. Comprehensive correlation analysis and mechanism explanation Based on an in-depth analysis of the above experiments and structural diagrams, it can be concluded that: (1) Time-dynamic effect: The compound drugs (T3, T4) showed obvious "early-late" differentiated effects. The T3 group (low concentration) showed the best SOD activity at 30 days. Figure 1 ) and the lowest disease index ( Figure 5 This is suitable for scenarios requiring rapid disease control; while the T4 group (high concentration) reached peak CAT activity at 60 days. Figure 2 This is more effective for controlling long-term diseases.

[0049] (2) Dose-response relationship: From Figure 1-3 It is evident that AzA and BABA exhibit a significant dose-dependent effect. In the T1 group (1 mM AzA), strong induction of CAT and POD was observed in the early stage (30 days), but this effect could not be maintained. However, after the combination (T3, T4), even with the AzA concentration halved (T3), stable enzyme activity was still maintained, demonstrating that BABA enhanced the duration of action of AzA.

[0050] (3) Synergistic activation of key enzyme systems: The three classes of antioxidant enzymes exhibit complementary activation modes. SOD ( Figure 1 It is mainly activated in the early stage (30 days) and is responsible for scavenging superoxide anions; CAT ( Figure 2 ) plays a dominant role in the later stage (60 days) in removing H2O2; POD ( Figure 3 These enzymes play a supporting role throughout the process. The compound preparation can coordinate the activities of these three enzymes to form a complete antioxidant defense network.

[0051] (4) Disease control mechanism: Figure 4 and Figure 5 With enzyme activity diagram ( Figure 1-3 The high correlation indicates that: Early stage (30 days): SOD activity ( Figure 1 ) and disease index ( Figure 5 ) showed a significant negative correlation (R 2=0.87); Later stage (60 days): CAT activity ( Figure 2 ) and incidence rate ( Figure 4 ) showed a significant negative correlation (R 2 =0.92); This proves that SOD is mainly responsible for early disease suppression, while CAT plays a key role in long-term disease resistance.

[0052] (5) Synergistic effect of compound formulation: Antioxidant enzyme activity in groups T3 and T4 ( Figure 1-3 ) and disease control effect ( Figure 4-5 The results showed that the combined effects of AzA and BABA were significantly better than those of single-agent treatments (T1 and T2), demonstrating a significant synergistic effect between AzA and BABA. Particularly noteworthy is that group T2 (BABA single agent) showed significantly higher SOD activity ( Figure 1 ) and POD activity ( Figure 3 While BABA showed poor performance in terms of absorption, its effectiveness was significantly enhanced when combined with AzA (T3, T4), suggesting that BABA may exert a synergistic effect by enhancing the absorption or signal transduction of AzA.

[0053] 5. Incidence survey and results analysis like Figure 4 and Figure 5 As shown, treatment with different concentration gradients of AzA and BABA compound drugs resulted in significantly lower morbidity and disease index compared to the control group (CK). Specifically: (1) Early effects (30 days after transplanting): The T3 group (0.5 mM AzA + 2.5 mM BABA) showed the most outstanding performance, with a disease index of only 0.8 (close to a disease-free state) and an incidence rate of about 5%. At the same time, the SOD activity of this treatment group reached a peak of 810 U / g FW. Figure 1 This demonstrates that the early enhancement of SOD activity is directly related to the rapid suppression of the disease. The T1 group (1 mMAzA single agent) had the lowest incidence rate (2%), but a higher disease index (approximately 0.2), indicating that although the single agent AzA can inhibit the spread of the disease, it is insufficient to control the severity of the disease on individual plants.

[0054] (2) Long-term effects (60 days after transplanting): Group T4 (1 mM AzA + 5 mM BABA) showed higher CAT activity (450 U / gFW). Figure 2 ) and POD activity (7700 U / g FW, Figure 3 The T3 group showed the best performance in all aspects, with lower incidence (48%) and disease index (7.2) than the CK group (58% and 7.5). Notably, the T3 group had a significantly lower disease index (3.0) at 60 days than other treatment groups, indicating that the low-concentration compound regimen has a unique advantage in controlling the severity of the disease.

[0055] In summary, the compound agent of the present invention forms a multi-layered defense mechanism by synergistically activating the antioxidant enzyme system in plants: ① Early prevention (30 days): SOD is the main component to quickly eliminate superoxide free radicals. The T3 group has the highest SOD activity during this stage, which corresponds to the lowest disease index. ② Continuous defense (60 days): CAT is the main component, effectively decomposing H2O2. The CAT activity in the T4 group reaches its peak, consistent with the lower long-term incidence rate. ③Auxiliary defense: POD plays an auxiliary role throughout the process, working with CAT to maintain redox balance.

[0056] Experimental data show that the combined treatment of AzA and BABA (T3, T4) is more effective than single-agent treatments (T1, T2) in coordinating the activities of SOD, CAT, and POD enzymes, forming a complete antioxidant defense network, thereby achieving efficient control of tobacco mosaic virus. In particular, the specific advantages shown by the T3 group in the early stages and the T4 group in long-term control provide a scientific basis for selecting the optimal treatment scheme according to different disease stages.

[0057] Although some preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0058] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this application and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A combination formulation comprising, in admixture, The active ingredient is composed of azelaic acid and β-amino butyric acid in a molar ratio of 1:(2-10), and the active ingredient concentration of the complex preparation when applied is 1-4 mM.

2. The complex formulation according to claim 1, characterized in that, The molar ratio of azelaic acid to β-amino butyric acid is 1:(4-6), and the active ingredient concentration of the complex preparation when applied is 1.5-3 mM.

3. The complex formulation according to claim 1, characterized in that, The molar ratio of azelaic acid to β-amino butyric acid is 1:

5.

4. Use of the complex preparation of claim 1, 2 or 3 in at least one of the following or for preparing a product with at least one of the following functions: (1) preventing and treating tobacco mosaic virus; (2) inhibiting tobacco mosaic virus or / and cucumber mosaic virus; (3) increasing the activity of antioxidant enzymes in plants; the antioxidant enzymes are at least one of SOD, CAT and POD; (4) increasing the resistance of plants to TMV or / and CMV.

5. Use according to claim 4, characterized in that, The plants include tobacco.

6. A method of controlling tobacco mosaic virus, characterized by, The complex preparation of claim 1, 2 or 3 is sprayed on tobacco plants 1-2 days before transplanting tobacco seedlings and 28-32 days after transplanting tobacco seedlings. The complex preparation of claim 1, 2 or 3 is sprayed on tobacco plants 1-2 days before transplanting tobacco seedlings and 28-32 days after transplanting tobacco seedlings.

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