Use of 5,8,11-eicosatrienoic acid in the prevention and treatment of plant fungal diseases

Abstract

The application discloses application of 5,8,11-eicosatrienoic acid in prevention and treatment of plant fungal diseases and belongs to the field of pesticide chemistry. The 5,8,11-eicosatrienoic acid mainly prevents and treats diseases caused by the oomycete class capsicum phytophthora, the compound is a long-chain unsaturated fatty acid, has an antifungal disease effect and can be used as a fungicide to prevent and treat plant diseases. The compound can effectively prevent pathogenic bacteria from invading plants by inhibiting mycelium growth, enhancing cell membrane permeability and interfering with the spore sac release process, and is further used for preventing and treating huge plant fungal diseases. Biological tests show that the compound has a significant concentration-dependent inhibitory effect on the mycelium growth of the capsicum phytophthora, and the half effective inhibitory concentration (EC50) is 60.2 µg / mL. At a concentration of 200 µg / mL, the inhibition rate is more than 83%, the 5,8,11-eicosatrienoic acid can effectively inhibit the release of the capsicum phytophthora spore sac, and the EC50 is 156.3 µg / mL. At a concentration of 200 µg / mL, the inhibition rate is morethan 77%.

Description

Technical Field

[0001] This invention relates to the field of pesticide chemistry, and more particularly to the application of a 5,8,11-hexadecanotrienoic acid in the control of plant fungal diseases. Background Technology Plant fungal diseases are a significant factor contributing to yield reductions in global agriculture. Among them, Phytophthora blight of peppers, a devastating soil-borne disease, severely damages several important economic crops, including peppers, tomatoes, and cucumbers. Currently, control of these diseases primarily relies on chemically synthesized fungicides. However, long-term use of these chemical fungicides not only leads to increased resistance in pathogens but also causes pesticide residues and environmental pollution. These problems seriously affect sustainable agricultural development and pose potential threats to the ecological environment and human health.

[0002] Unsaturated fatty acids are widely found in plants, animals, and microorganisms. Recent studies have shown that some unsaturated fatty acids possess certain antibacterial activities. Nevertheless, the potential of long-chain polyunsaturated fatty acids with specific structures, especially compounds with a C26 carbon chain and specific double bond positions (such as 5,8,11-trienes), in controlling plant pathogenic fungi, particularly oomycete diseases, has not been fully recognized or developed. These compounds may possess unique antibacterial mechanisms, potentially providing new ideas and methods for the control of plant diseases.

[0003] In existing technologies, there are no reports on the effective inhibition of the growth and sporangium release of plant pathogenic fungi such as Phytophthora capsici. Therefore, exploring the application of 5,8,11-hexadecanoic acid in the control of plant fungal diseases is not only of significant theoretical importance, but may also provide new avenues for developing novel and environmentally friendly plant disease control agents. This is of great significance for reducing the use of chemical fungicides, mitigating the risk of pathogen resistance, and protecting the ecological environment. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides the application of 5,8,11-hexadecanoic acid in the control of plant fungal diseases, providing a novel active ingredient for the development of highly efficient, low-toxicity, and environmentally friendly new biological pesticides.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides the application of 5,8,11-hexadecanetrienoic acid and its salts or esters in the prevention and control of plant fungal diseases.

[0006] Furthermore, the fungal diseases include those caused by Phytophthora capsici, a fungus belonging to the Oomycetes class.

[0007] Further, the salts or esters of the 5,8,11-hexadecanoic acid comprise compounds with the following structural formula: HOOC-(CH2)3-CH=CH-CH2-CH=CH-CH2-CH=CH-(CH2)9-CH3, Na +- OOC-(CH2)3-CH=CH-CH2-CH=CH-CH2-CH=CH-(CH2)9-CH3 or CH3OOC-(CH2)3-CH=CH-CH2-CH=CH-CH2-CH=CH-(CH2)9-CH3.

[0008] The present invention also provides a method for the application of 5,8,11-hexadecanotrienoic acid in the prevention and control of plant fungal diseases, wherein a pesticide composition is prepared using 5,8,11-hexadecanotrienoic acid or its salts or esters as active ingredients.

[0009] Furthermore, the pesticide composition also includes a carrier and adjuvants.

[0010] Furthermore, the formulation of the pesticide composition is selected from one of wettable powder, water-dispersible granules, suspension concentrate, emulsifiable concentrate, water-in-oil emulsion, or microcapsule.

[0011] Furthermore, the wettable powder comprises: 10% 5,8,11-hexadecanoic acid, 5% dispersant, 3% wetting agent, and 82% filler, wherein the dispersant and wetting agent are auxiliary agents, and the filler is a carrier.

[0012] Furthermore, the dispersant is sodium lignosulfonate, the wetting agent is sodium dodecyl sulfate, and the filler is kaolin.

[0013] Furthermore, the 5,8,11-hexadecanoic acid and its salts or esters are obtained through chemical synthesis, extraction from natural products, or separation and purification after microbial fermentation.

[0014] Compared with the prior art, the present invention has at least the following advantages and technical effects: 1. Expansion of New Applications: This invention reveals for the first time a novel application of compound 5,8,11-hexadecanoic acid (HTA) in the control of plant fungal diseases, particularly Phytophthora in pepper. This discovery not only opens up new directions for the application of unsaturated fatty acids in agriculture but also provides new ideas and methods for solving problems such as drug resistance, pesticide residues, and environmental pollution caused by traditional chemical fungicides.

[0015] 2. Clear Mechanism of Action and Significant Efficacy: HTA has a clear mechanism of action against Phytophthora capsici, effectively inhibiting mycelial growth, disrupting cell membrane structure, and suppressing spore dispersal. These multi-faceted mechanisms of action make HTA exhibit significant efficacy in controlling plant fungal diseases, providing a more efficient and reliable means of disease control for agricultural production.

[0016] 3. Development of Environmentally Friendly Formulations and Green Pesticides: The pesticide composition provided by this invention uses HTA as the active ingredient and can be formulated into various environmentally friendly formulations, such as emulsifiable concentrates, suspension concentrates, and water-dispersible granules. These environmentally friendly formulations are not only easy to use and store, but also reduce environmental pollution, providing a core material basis for the development of new green biological pesticides, helping to promote sustainable agricultural development, reduce the use of chemical pesticides, and protect the ecological environment. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a schematic diagram of the chemical structure of compound HTA; Figure 2 Photographs showing the inhibitory effect of HTA on the mycelial growth of Phytophthora capsici in Example 2; Figure 3 The images are fluorescence micrographs of *Phytophthora capsici* hyphae treated with HTA-free buffer, buffer at a concentration of 60 µg / mL, and buffer at a concentration of 160 µg / mL in Example 3, stained with propidium iodide (PI). Figure 4 This is a diagram illustrating the inhibitory effect of HTA on sporangium release from Phytophthora capsici in Example 4. Figure 5 The bar graph shows the inhibition rate of HTA on sporangium release of Phytophthora capsici in Example 4. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] This invention provides an application of 5,8,11-hexadecanetrienoic acid and its salts or esters in the prevention and control of plant fungal diseases.

[0025] In some embodiments of the present invention, the fungal diseases include those caused by Phytophthora capsici, a fungus belonging to the class Oomycetes.

[0026] The mechanisms of the application include: inhibiting the growth of pathogenic fungal hyphae, disrupting the integrity of hyphal cell membranes, and / or inhibiting the release of sporangia.

[0027] In some embodiments of the present invention, the salts or esters of the 5,8,11-hexadecanoic acid comprise compounds with the following structural formula: HOOC-(CH2)3-CH=CH-CH2-CH=CH-CH2-CH=CH-(CH2)9-CH3, Na +- OOC-(CH2)3-CH=CH-CH2-CH=CH-CH2-CH=CH-(CH2)9-CH3, CH3OOC-(CH2)3-CH=CH-CH2-CH=CH-CH2-CH=CH-(CH2)9-CH3.

[0028] This invention also provides a method for using 5,8,11-hexadecanoic acid in the prevention and control of plant fungal diseases, wherein a pesticide composition is prepared using 5,8,11-hexadecanoic acid or its salts or esters as active ingredients.

[0029] In some embodiments of the present invention, the pesticide composition further includes a carrier and adjuvants.

[0030] In some embodiments of the present invention, the formulation of the pesticide composition is selected from one of wettable powder, water-dispersible granules, suspension concentrate, emulsifiable concentrate, water-in-oil emulsion, or microcapsule.

[0031] In some embodiments of the present invention, the wettable powder comprises: 10% 5,8,11-hexadecanoic acid, 5% dispersant, 3% wetting agent, and 82% filler, wherein 5,8,11-hexadecanoic acid is the active ingredient, the dispersant and wetting agent are auxiliary agents, and the filler is a carrier.

[0032] In some embodiments of the present invention, the dispersant is sodium lignosulfonate, the wetting agent is sodium dodecyl sulfate, and the filler is kaolin.

[0033] In some embodiments of the present invention, the 5,8,11-hexadecanoic acid is obtained by chemical synthesis, extraction from natural products, or separation and purification after microbial fermentation.

[0034] Phytophthora capsici ( Phytophthora capsici LT263 was isolated from the soil of the experimental field of Huazhong Agricultural University and preserved in the Fermentation Engineering Research Laboratory of Huazhong Agricultural University.

[0035] Example 1 Preparation and identification of compound 5,8,11-hexadecanetriene (HTA) As an example preparation method: Extract Pseudomonas aeruginosa from the Fermentation Engineering Research Laboratory of Huazhong Agricultural University. Pseudomonas aeruginosa Glycerol tubes of strain JD3-20310 were streaked onto LB agar plates (containing 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 2 g / L agar, pH 7.0±0.2) and incubated in the dark at 37 °C for 12–24 h. Single colonies of strain JD3-20310 were picked from the LB agar plates and inoculated into 5 mL of LB liquid medium, and cultured overnight at 37 °C with shaking to obtain a seed culture. The seed culture was then transferred at a 1% inoculation ratio to 250 mL Erlenmeyer flasks containing 40 mL of LB liquid medium and cultured at 37 °C with shaking at 180 r / min for 4 days.

[0036] Centrifuge 35 L of fermentation broth at 4 ℃ and 7000 r / min for 1 h, and collect the supernatant. Add 2 times the volume of n-butanol (70 L) to the fermentation supernatant and extract three times. Combine the organic phases. Concentrate the organic phase to dryness using a rotary evaporator at 65 ℃ and 0.08 MPa to obtain the crude extract. Scrape the crude fermentation extract with a spatula and place it in a 50 mL centrifuge tube for storage at room temperature.

[0037] A small amount of crude extract was dissolved in methanol and filtered through a 0.22 μL organic filter membrane. The crude extract was then analyzed by high-performance liquid chromatography (HPLC). The HPLC program is shown in Table 1-1. The chromatographic column was an Agilent C18 column (4.6 mm × 150.0 mm, 5 μm), pump A (ultrapure water + 0.1% trifluoroacetic acid), pump B (liquid-grade acetonitrile), the detection wavelength was 254 nm, the injection volume was 20 μL, and the flow rate was 1.0 mL / min.

[0038] To improve the purity of the crude extract, methanol dissolution and purification were employed: At 45 ℃ and 0.08 MPa, a small amount of methanol (5-10 mL) was added to dissolve the crude extract, which was then evaporated to dryness. This process was repeated six times to remove residual proteins and small molecule salts. A small amount of the crude extract methanol solution (approximately 2-3 mL) was taken and filtered through a 0.22 μm organic filter membrane. High-performance liquid chromatography (HPLC) was then used to detect changes in the peak shape of the preliminarily purified crude extract (the HPLC procedure and conditions were the same as above). The crude extract solution was then evaporated to dryness by rotation, scraped with a spatula, and placed in a 50 mL centrifuge tube. The tube was stored at room temperature to obtain the crude HTA extract. The components of the crude HTA extract were separated by gel column chromatography: Sephadex LH-20 gel (Pharmacia) was soaked in methanol for 24 h to allow it to fully swell, and then sonicated for 15 min to remove air bubbles. Using a 1.5 cm × 60 cm glass chromatography column, the swollen gel was slowly poured in along the glass rod and allowed to settle naturally to a filling height of about 50 cm. The two column volumes were then equilibrated with methanol. The purified crude extract was fully dissolved in 5 mL of methanol and filtered through a 0.22 μm sterile filter membrane. The sample solution was slowly added dropwise to the top of the gel column, allowing it to naturally penetrate the gel bed. Dichloromethane:methanol = 1:1 (v / v) was used as the eluent at a flow rate of 1.5 mL / min. The eluent was collected at 12 mL / tube, for a total of 70 tubes. The samples were analyzed by TLC (developing solvent: dichloromethane:methanol = 8:1, v / v). Adjacent 2-3 tubes were combined (27 samples in total), labeled A0-A26.

[0039] Fragments A0-A26 were diluted with methanol, filtered through a 0.22 μm filter membrane, and then analyzed by HPLC (under the same conditions as above). Based on the TLC detection combined with the HPLC results, fractions with similar chemical types, mobilities, and elution times were grouped together and labeled as B1-B5.

[0040] Further separation of crude extract components was performed using silica gel column chromatography: The elution system for components B2-B4 was optimized using a dichloromethane-methanol (65:1→8:1, v / v) solvent system. The optimal initial elution ratio was determined by TLC spot migration analysis (Rf≈0.3), as shown in Tables 1-2. A total of 221 subfractions were obtained from the three components after silica gel column chromatography. Based on the similarity of TLC migration values, these were merged into 14 characteristic components, named L1-L3 (origin of component B2), 1-5 (origin of component B3), and D1-D6 (origin of component B4), respectively. Dissolve 1 g of the sample to be separated in 2 mL of dichloromethane. Add 3 g of 100-200 mesh silica gel powder to a rotary evaporator flask, stir well, and then evaporate the solvent to dryness using a rotary evaporator (plug the flask opening with cotton wool) to ensure the sample is fully adsorbed onto the silica gel for column chromatography separation. Weigh 30 g of 200-300 mesh silica gel into a beaker, add an appropriate amount of dichloromethane, and stir until a paste forms. Slowly pour the silica gel suspension into the chromatography column using a funnel. Open the stopcock of the chromatography column and gently tap the outer wall of the column from bottom to top to ensure the silica gel is as uniform and compact as possible. This experiment uses dry loading. Leave a 2-3 cm eluent layer at the top of the column bed, and slowly and evenly sprinkle it onto the silica gel surface during loading. After loading, add a small amount of quartz sand to prevent sample leakage. Prepare different ratios of dichloromethane:methanol (in ascending polarity) for gradient elution of different components and collect each fraction.

[0041] The target fraction was collected, and the D1 and L3 components were further separated and purified using semi-preparative high-performance liquid chromatography (semi-preparative HPLC). First, the absorption peak positions of the D1 and L3 components were scanned using a multi-wavelength ultraviolet detection system (210 nm, 254 nm, 280 nm, 320 nm, 380 nm), and the optimal detection wavelength was determined based on the peak shape characteristics and response values ​​at each wavelength. Then, the mobile phase ratio was optimized by calculating the chromatographic peak resolution. Finally, preparative separation was performed under the selected chromatographic conditions, and the high-purity monomeric compounds were collected, evaporated to dryness under reduced pressure, and high-purity HTA was obtained.

[0042] High-resolution mass spectrometry (HR-MS) and nuclear magnetic resonance (NMR) 1 H NMR, 13Its structure was identified by C10 NMR, and the spectral data were consistent with those reported in the literature (Song et al., 2013, Journal of Lipid Research), confirming it as 5,8,11-hexadecanoic acid. The structural formula is shown below. Figure 1 .

[0043] Table 1-1 HPLC Detection Procedure Table 1-2 Gradient elution ratios of components B2-B4 Example 2 Determination of the inhibitory activity of HTA on the mycelial growth of Phytophthora capsici The mycelial growth rate method was used. Different concentrations of HTA (dissolved in methanol) were uniformly mixed into potato dextrose agar (PDA) medium cooled to approximately 50°C to prepare drug-containing plates with final concentrations of 5, 10, 20, 40, 80, 160, and 200 µg / mL. An equal volume of methanol served as a control (CK). A 6 mm diameter *Phytophthora capsici* spore was inoculated in the center of each plate. Phytophthora capsici After 5 days of incubation in the dark at 28°C, the diameter of the colony (LT263) was measured using the cross-cross method, and the mycelial growth inhibition rate was calculated.

[0044] See results Figure 2 As can be seen, the control plate exhibited abundant mycelium, while the colony diameter decreased significantly with increasing HTA concentration. HTA showed a significant concentration-dependent inhibitory effect on the mycelial growth of *Phytophthora capsici*, with a half-maximal inhibitory concentration (EC50) of 60.2 µg / mL. At a concentration of 200 µg / mL, the inhibition rate reached over 83%.

[0045] Example 3 Effects of HTA on cell membrane permeability of Phytophthora capsici mycelia Propidium iodide (PI) staining was used. Vigorously growing hyphae of *Phytophthora capsici* were treated with buffers containing HTA (final concentrations of 60 µg / mL and 160 µg / mL) for 2 hours and 4 hours, respectively, with a buffer without HTA serving as a control. After treatment, PI staining solution (final concentration 2 µg / mL) was added and stained in the dark for 30 minutes. After washing with PBS, the samples were observed under a fluorescence microscope (excitation wavelength 535 nm, emission wavelength 617 nm).

[0046] The results are as follows Figure 3As shown, the hyphae in the control group showed no red fluorescence, while those in the HTA-treated group exhibited significant red fluorescence (positive for PI staining). After 2 hours of treatment with 160 µg / mL HTA, a large number of hyphae showed red fluorescence, indicating that the integrity of the hyphal cell membrane was disrupted and permeability significantly increased. The 60 µg / mL treatment group also showed fluorescence signals after a certain period, demonstrating that its membrane-damaging effect is concentration- and time-dependent.

[0047] Example 4 HTA assay for the inhibitory activity of Phytophthora capsici sporangium release Phytophthora capsici mycelium was placed in V8 liquid medium to induce sporangium production. Mature sporangia were then transferred to solutions containing different concentrations of HTA (40, 80, 160, 200 µg / mL) and treated for 2 hours. After low-temperature-room-temperature cycling stimulation, the proportion of zoospores released from the sporangia was counted under a microscope, and the inhibition rate was calculated.

[0048] See results Figure 4 and Figure 5 HTA effectively inhibits sporangium release, with an EC50 of 156.3 µg / mL. At a concentration of 200 µg / mL, the inhibition rate exceeds 77%.

[0049] Example 5 Application of field-grown Phytophthora in pepper control A wettable powder containing HTA was prepared by weight percentage as follows: 10% HTA, 5% dispersant (sodium lignosulfonate), 3% wetting agent (sodium dodecyl sulfate), and filler (kaolin) to bring the total to 100%. All components were thoroughly mixed and pulverized using an air jet mill until the average particle size was less than 10 micrometers, thus obtaining a 10% HTA wettable powder. This formulation was diluted 500 times with water to 200 ppm before use, and applied as a foliar spray or root drenching treatment in a 5-acre field of chili peppers. The incidence of Phytophthora blight was reduced by 76% in the foliar spray group and by 64% in the root drenching group.

[0050] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of a 5,8,11-hexadecanoic acid and its salts or esters in the control of plant fungal diseases.

2. The application of 5,8,11-hexadecanoic acid and its salts or esters according to claim 1 in the control of plant fungal diseases, characterized in that, The fungal diseases mentioned include those caused by Phytophthora capsici, a fungus belonging to the Oomycetes class.

3. The application of 5,8,11-hexadecanoic acid and its salts or esters according to claim 1 in the control of plant fungal diseases, characterized in that, Salts or esters of 5,8,11-hexadecanoic acid include compounds with the following structural formula: HOOC-(CH2)3-CH=CH-CH2-CH=CH-CH2-CH=CH-(CH2)9-CH3, Na +- OOC-(CH2)3-CH=CH-CH2-CH=CH-CH2-CH=CH-(CH2)9-CH3 or CH3OOC-(CH2)3-CH=CH-CH2-CH=CH-CH2-CH=CH-(CH2)9-CH3.

4. A method for controlling plant fungal diseases, characterized in that, A pesticide composition is prepared using the 5,8,11-hexadecanetrienoic acid and its salts or esters as active ingredients as described in claim 1.

5. The method according to claim 4, characterized in that, The pesticide composition also includes a carrier and adjuvants.

6. The method according to claim 5, characterized in that, The pesticide composition is selected from one of the following formulations: wettable powder, water-dispersible granules, suspension concentrate, emulsifiable concentrate, water-in-oil emulsion, or microcapsule.

7. The method according to claim 6, characterized in that, The wettable powder comprises, by weight percentage: 10% 5,8,11-hexadecanoic acid, 5% dispersant, 3% wetting agent, and 82% filler, wherein the dispersant and wetting agent are additives, and the filler is a carrier.

8. The method according to claim 7, characterized in that, The dispersant is sodium lignosulfonate, the wetting agent is sodium dodecyl sulfate, and the filler is kaolin.

9. The method according to any one of claims 4-8, characterized in that, The 5,8,11-hexadecanoic acid and its salts or esters are obtained through chemical synthesis, extraction from natural products, or separation and purification after microbial fermentation.

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