Trans-anethole nanoemulsion, preparation method and application thereof

Abstract

The application discloses trans-anethole nanoemulsion as well as a preparation method and application thereof, and belongs to the technical field of plant protection. The preparation of the trans-anethole nanoemulsion (TAN-NE) comprises the following steps: mixing polyethylene glycol octylphenyl ether, castor oil and anhydrous ethanol according to a mass ratio of 1:3:12 to prepare mixed surfactants; according to the mass percentage, 12.40% of trans-anethole is added into 28.93% of the mixed surfactants, and deionized water is supplemented to 100% to obtain the TAN-NE. The TAN-NE has a significantly better proliferation inhibition effect on Ralstonia solanacearum than free trans-anethole TAN, and has strong bacteriostatic activity. Indoor pot experiments show that on the 15th day after inoculation, the prevention effect of the TAN-NE is better than the treatment effect, and both are better than TAN and a commercial pesticide, thioconazole, and the TAN-NE has good biological safety to a soil environment.

Description

Technical Field

[0001] This invention belongs to the field of plant protection technology, and relates to a trans-anessenolene nanoemulsion, its preparation method, and its application. Background Technology

[0002] Ralstonia solanacearum ( Ralstonia solanacearum Bacterial wilt caused by CQPS-1 is a highly damaging soil-borne bacterial disease. It is characterized by its strong pathogenicity, rapid spread, and wide host range. Ralstonia solanacearum survives in the soil environment for extended periods, primarily infecting crop roots. It then migrates upwards along the xylem vessels, carrying water and nutrients with it. After multiplying extensively within the plant, it secretes extracellular polysaccharides (EPS) that block the vascular bundles, thus affecting plant growth and development.

[0003] trans-anesinene ( trans Trans-anethole (TAN), a typical plant-derived active substance, exhibits multiple antibacterial physiological activities against Ralstonia solanacearum. It can significantly inhibit the proliferation and growth of the pathogen, disrupt its biofilm structure, and regulate its motility. Simultaneously, it interferes with key physiological processes such as extracellular polysaccharide synthesis and intracellular ATP energy metabolism, demonstrating excellent potential for developing green plant-derived fungicides. However, trans-anethole suffers from poor water solubility, easy volatilization and loss, easy decomposition and degradation under light conditions, and weak systemic translocation properties. Direct application results in unstable field efficacy and a short duration of action, failing to meet the long-term control needs of field diseases. Furthermore, under the extensive application of conventional chemical pesticides, approximately 55%–60% of the pesticides cannot accurately target the pathogens, easily being lost and dispersed through pesticide drift, volatilization, raindrop bouncing, and surface runoff. This not only significantly reduces pesticide utilization but also easily leads to environmental risks such as soil residues, water pollution, and microecological imbalance. Summary of the Invention

[0004] To address the aforementioned technical problems and deficiencies, this invention provides a trans-anisene nanoemulsion, its preparation method, and its application.

[0005] In a first aspect, the present invention provides a trans-anessenol nanoemulsion, wherein, by mass percentage, the trans-anessenol nanoemulsion comprises: 12.40%~18.35% trans-anessenol, 18.35%~28.93% mixed surfactant, and the balance being deionized water;

[0006] The mixed surfactant is composed of polyethylene glycol octylphenyl ether, castor oil and anhydrous ethanol; The mass ratio of polyethylene glycol octylphenyl ether, castor oil, and anhydrous ethanol is 1:3:12.

[0007] Furthermore, in the trans-anessenolene nanoemulsion provided by the present invention, the trans-anessenolene nanoemulsion comprises, by mass percentage: 12.40% trans-anessenolene, 28.93% mixed surfactant, and the balance being water.

[0008] Secondly, the present invention provides a method for preparing the above-mentioned trans-anetifene nanoemulsion, comprising: first, fully mixing polyethylene glycol octylphenyl ether with castor oil, then adding anhydrous ethanol and mixing evenly to obtain a mixed surfactant; subsequently, adding trans-anetifene to the mixed surfactant and stirring evenly, and adding deionized water dropwise under continuous stirring to obtain the trans-anetifene nanoemulsion.

[0009] Thirdly, this invention provides the application of trans-anessenolene nanoemulsion in the prevention and control of bacterial wilt in plants.

[0010] Furthermore, in the application provided by the present invention, the pathogen of bacterial wilt is Ralstonia solanacearum.

[0011] Furthermore, in the application provided by this invention, the plant is tobacco.

[0012] Furthermore, in the application provided by the present invention, the trans-anisene nanoemulsion reduces the pathogenicity of Ralstonia solanacearum by regulating the formation of biofilm, cell motility, extracellular polysaccharide production capacity, and expression of infection-related genes of Ralstonia solanacearum.

[0013] Furthermore, in the application provided by this invention, the infection characteristic-related genes include: lecM , cheW , fliA , xpsR and epsE.

[0014] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: (1) This invention successfully constructed a structurally stable and high-performance water-in-oil trans-anetifene nanoemulsion (TAN-NE) using low-energy emulsification technology. The average particle size was 20.68±0.50 nm, the polydispersity index was 0.15±0.00, and the zeta potential was -0.15 mV. Transmission electron microscopy showed a uniform spherical distribution, and infrared spectroscopy confirmed that it was a stable system based on molecular self-assembly. After storage at 4℃ and 25℃ for 28 days, the system maintained good physical stability and exhibited excellent resistance to photodegradation.

[0015] (2) The low-temperature emulsification method used in this study achieved efficient encapsulation of volatile active ingredients (encapsulation rate >90%) under mild conditions through a phase transition mechanism. Structural characterization results showed that TAN-NE formed a thermodynamically stable self-assembled structure through the orderly arrangement of surfactant molecules at the oil-water interface. Nanoemulsification enhanced the biological effects of trans-anesandene (TAN) from multiple dimensions. At the level of physical solubilization, the hydrophobic active ingredient was transformed into a nanoscale dispersion system, which significantly improved its apparent solubility and bioavailability. At the level of pharmacokinetics, in vitro release studies showed that the system has sustained-release properties and can maintain a long-term effective concentration. At the level of biological effects, the inhibitory activity against Ralstonia solanacearum was significantly enhanced. In pot experiments, the preventive effect (71.74%) and therapeutic effect (41.75%) were significantly better than the original drug and the commercial control drug (thiabendazole copper). Mechanism analysis showed that nanoscale droplets can enhance cell membrane permeability, promote accumulation at the target site, and effectively protect the active ingredient from degradation by environmental factors.

[0016] (3) Regarding ecological safety, acute toxicity tests showed that no significant lethal effect was observed in the model organism *Eisenia fetida* at the recommended concentration; plant compatibility tests showed no inhibitory effect on tobacco seed germination and seedling growth. More importantly, high-throughput sequencing analysis revealed that the α-diversity index of bacterial and fungal communities in the treated soil remained stable, while the β-diversity fluctuations were within the range of natural variation, indicating that the nano-formulation did not significantly disturb the soil microbial community structure. This study successfully developed a trans-anisene nanoemulsion system with both high bioactivity and good environmental compatibility. This formulation not only effectively solved the delivery problem of natural hydrophobic compounds through nanotechnology, but its low-energy preparation process also conforms to the development concept of green chemistry. This study provides important theoretical support and technical paradigm for the research and development of green pesticides based on natural products, which is of positive significance for promoting the sustainable development of agricultural production. Future research should focus on the optimization of the large-scale production process of this formulation, the systematic assessment of its environmental behavior, and the verification of its application effects in different agricultural ecosystems, laying a solid foundation for its practical application. Attached Figure Description

[0017] Figure 1 This is a pseudo-ternary phase diagram for single and compound surfactants. (a~d) represent single surfactants: polyethylene glycol octylphenyl ether (Triton X-100), castor oil, isotretinoin polyoxyethylene ether, and Tween 80, respectively. (e~h) represent compound surfactants composed of polyethylene glycol octylphenyl ether and castor oil, with mass ratios of 1:1, 1:3, 3:1, and 5:1, respectively.

[0018] Figure 2The optimal ratio screening results for TAN-NE are shown. Among them, (a) is the pseudo-ternary phase diagram of the preferred mixed surfactant; and (b) is the Tyndall effect of TAN-NE obtained by mixing the surfactant and TAN at different mass ratios.

[0019] Figure 3 The diagram shows the structural characterization of TAN-NE. (a) Diagram of TAN-NE preparation method; (b) Particle size and type; (c) Zeta potential; (d) Transmission electron microscopy image; (e) Infrared spectrum.

[0020] Figure 4 The graph shows the analysis results of TAN-NE's resistance to degradation and volatility. Among them, (a) DSC thermogram; (b) volatility; (c) photolysis; (d) thermal stability; and (e) stability diagram.

[0021] Figure 5 The figure shows the stability analysis results of TAN-NE. (a) pH stability; (b) ionic stability; (c) storage stability; (d) centrifugal stability.

[0022] Figure 6 Figure 1 shows the antibacterial activity of TAN-NE against Ralstonia solanacearum. (a) Minimum inhibitory concentration (MIC); (b) Minimum bactericidal concentration (MBC); (c) Growth curve of Ralstonia solanacearum; (d) Survival rate of Ralstonia solanacearum; (e-f) Scanning electron microscopy and transmission electron microscopy images of the cell morphology of Ralstonia solanacearum.

[0023] Figure 7 Figure 1 shows the results of TAN-NE's study on the infection characteristics of Ralstonia solanacearum. (a) Biofilm formation; (b) Effect of motility; (c) Extracellular polysaccharide content; (d) Expression of infection-related genes.

[0024] Figure 8 The biosafety assessment of TAN-NE was conducted. (a) Seed germination rate; (b) Survival of adult earthworms after TAN-NE treatment; (c) Schematic diagram of TAN-NE root irrigation and tobacco seedling growth on day 15; (d-h) Plant height, maximum leaf length, maximum leaf width, fresh weight, and dry weight of tobacco seedlings, in that order. Figure 9 The effects of TAN-NE on the prevention and treatment of bacterial wilt of tobacco were investigated. The results included: (a) disease index of the preventive treatment; (b) relative efficacy of the preventive treatment; (c) bacterial wilt symptoms of the preventive treatment on day 15 after Ralstonia solanacearum inoculation; (d) disease index of the treatment treatment; (e) relative efficacy of the treatment treatment; and (f) bacterial wilt symptoms of the treatment treatment on day 15 after Ralstonia solanacearum inoculation.

[0025] Figure 10The effects of TAN-NE on rhizosphere bacterial communities are shown in the following figures: (a) principal component analysis; (b) Chao index; (c) Shannon index; (d) Venn diagram of bacterial OTUs; (e) relative abundance at the phylum level in rhizosphere bacterial communities; (f) cluster heatmap of species richness; and (g) bar chart of LDA value distribution.

[0026] Figures 5-9 Different lowercase letters in the text represent significant differences between groups. Detailed Implementation

[0027] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods and detection methods described in each embodiment are conventional methods; unless otherwise specified, the reagents and materials can be purchased commercially.

[0028] The following examples involve Ralstonia solanacearum (Ralstonia solanacearum) Ralstonia solanacearum CQPS-1 was isolated from tobacco plants infected with bacterial wilt in Runxi Township, Pengshui Tujia Autonomous County, Chongqing, and is a highly pathogenic strain of bacterial wilt.

[0029] Example 1 This embodiment provides a method for preparing trans-anessenolene nanoemulsions.

[0030] 1. Screening of Single Surfactants: Using Tween 20, Tween 80, Tween 85, E-1306 (isotridecyl polyoxyethylene ether), Span 80, EL-40 (castor oil), NP-4 (alkylphenol polyoxyethylene ether-4), Triton X-100 (polyethylene glycol octylphenyl ether), and APG (alkyl glycoside) as single surfactants, trans-anethole (TAN) was weighed to the surfactants at mass ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1. TAN was slowly added to the surfactants, with thorough stirring until homogeneous during the addition process. Subsequently, while stirring, distilled water was continuously added dropwise to the mixed solution, and the total mass of distilled water (phase transition point) was recorded when the mixture changed from clear and transparent to viscous and turbid, and then back to clear and transparent. A pseudo-ternary phase diagram was drawn with the masses of TAN, surfactant, and distilled water as vertices, and the single surfactant with the largest emulsification area and the most phase transition points was selected for the preparation of trans-anetifrenene nanoemulsion (TAN-NE).

[0031] 2. Screening of compound surfactants: Triton X-100 and E-1306, EL-40 and Tween 80, and Triton X-100 and EL-40 were compounded at mass ratios of 1:1, 1:3, and 1:5, respectively, to obtain compound surfactants. TAN was then weighed with the compound surfactants at mass ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, and the process of preparing TAN-NE from a single surfactant was repeated to screen for compound surfactants.

[0032] 3. Screening of co-surfactants: Glycerol, anhydrous ethanol, polyethylene glycol (PEG-400), n-butanol, n-octanol, and n-pentanol were selected as co-surfactants. The compound surfactant Triton X-100:E-L40 (mass ratio 3:1) was mixed with the co-surfactants at a mass ratio of 3:1. After thorough mixing, the mixture was centrifuged at 3000 r / min for 15 min to obtain a mixed surfactant. The co-surfactant selected from the mixed surfactants that were clear, transparent, had good flowability, and showed no stratification was chosen.

[0033] 4. Determination of Km value: The co-surfactants n-octanol, n-pentanol, and anhydrous ethanol were mixed with the compound surfactant (Triton X-100:EL-40 = 3:1) at mass ratios of 1:1, 2:1, 3:1, and 4:1, respectively, and centrifuged at 3000 r / min for 15 min. Using the compound surfactant (Triton X-100:EL-40 = 3:1) as a control group, the optimal Km value was determined based on clarification transparency, flowability, and stratification.

[0034] 5. Optimal Ratio of TAN-NE: Mix TAN, the compound surfactant (Triton X-100:EL-40 = 3:1), and the co-surfactant (n-octanol) thoroughly. Under magnetic stirring, slowly add distilled water to the phase transition point. Calculate the mass fraction of TAN, the mixed surfactant (compound surfactant + co-surfactant), and distilled water at the phase transition point and plot a pseudo-ternary phase diagram to determine the drug loading of TAN-NE.

[0035] Depend on Figure 1As shown in (a) to (d), the pseudo-ternary phase diagram of TAN-NE prepared with Triton X-100 as the surfactant shows 5 points, indicating that Triton X-100 can form nanoemulsions (TAN-NE) with TAN at mass ratios of 1:9, 2:8, 7:3, and 6:4. TAN-NE is prepared with EL-40 and TAN at mass ratios of 9:1, 8:2, and 7:3. TAN-NE is also prepared with Tween 80 and E-1306 and TAN at mass ratios of 9:1 and 8:2. The pseudo-ternary phase diagrams of the four emulsifiers show different emulsion areas. The nanoemulsion area formed by Triton X-10 is larger than that formed by EL-40, E-1306, and Tween 80. Therefore, Triton X-100 and EL-40 are selected as surfactants for preparing TAN-NE.

[0036] Depend on Figure 1 As shown in (e) to (h), Triton X-100 and EL-40 (T:E) at mass ratios of 1:1, 1:3, 3:1, and 5:1 can all form nanoemulsions with TAN, showing 3, 3, 4, and 4 points on the pseudo-ternary phase diagram, respectively. The emulsifying effect of the compound surfactant composed of Triton X-100 and EL-40 is related to the concentration of Triton X-100; the higher the concentration of Triton X-100, the better the emulsifying effect. Among them, the emulsified region areas at T:E mass ratios of 3:1 and 5:1 are comparable and much larger than those at 1:1 and 1:3. Therefore, a T:E compound ratio of 3:1 was selected as the compound surfactant for preparing TAN-NE.

[0037] Anhydrous ethanol, n-butanol, n-pentanol, and n-octanol can all form a non-stratified, pale yellow, transparent, and well-flowing solution with the compound surfactant (Triton X-100:EL-40=3:1). When polyethylene glycol and glycerol are mixed with the compound surfactant, the solution separates, the flowability decreases, and the viscosity increases.

[0038] The compound surfactant, when mixed with n-octanol, n-pentanol, and anhydrous ethanol at mass ratios of 1:1, 1:2, 1:3, and 1:4, did not separate into layers after centrifugation. The solutions obtained by mixing the compound surfactant with n-octanol, n-pentanol, and anhydrous ethanol at mass ratios of 1:2, 1:3, and 1:4 were all yellow and transparent, while the solutions obtained by mixing at a mass ratio of 1:1 were all pale yellow. The fluidity of the compound surfactant varied with different proportions of n-octanol and n-pentanol. The fluidity of n-octanol and n-pentanol showed a "V" shape with increasing co-surfactant levels, meaning the fluidity first decreased and then increased. The fluidity from highest to lowest was 1:1 > 1:2 = 1:4 > 1:3 > compound surfactant. When anhydrous ethanol was used as the co-surfactant, the difference in fluidity between different proportions was not significant. Therefore, anhydrous ethanol was chosen as the co-surfactant, with a compound surfactant:anhydrous ethanol ratio of 1:3.

[0039] When the mass ratio of mixed surfactant (compound surfactant: anhydrous ethanol = 1:3):TAN was 9:1, 8:2, 7:3, 6:4, and 5:5, a phase transition point was observed upon addition of distilled water. The drug loading of the nanoemulsion increased with increasing TAN content. The drug loading was 18.35% for the mixed surfactant:TAN mass ratio of 5:5, followed by 17.32% and 12.40% for 6:4 and 7:3, respectively. Figure 2 As shown in (a), the emulsified region area of ​​the mixed surfactant is larger than that of the single surfactant (Triton X-100, EL-40, E-1306 and Tween 80), indicating that the co-surfactant and the compound surfactant have a synergistic effect.

[0040] Figure 2As shown in (b), when a beam of green light irradiates the mixed surfactant:TAN at mass ratios of 1:9, 2:8, 3:7, and 4:6, no green light is formed in the solution (no Tyndall effect occurs), while a beam of green light is produced at mass ratios of 5:5, 6:4, 7:3, 8:2, and 9:1. This indicates that mixed surfactant:TAN mass ratios of 5:5, 6:4, 7:3, 8:2, and 9:1 can form nanoemulsions. Mixed surfactant:TAN mass ratios of 5:5, 6:4, and 7:3 form nanoemulsions, while 8:2 and 9:1 form microemulsions. In this embodiment, the suitable ratio of TAN-NE, by mass percentage, is: 12.40%~18.35% TAN, 18.35%~28.93% mixed surfactant, and deionized water to bring the total to 100%. In this study, the mass ratio of Triton X-100, EL-40, and anhydrous ethanol in the mixed surfactant was 1:3:12. A higher proportion of co-surfactant can easily cause environmental problems when applied in the field. Therefore, TAN-NE, formed by a mass ratio of mixed surfactant to TAN of 7:3 and a drug loading of 12.4%, was selected for further research.

[0041] Example 2 This embodiment provides the physicochemical properties and stability analysis of trans-anessenolene nanoemulsion.

[0042] Particle size and potential analysis revealed that ( Figure 3 In (b) and (c) of the study, the particle size of TAN-NE was 20.68 ± 0.50 nm, and the dispersion index (PDI) was 0.15 ± 0.00. The potential of the TAN stock solution (TAN) in water showed that the surface of TAN carried a negative charge, with a potential of -13.64 mV. At the same concentration (TAN concentration in water was 100 mg / L), the surface of TAN-NE also carried a negative charge, with a potential of -0.15 mV. However, the potential of TAN-NE was lower than that of TAN, indicating that TAN was encapsulated within a nonionic surfactant, thus shielding the charge. These results indicate that the stability of the nanoemulsion does not depend on electrostatic repulsion but on strong steric stabilization, consistent with O / W type nanoemulsions with nonionic surfactants as stabilizers, suggesting that TAN-NE has good dispersibility in water. TEM observation showed that... Figure 3 In (d), TAN-NE consists of well-defined spherical or near-spherical particles with good dispersibility and no obvious agglomeration.

[0043] In the FTIR spectrum ( Figure 3 In (e) of the TAN, there are three characteristic peaks, namely at 839.0 cm⁻¹. -1 The presence of a strong, broad peak indicates the out-of-plane bending vibration of the =CH group in the para-substituted benzene ring. (966.3 cm⁻¹)-1 A strong spike exists at this point, which is the out-of-plane bending vibration of the inverse double bond C=C. Additionally, there is a peak at 1247.9 cm⁻¹. -1 and 1037.7cm -1 A strong peak is observed at this location, indicating the C=O stretching vibration of the aromatic methoxy group. Three characteristic peaks are present at EL-40, namely at 1745.6 cm⁻¹. -1 A very strong C=O stretching vibration peak of the ester carbonyl group is present at 1156.9 cm⁻¹, indicating it is an ester-type fat; -1 A relatively broad and strong peak is observed at this location, indicating the presence of the ester group (COC). Additionally, a peak is present at 3350.3 cm⁻¹. -1 A broad peak is observed at [cm], indicating the stretching vibration of the hydroxyl group (OH), a characteristic feature of ricinoleic acid. Two characteristic peaks are observed in the Triton X-100, one at 1156.9 cm⁻¹. -1 There is an extremely strong and broad peak nearby, which is a polyethylene glycol chain. Simultaneously, at 2925 cm⁻¹... -1 and 2850cm -1 The strong doublet is a typical characteristic of long alkyl chains. In the FTIR spectrum of TAN-NE, a peak was observed at 839.0 cm⁻¹. -1 A strong, broad peak also exists at this location, indicating the out-of-plane bending vibration of the =CH group of the para-substituted benzene ring; 954.7 cm⁻¹ -1 A strong peak is present at 1247.9 cm⁻¹, which is the out-of-plane bending vibration of the trans double bond C=C. This peak is shifted in TAN-NE, possibly due to intermolecular interactions between TAN and the hydroxyl groups (-OH) in EL-40 and Triton X-100 molecules, forming weak hydrogen bonds (CH···O). TAN-NE also shows a peak at 1247.9 cm⁻¹. -1 and 1037.7cm -1 Aromatic methoxy C=O stretching vibrations were observed at the peak, and the peak did not shift. These results indicate that the formation mechanism of TAN-NE can be attributed to a molecular self-assembly process involving physical interactions between components, such as hydrogen bonding and steric hindrance, without any breaking or formation of chemical bonds. Figure 3 (a) in the middle.

[0044] Figure 4Figure (a) shows the DSC heating changes of TAN-NE, TAN, and the mixed surfactant. The heating curves show two endothermic peaks. The endothermic peak of TAN-NE is shifted to the left relative to TAN, and the heating curves do not overlap, indicating that the emulsification process affected the melting point of the nanoemulsion, and that TAN is not a free oil phase but was successfully encapsulated. Furthermore, the migration of the low-temperature peak from 23.27℃ to -4.81℃ may be due to the melting or crystallization transformation of the low-melting-point component. The dramatic migration of the high-temperature peak (275.80℃ to 141.45℃) may be due to the evaporation of water molecules in the O / W type TAN-NE (boiling point 100℃). In actual storage and application (temperatures far below 141.45℃), this indicates that TAN-NE has good thermal stability.

[0045] like Figure 4 As shown in (b), TAN-NE can effectively reduce the volatilization rate of TAN. After prolonged high-temperature treatment for 12 hours, the volatilization rate of TAN-NE was 16.33%, significantly lower than that of TAN (58.67%). Photodegradation is also a key factor restricting pesticide application, such as... Figure 4 As shown in (c), there are certain differences in the retention rates of TAN-NE and TAN samples stored continuously for 14 days under light and dark conditions. The retention rate of TAN samples changed significantly between light and dark conditions after 14 days, with retention rates of 77.50% and 47.56% on day 14, respectively. In contrast, the retention rate of TAN-NE samples did not differ much between light and dark conditions after 14 days, with retention rates of 92.28% and 91.33% on day 14, respectively. This indicates that TAN-NE has a strong resistance to photodegradation.

[0046] like Figure 4 As shown in (d), after treatment at 4℃, 25℃, 35℃, and 50℃ for 30 min, no significant color change, solution stratification, or precipitation was observed in TAN-NE. At 75℃, TAN-NE became more transparent and showed stratification at the bottom, while at -20℃, the TAN-NE solution solidified and turned white. After standing for 30 min (cooling to room temperature), except for the TAN-NE treated at -20℃ which showed white flocculent matter, the color of TAN-NE at the other treatment temperatures remained the same. Furthermore, after shaking for 1 min, the TAN-NE treated at -20℃ regained stability, which may be due to spontaneous emulsification, indicating that TAN-NE has good cold and hot storage stability. The mechanism diagram of TAN-NE's good stability is shown below. Figure 4 As shown in (e) in the diagram.

[0047] Depend on Figure 5As shown in (a), the particle size of TAN-NE exhibits two distinct trends with increasing pH: from pH 5 to 7, the particle size gradually increases, reaching its maximum at pH 7 (167.53 nm); from pH 7 to 9, the particle size initially decreases and then increases, but remains below 167.53 nm. Furthermore, the dispersion index (PDI) of TAN-NE fluctuates at different pH levels. The PDI values ​​for pH 5, 6, 7, 8, and 9 are 0.107, 0.160, 0.167, 0.196, and 0.187, respectively, while the PDI for untreated TAN-NE is 0.152. All PDI values ​​are less than 0.2, indicating that TAN-NE possesses good pH stability.

[0048] from Figure 5 As shown in (b), at ion concentrations of 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, and 0.5 mol / L, the particle sizes of TAN-NE were 14.78 nm, 15.14 nm, 17.00 nm, 15.70 nm, and 16.44 nm, respectively. While not significantly increasing, the particle sizes decreased, which may be due to the 100-fold dilution of TAN-NE. Furthermore, at different ion concentrations, the PDI of TAN-NE was 0.107, 0.160, 0.168, 0.196, and 0.187, respectively, all less than 0.2, indicating that TAN-NE possesses good ionic stability.

[0049] from Figure 5 As shown in (c), TAN-NE maintained good stability at 0, 10, 20, and 30 days after preparation, with particle sizes of 20.68 nm, 16.57 nm, 16.30 nm, and 16.72 nm, respectively. The particle sizes at 10, 20, and 30 days after storage were smaller than those on the day of preparation. This may be because, in the initial stage of preparation, emulsifier molecules may continue to adsorb and optimize the interfacial arrangement, causing slight droplet compaction and resulting in a slight decrease in particle size. Furthermore, the particle size variation of TAN-NE was less than 5%, indicating that the nanoemulsion did not undergo aggregation or Ostwald ripening within 30 days.

[0050] Depend on Figure 5 As shown in (d), under the condition that the solution contains the same concentration of TAN (100 mg / L), the OD of TAN-NE before centrifugation (T0) is higher than that of TAN-NE. 340 There was no significant difference in nm values, while TAN showed a significant difference in OD after centrifugation (T1). 340 The nm value decreased significantly. Calculations show that the TAN centrifugation stability parameter K... E The TAN-NE centrifugal stability parameter K is 48.74 ± 8.85. EThe value was 7.08±1.22. The smaller the value of the centrifugal stability parameter, the more stable the nanoemulsion, indicating that TAN-NE has good centrifugal stability.

[0051] Example 3 This embodiment provides the effect of trans-anessenolene nanoemulsion on the activity of Ralstonia solanacearum.

[0052] 1. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC): The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of TAN-NE against Ralstonia solanacearum were determined using the two-fold dilution method. TAN-NE was prepared into concentrations of 25 mg / L, 35 mg / L, 45 mg / L, 55 mg / L, 65 mg / L, and 75 mg / L using sterile liquid B medium (10 g / L bacterial peptone, 1 g / L yeast extract, 1 g / L casein amino acids, and 5 g / L glucose). 75 mg / L mixed surfactant (MS) and sterile water (CK) were used as controls. 200 µL of TAN-NE-containing culture was pipetted into 96-well polystyrene microtiter plates, with 6 wells allocated for each concentration. Then, 1 µL of Ralstonia solanacearum (OD200) was added to each well. 600 =0.8~1.0), 1µL TTC solution, sealed and transferred to a 30℃ incubator, and OD was measured using a microplate reader at 24h and 48h. 600 .

[0053] like Figure 6 In the figures (a) to (b), TAN-NE represents the MIC and MBC of Ralstonia solanacearum. Compared with CK (sterile water), when the effective concentration of TAN in TAN-NE is higher than 25 mg / L, the growth of Ralstonia solanacearum is significantly inhibited. P<0.05 At 24h and 48h, when the effective TAN concentration in TAN-NE was 45 mg / L and 65 mg / L respectively, Ralstonia solanacearum showed almost no growth, and its OD... 600 The nm values ​​were 0.212 and 0.067, respectively, while those of CK were 1.15 and 3.07, respectively. MS (mass concentration of 75 mg / L) also showed certain antibacterial activity, indicating that anhydrous ethanol, Triton X-100 and castor oil had a synergistic effect.

[0054] 2. Growth and Survival Rate: The growth of Ralstonia solanacearum on Ralstonia solanacearum was evaluated using a liquid co-culture method. TAN-NE was prepared into liquid culture medium B containing TAN concentrations of 25 mg / L, 35 mg / L, 45 mg / L, 55 mg / L, and 65 mg / L. The control group consisted of 65 mg / L mixed surfactant and 0.1% dimethyl sulfoxide (DMSO). Then, 50 µL of Ralstonia solanacearum (OD200) was added to each culture medium. 600=0.8~1.0), and cultured at 30℃ and 180 r / min for 24 h. Starting from 3 h, the absorbance (OD) of the samples was measured every 3 h. 600 nm).

[0055] like Figure 6 As shown in (c), different concentrations of TAN-NE all affected the growth of Ralstonia solanacearum. Under culture conditions with TAN concentrations of 25–45 mg / L, the growth of Ralstonia solanacearum followed an "S"-shaped curve. However, when the TAN concentration was higher than 55 mg / L, the growth of Ralstonia solanacearum was completely inhibited, with an IC50 value of 100%. 50 It is 29.93 mg / L. (For example...) Figure 6 As shown in (d), after co-culturing TAN-NE, TAN, and MS with Ralstonia solanacearum for 24 h, the cell viability of TAN-NE, TAN, and MS was 0, 3.14%, and 40.89%, respectively.

[0056] 3. Cell morphology: such as Figure 6 As shown in (e), *Ralstonia solanacearum* treated with CK (0.1% DMSO) showed no abnormalities; the cell surface was smooth and maintained its intact morphology. However, *Ralstonia solanacearum* treated with MS, TAN, and TAN-NE all showed varying degrees of damage. TAN-NE-treated *Ralstonia solanacearum* cells exhibited significant cell breakage, with extreme wrinkling, unevenness, and collapse of the cell surface. TAN-treated cells also showed obvious damage, cell wrinkling, unevenness, and collapse, but the severity of these symptoms was lower than that of TAN-NE. Figure 6 In (f), the Ralstonia solanacearum cells treated with CK remained intact, without cell leakage or plasmolysis. The Ralstonia solanacearum cells treated with MS showed a small number of incomplete cells and cell leakage, but no plasmolysis. After treatment with TAN-NE and TAN, some Ralstonia solanacearum cell membranes became discontinuous, broken, and detached from the cell wall (plasmolysis), and the cytoplasm became sparse, resulting in leakage of cellular material. The nanoemulsion TAN-NE showed a more significant effect than TAN.

[0057] Example 4 This embodiment provides the effect of trans-anessenol nanoformulation on the pathogenicity of Ralstonia solanacearum.

[0058] 1. Biofilm: The effect of TAN-NE on Ralstonia solanacearum biofilm was determined by the crystal violet method. 40 μL of Ralstonia solanacearum (OD200) was added to an 8 mL glass tube. 600=0.8~1.0), and TAN and TAN-NE were added separately to make the TAN concentration in the glass tube 50 mg / L. The tube was incubated statically at 30°C for 24 h. A mixed surfactant (MS) and 0.5% dimethyl sulfoxide (DMSO, CK) were used as controls. After 24 h, the culture medium was removed, and 8 mL of 0.1% crystal violet was added, and the tube was stained statically at room temperature for 30 min. After staining, the crystal violet was removed, and residual crystal violet was washed with 5 mL of sterile water and dried at room temperature for 30 min. Then, 5 mL of 95% ethanol was added, and the tube was incubated statically at room temperature for 30 min. Finally, the absorbance (OD) was measured using a microplate reader. 530 ).

[0059] 2. Extracellular polysaccharides: Take 10 mL of fresh Ralstonia solanacearum suspension (OD200). 600 =0.8~1.0), and remove extracellular polysaccharides (EPS) by centrifugation at 5000×g for 15 min. Dilute the bottom bacterial cake to OD using M63 medium (13.6 g / L potassium dihydrogen phosphate, 2.0 g / L ammonium sulfate, 0.5 mg / L ferrous sulfate heptahydrate, 1 mL magnesium sulfate heptahydrate (1 mol / L), 10 mL 20% glucose). 600 =0.2, and TAN and TAN-NE were added separately to make the TAN concentration in the culture medium 50 mg / L. The culture was incubated at 30℃ and 180 rpm for 12 h in a constant temperature shaker. Afterwards, the culture was centrifuged at 12000 rpm for 5 min, and 0.2 mL of the supernatant was transferred to a 2 mL enzyme-free centrifuge tube. 0.8 mL of ethanol was added and mixed well, and the mixture was incubated at 4℃ in the dark for 1 h. After incubation, the culture was centrifuged at 12000 rpm for 5 min to remove the supernatant, and 1 mL of 80% ethanol was added and mixed well. The mixture was centrifuged at 12000 rpm for 5 min, and the supernatant was discarded. The remaining supernatant was gently aspirated with a pipette (and blotted dry on absorbent paper) to collect the bacterial pellet. After complete removal, 2 mL of ddH2O was added to the centrifuge tube, and the tube was heated at 95-100℃ for about 5 min to completely dissolve the precipitate. The resulting liquid is the EPS test solution. The EPS detection method refers to the instructions for the G0593W polysaccharide content kit, with 3 biological replicates set up for each strain.

[0060] 3. Motility: Prepare a semi-solid SMM medium containing 50 mg / L TAN (3.4 g / L potassium dihydrogen phosphate, 0.5 g / L ammonium sulfate, 0.03 g / L magnesium sulfate, 0.721 mg / L ferrous sulfate heptahydrate, 3 g / L agar, 3.74 g / L sodium glutamate). After inverting and cooling the medium, vertically add 5 μL of Ralstonia solanacearum (OD200) to the culture dish. 600=0.1), and sealed in a 30°C incubator for upright incubation. After 72 h, the motility diameter (white band around the colony) of Ralstonia solanacearum was measured. A mixed surfactant (MS) and 0.5% dimethyl sulfoxide (DMSO) were used as controls. Each treatment was repeated three times.

[0061] 4. Expression of pathogenic genes: 50 µL of Ralstonia solanacearum suspension cultured overnight (OD200) was used to express pathogenic genes. 600 =1.0) was added to 10 ml of LM63 medium and cultured at 30°C and 180 rpm / min for 12 h. Then, TAN and TAN-NE were added separately to make the TAN concentration in the medium 50 mg / L. MS and 0.1% DMSO were used as controls, and the culture was carried out for 1 h. The bacterial cells were then collected by centrifugation, and RNA was extracted using the TRIzol method. After reverse transcription to extract cDNA, biofilm-related genes were selected. lecM extracellular polysaccharide-related genes epsE and xpsR Exercise-related genes cheW and fliA Gene expression level analysis was performed, among which serC It is an internal reference gene.

[0062] like Figure 7 As shown, compared to the control (CK), TNA-NE, TAN, and MS all affected the biofilm formation, motility, and extracellular polysaccharide production of Ralstonia solanacearum. Among them, TAN-NE and TAN significantly affected the biofilm formation, motility, and extracellular polysaccharide production of Ralstonia solanacearum. P<0.05 ).like Figure 7 In (a), at the MIC (25 mg / L) concentration, the OD of TAN-NE 530 nm is 0.183, while TAN's OD 530 The nm value was 0.227, indicating that TAN-NE had a significantly better biofilm inhibitory effect on Ralstonia solanacearum than TAN. Similarly, after 48 hours of stress from TAN-NE, TAN, and MS, both TAN-NE and TAN significantly reduced the motility of Ralstonia solanacearum, while MS had no significant effect. Specifically, after 48 hours of stress from TAN-NE, TAN, and MS, the motility diameters of Ralstonia solanacearum were 0.50 cm, 1.34 cm, and 2.74 cm, respectively, while the motility diameter of the control group (CK) was 2.90 cm. The motility diameters of Ralstonia solanacearum after TAN-NE and TAN stress decreased by 82.76% and 53.68% compared to the CK, respectively, indicating that TAN-NE further enhanced the inhibitory effect of TAN on the motility of Ralstonia solanacearum. Figure 7(b)). In addition, both TAN-NE and TAN significantly inhibited the production of EPS by Ralstonia solanacearum, with EPS concentrations of 0.15 mg / mL and 0.24 mg / mL, respectively, while MS and CK concentrations were 0.70 mg / mL and 0.74 mg / mL, respectively. Figure 7 (c)). Under TAN-NE and TAN stress, the EPS of Ralstonia solanacearum decreased by 5.75%, 67.86%, and 79.48% compared with CK, respectively.

[0063] The expression of genes related to the infectivity of *Ralstonia solanacearum* under TAN-NE stress was determined using RT-qPCR. Figure 7 As shown in (d), the infection characteristic genes of Ralstonia solanacearum treated with CK were identified. lecM , cheW , fliA , xpsR and epsE The expression levels were 1.03, 1.26, 1.29, 1.43, and 1.29, respectively, indicating the expression of the infection characteristic genes of Ralstonia solanacearum treated with MS. lecM , cheW , fliA , xpsR and epsE The expression levels were 0.81, 1.21, 1.20, 1.34, and 1.25, respectively, indicating the expression of TAN-treated Ralstonia solanacearum infection characteristic genes. lecM , cheW , fliA , xpsR and epsE The expression levels were 0.59, 0.97, 0.78, 0.97, and 0.80, respectively, while the expression levels after TAN-NE treatment were 0.46, 0.59, 0.68, 0.65, and 0.58. Compared with the control group, TAN-NE treatment significantly reduced the expression levels of the control group. lecM , cheW , fliA , xpsR and epsE Gene expression levels decreased to 0.45, 0.46, 0.53, 0.46, and 0.45 times that of CK, respectively, while TAN treatment resulted in significantly lower levels. lecM , cheW , fliA , xpsR and epsE Gene expression levels decreased to 0.57, 0.77, 0.61, 0.68, and 0.62 times that of CK, respectively. In conclusion, the nanoemulsion TAN-NE further enhanced the effect of TAN on the infection characteristics of Ralstonia solanacearum, exhibiting a certain synergistic effect.

[0064] Example 5 This embodiment provides the biosafety of trans-anessenol nanoemulsions.

[0065] 1. Seed germination and plant growth of Yunyan 87: Yunyan 87 tobacco seeds were disinfected with 2.5% NaClO solution for 5 min, and then rinsed 4 times with sterile water to remove residual NaClO. Twenty-five seeds were placed in a petri dish (8.5cm × 8.5cm) containing filter paper, and 4 mL of TAN-NE solution containing 40 mg / L, 80 mg / L, 160 mg / L, 320 mg / L, and 640 mg / L TAN were added. A control group (CK) containing 640 mg / L TAN, a mixed surfactant, and water was used. The plants were then transferred to an artificial climate chamber at 28℃ and relative humidity of 75% (14 h light) / 28% (10 h darkness) for 6 days. Seed germination for each treatment was then recorded. Each treatment was replicated in triplicate (3 petri dishes).

[0066] Approximately 6 weeks after transplanting, tobacco seedlings were transferred to a specialized seedling substrate and cultured for 24 hours. Then, 30 mL of a solution containing 100 mg / L TAN and TAN-NE was applied to the roots of the seedlings. Water was used as a control. Ten seedlings were used in each treatment. The seedlings were then placed in a greenhouse at 28℃ with a relative humidity of 75% (14 hours of light) / 28% (10 hours of darkness) for 15 days. The fresh weight, dry weight, plant height, maximum leaf length, and maximum leaf width of the seedlings were then measured.

[0067] 2. Earthworm toxicity assay: 3 mL of TAN-NE containing TAN concentrations of 80 mg / L, 160 mg / L, 320 mg / L, 640 mg / L, 960 mg / L, and 1280 mg / L were added to beakers (8.5 cm × 10.5 cm) containing filter paper. Adult earthworms (n=3) were then placed on the filter paper and incubated in the dark for 24 hours. Afterward, the survival rate of the earthworms was observed; if they moved when gently picked up with tweezers, they were alive; otherwise, they were dead. A 640 mg / L mixed surfactant and water (CK) were used as controls. Each treatment involved 15 earthworms and was repeated four times.

[0068] By evaluating the acute toxicity of Yunyan 87 tobacco seeds to germination, earthworms, and its safety for plant growth, such as... Figure 8 As shown in (a), the germination rates of tobacco seeds treated with TAN-NE at concentrations of 40 mg / L, 80 mg / L, and 160 mg / L were 97.78%, 95.74%, and 91.88%, respectively, showing no significant difference compared to the control (97.81%). However, when the TAN concentration in the TAN-NE was 320 mg / L and 640 mg / L, the germination of tobacco seeds was inhibited, with germination rates of only 51.11% and 10.98%, respectively, significantly lower than the 97.81% of the control. Figure 8As shown in (b), TAN-NE can kill adult earthworms even when the effective concentration of TAN is higher than 640 mg / L. SPSS calculations show that the regression equation is y = 5.19x - 14.89, and the toxicity to earthworms is LC. 50 The concentration was 744.07 mg / L. Based on the toxicity classification, the LC50 of TAN-NE for adult earthworms was... 50 The concentration is much higher than 10 mg / L, indicating that TAN-NE is a green and environmentally friendly pesticide.

[0069] A schematic diagram of TAN-NE root irrigation and the growth of tobacco seedlings on the 15th day after irrigation are shown below. Figure 8 As shown in (c) in the diagram. Figure 8 From (d) to (h), it can be seen that the plant height, maximum leaf length, maximum leaf width, fresh weight, and dry weight of TAN-NE and TAN were significantly higher than those of CK. p< 0.05 Furthermore, TAN-NE has a certain improving effect compared to TAN. For example, the plant height, maximum leaf length, maximum leaf width, fresh weight, and dry weight of TAN-NE were 6.58cm, 14.72cm, 7.90cm, 6.83g, and 0.59g, respectively, while those of TAN were 6.28cm, 13.37cm, 6.89cm, 6.54g, and 0.57g, respectively. Compared with TAN, these figures represent increases of 4.56%, 9.17%, 12.78%, 4.22%, and 4.71%, respectively, and increases of 19.00%, 20.99%, 19.11%, 31.43%, and 31.14%, respectively. It is evident that TAN-NE has a significant growth-promoting effect.

[0070] Example 6 This embodiment provides the preventive and therapeutic effects of trans-anessenolene nanoemulsion on tobacco bacterial wilt.

[0071] The efficacy of TAN-NE in controlling tobacco bacterial wilt was evaluated using root drenching inoculation.

[0072] (1) Preventive treatment: Select tobacco seedlings of uniform size (about 6 weeks old), and irrigate the base of the seedlings with 30 mL of TAN-NE (100 mg / L TAN) and 100 mg / L TAN. Use 100 mg / L thiabendazole copper (TCO) as a positive control and water as a negative control (CK). 24 hours after the irrigation, inoculate the base of the seedlings with 10 mL of Ralstonia solanacearum suspension (OD200). 600 =0.1).

[0073] (2) Treatment: The TAN-NE treatment for tobacco bacterial wilt was performed by inoculating the seedlings with Ralstonia solanacearum and then drenching the roots with a root fungicide. The dosage and concentration of the fungicide were the same as those for prevention. The seedlings were then placed in a greenhouse at 28°C, 75% humidity, and a 14h / 10h light cycle. The disease index was assessed after the control group began to show symptoms. The disease severity rating for tobacco bacterial wilt was 0-4 (where 0 was no symptoms; 1 was 1-25% leaf wilting; 2 was 26-50% leaf wilting; 3 was 51-75% leaf wilting; and 4 was 76-100% leaf wilting). Each treatment included at least 12 seedlings and was repeated 3 times. The disease index and relative control effect were calculated using the following formula: Disease index =

[0074] In the formula, n i v represents the number of plants with the corresponding disease index. i The disease index is (0, 1, 2, 3, 4), and N is the total number of plants in each treatment.

[0075] Relative prevention and control effect = % In the formula, T is the disease index of the treatment group and CK is the disease index of the control group.

[0076] TAN-NE exhibits a good size effect, which can promote the water solubility of TAN, thereby enhancing the control effect of TAN. For example... Figure 9 As shown, TAN-NE improved the control effect of TAN on tobacco bacterial wilt. Specifically, 100 mg / L of TAN-NE, TAN, and TCO all delayed the occurrence of bacterial wilt (e.g., Figure 9 (a) in the figure, the inoculum was delayed by 7 days, 4 days, and 3 days, respectively. Throughout the experimental period, the disease index of TAN-NE was lower than that of TAN and TCO treatments, and at day 15 post-inoculation, the disease index of TAN-NE was significantly lower than that of TAN and TCO, at 21.67, 36.67, and 44.17, respectively. Figure 9 As shown in (b), the relative efficacy of TAN-NE was significantly higher than that of TAN and TCO. At day 15 post-inoculation, the relative efficacy of TAN-NE, TAN, and TCO against tobacco bacterial wilt were 71.74%, 52.17%, and 42.39%, respectively. Figure 9 As shown in (c), the tobacco seedlings treated with TAN-NE grew well, indicating that formulating TAN into a nanoemulsion can improve its efficacy.

[0077] The therapeutic effect of TAN-NE on tobacco bacterial wilt was evaluated using a non-invasive root inoculation method, which involved drenching the roots of tobacco seedlings with the agent 24 hours after inoculation. The results were as follows: Figure 9As shown in (d), compared to the control (CK), all treatments reduced the disease index of tobacco bacterial wilt. The disease index of different treatments differed from that of the preventive treatment; the disease index after TAN treatment was actually higher than that after TCO treatment, but all were lower than the disease index of TAN-NE. Figure 9 In (e) of the study, the nanoemulsion TAN-NE showed the best therapeutic effect. At days 5 and 7 post-inoculation, the relative efficacy of TAN-NE was 95.65% and 76.00%, respectively; the relative efficacy of TCO was 91.31% and 70.00%, while that of TAN was 86.96% and 48.00%. At day 15 post-inoculation, the relative efficacy was 41.75%, while the relative efficacy of TCO and TAN were 33.98% and 30.10%, respectively. Figure 9 As shown in (f), the therapeutic effect of TAN is slightly lower than that of TCO, but the therapeutic and preventive effects of TAN-NE are higher than those of the positive agents TCO and TAN, which suggests that TAN-NE may have an effect of systemic conduction.

[0078] Principal coordinate analysis (PCoA) using the Bray-Curtis distance algorithm was used to analyze the rhizosphere bacterial composition of TAN-NE and CK. Figure 10 In (a) of the study, the bacterial communities of TAN-NE and CK were distributed in different regions and were clearly separated. Anosim analysis showed that there were significant differences in the rhizosphere bacterial community composition between TAN-NE and CK. R=0.875 , p=0.022 The Chao index and Shannon index are indicators used to measure species richness and diversity, respectively. Figure 10 As shown in (b) to (c), the Chaos and Shannon values ​​in the rhizosphere of the TAN-NE treatment were higher than those of the CK treatment, indicating that TAN-NE can improve the richness and diversity of rhizosphere bacteria. OTU statistical analysis of bacterial microorganisms in the TAN-NE and CK groups ( Figure 10 In (d), a total of 4889 bacterial OTUs were generated, of which 2653 were from TAN-NE, which was higher than 2236 from CK, further indicating that TAN-NE treatment produced more rhizosphere bacterial species than CK.

[0079] At the phylum level (relative abundance >1%), the eight dominant bacterial genera shared by TAN-NE and CK are Pseudomonadota, Gemmatimonadota, Bacteroidota, Acidobacteriota, Chloroflexota, Actinomycetota, Planctomycetota, and Cyanobacteriota (see...). Figure 10 (e) in the middle). Further analysis of the species abundance clustering heatmap revealed ( Figure 10 In (f) of the TAN treatment group, Ralstonia solanacearum ( Ralstonia The abundance of ) was significantly lower than that of CK ( p<0.05 This indicates that TAN-NE can inhibit the growth of Ralstonia solanacearum. Furthermore, TAN-NE significantly enriched Bacillus spp. (rhizobium). Gemmatimonas ), Sphingosine Bacteria ( Sphingomonas ), Neosphingosine Bacteria ( Novosphingobiums ), Sphingosine Box Bacteria ( Sphingopyxis ), Sphingomyelin spp. ( Sphingobium Further analysis of LEfSe ( p<0.05 (LDA score > 3.8), TAN-NE and CK had 15 and 10 dominant groups respectively. At the genus level, CK was significantly enriched in Ralstonia solanacearum ( Ralstonia Flavobacterium ( flavobacterium TAN-NE is mainly enriched in Bacillus spp. ( Gemmatimonas ), Sphingolipids ( Sphingobium It is evident that TAN-NE can reduce the number of rhizosphere pathogens, thereby reducing the occurrence of diseases. Figure 10 (g) in the middle. Additionally, Sphingobium It is a functionally specialized genus of bacteria with strong degradation capabilities, capable of degrading surfactants and co-surfactants in TAN-NE, which also indicates that TAN-NE is relatively safe for the environment after application.

[0080] The embodiments described above are some, but not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art through related deductions and substitutions based on the inventive concept, without inventive effort, are within the scope of protection of the present invention.

Claims

1. A trans-anisene nanoemulsion, characterized in that, The trans-anestigmine nanoemulsion comprises, by weight percentage: 12.40%~18.35% trans-anestigmine, 18.35%~28.93% mixed surfactant, and the balance being water; The mixed surfactant is composed of polyethylene glycol octylphenyl ether, castor oil and anhydrous ethanol; The mass ratio of polyethylene glycol octylphenyl ether, castor oil, and anhydrous ethanol is 1:3:

12.

2. The trans-anisene nanoemulsion according to claim 1, characterized in that, The trans-anestinene nanoemulsion comprises, by weight percentage: 12.40% trans-anestinene, 28.93% mixed surfactants, and the balance being deionized water.

3. The method for preparing the trans-anisene nanoemulsion according to any one of claims 1 to 2, characterized in that, include: First, polyethylene glycol octylphenyl ether and castor oil are thoroughly mixed, and then anhydrous ethanol is added and mixed evenly to obtain a mixed surfactant. Then, trans-anisene is added to the mixed surfactant and stirred evenly. Deionized water is added dropwise under continuous stirring to obtain a trans-anisene nanoemulsion.

4. The application of the trans-anestinene nanoemulsion according to any one of claims 1 to 2 in the prevention and control of bacterial wilt in plants.

5. The application according to claim 4, characterized in that, The pathogen causing bacterial wilt is Ralstonia solanacearum.

6. The application according to claim 4, characterized in that, The plant in question is tobacco.

7. The application according to claim 5, characterized in that, The trans-anisene nanoemulsion reduces the pathogenicity of Ralstonia solanacearum by regulating the formation of biofilms, cell motility, extracellular polysaccharide production, and expression of infection-related genes.

8. The application according to claim 7, characterized in that, The infection-related genes include: lecM , cheW , fliA , xpsR and epsE.

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