Plant disease fungicides and their applications with medium-chain 3-hydroxy fatty acids as active ingredients

Abstract

This invention provides a fungicide for plant diseases using medium-chain 3-hydroxy fatty acids as the active ingredient, belonging to the field of biopesticide technology. Chemically synthesized fungicides often suffer from drawbacks such as high toxicity, easy development of drug resistance, and environmental pollution. Medium-chain 3-hydroxy fatty acids are naturally occurring substances and are safe for humans and the environment. This invention uses medium-chain 3-hydroxy fatty acids as the main active component, adding adjuvants to prepare water-in-oil emulsions, microemulsions, aqueous solutions, or soluble powders. The fungicide containing medium-chain 3-hydroxy fatty acids can directly kill various plant disease fungi, and also has a certain preventive effect against fungal plant diseases. It is a broad-spectrum, green, highly efficient, safe, and convenient antifungal biopesticide.

Description

Technical Field

[0001] This invention belongs to the field of agricultural technology, specifically relating to a fungicide for plant diseases based on biologically derived compounds and its application. Background Technology

[0002] Fungi are a major cause of plant diseases. In important grain crops like rice and wheat, fungal diseases such as rice blast and Fusarium head blight can lead to severe yield reductions or even total crop failure. In fruit and vegetable cultivation, powdery mildew caused by powdery mildew and wilt caused by Fusarium are highly prevalent diseases, especially when crops are continuously planted in the same field. Due to the widespread use of chemical pesticides, fungal resistance is increasing, and pesticide efficacy is declining year by year. As a result, many fungal diseases lack effective control measures, causing significant losses to agricultural production. Furthermore, many chemically synthesized pesticides are highly toxic to humans. Given the strict national regulations limiting the total application of chemical pesticides, the development of broad-spectrum antibacterial biological agents that are safe for human use has significant application value and broad application prospects.

[0003] Hydroxy fatty acids are a class of naturally occurring compounds, mainly derived from microbial fermentation products or insect secretions (such as hydroxydecenoic acid in royal jelly secreted by bees). They are highly safe for human use and very environmentally friendly. Early reports indicated that 12,13,17-trihydroxy-9(Z)-octadecenoic acid, a long-chain fatty acid containing multiple hydroxyl groups, exhibits weak inhibitory activity against some plant pathogens, including powdery mildew (Erysiphe graminis), puccinia recondita, Phytophthora infestans, and Botrytis cinerea. However, the monohydroxy and dihydroxy 10-hydroxystearic acid and 7S,10S-dihydroxy-8(E)-octadecenoic acid showed no antibacterial activity (Hou CT & Forman RJ. J Ind Microbiol Biotechnol, 2000, 24, 275-276). 3-Hydroxydecanoic acid has been shown to have poor antibacterial effects against bacteria (S. aureus, E. coli) and yeasts (C. albicans, S. cerevisiae), failing to completely inhibit their growth even at high concentrations of 2000 mg / L (Xiao Haifeng, Master's Thesis, Zhejiang University, 2023). However, 3-Hydroxydecanoic acid and 3-Hydroxyoctanoic acid have some inhibitory effects on Aspergillus fumigatus and Microsporum gypseum (Pekmezovic et al. Antibiotics 2021, 10, 737). 10-Hydroxydecanoic acid has an antifungal effect against Malassezia furfur and is therefore used for the treatment and prevention of acne (CN202310655319.7, CN202010455320.1). However, the bactericidal effects of hydroxy fatty acids with different structures vary greatly on different bacterial species, and the mechanism by which their structure affects antibacterial activity remains unclear.

[0004] More importantly, existing studies have only tested the bactericidal effects of hydroxy fatty acids on microorganisms cultured in vitro in petri dishes; their effectiveness against pathogen-infected plants has not been reported. In fact, effectiveness against microorganisms in vitro does not equate to effectiveness against infected plants. A typical example is tricyclazole, a commonly used pesticide for controlling rice blast, whose median inhibitory concentration (EC50) against blast fungus in vitro is... 50The concentration was approximately 200 mg / L (Liu Xianyuan, Heilongjiang Agricultural Sciences, 2011); however, it had almost no therapeutic effect on already infected rice plants. Furthermore, in the application of plant antifungals, drugs do not necessarily possess both preventative and curative effects. For example, M-PEDE (a mixture of long-chain fatty acids with a carbon chain length of 16-20) produced in the United States only showed curative control of powdery mildew, without any observed preventative control. Therefore, due to the complexity of fungal infections in plants, the effectiveness of compounds in controlling fungal infections requires rigorous experimental verification.

[0005] Furthermore, similar to other fatty acid compounds, hydroxy fatty acids are water-insoluble solids / liquids at low pH, but transform into fatty acid salts and become water-soluble at higher pH. Existing studies on the antibacterial properties of hydroxy fatty acids have all used pure products for experiments; whether they can be prepared into various formulations and whether there are differences in antibacterial effects among different formulations have not been reported. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by designing a fungicide for plant diseases and providing its formulation, preparation method, and uses.

[0007] The technical solution adopted in this invention is as follows:

[0008] This plant disease fungicide contains one or more medium-chain 3-hydroxy fatty acids or potassium salts of medium-chain 3-hydroxy fatty acids, mixed in any proportion. Specifically, the medium-chain 3-hydroxy fatty acid mentioned in this invention refers to a 3-hydroxy fatty acid with a carbon chain length of 8-14, typically one or more of 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid, 3-hydroxylauric acid, and 3-hydroxytetradecanoic acid. Its formulation is an emulsion, microemulsion, aqueous solution, or soluble powder.

[0009] The water-in-oil emulsion is made from the following raw materials by weight percentage: 10-45% medium-chain 3-hydroxy fatty acids, 1-10% oily solvents, 1-10% surfactants, and the balance being water and pH adjusters.

[0010] The oily solvent is one or more of soybean oil, oleic acid, or linoleic acid. The pH adjuster is sulfuric acid or potassium hydroxide.

[0011] The preparation process of the water-in-oil emulsion is as follows: Weigh an appropriate amount of medium-chain 3-hydroxy fatty acid, add one or more of soybean oil, oleic acid or linoleic acid, stir thoroughly to form an oily liquid, add water and surfactant, and use an appropriate amount of sulfuric acid or potassium hydroxide to adjust the pH to 4-7. Mix in a high-speed homogenizer at 12000rpm for 30-60 minutes to obtain a uniform milky white water-in-oil emulsion.

[0012] The microemulsion is made from the following ingredients by weight percentage: 1-10% medium-chain 3-hydroxy fatty acids, 1-10% propylene glycol, ethylene glycol or glycerol, 5-20% surfactant, and the balance being water and pH adjuster.

[0013] The polyol is one or more of propylene glycol, ethylene glycol, and glycerol.

[0014] The preparation process of microemulsion is as follows: Weigh an appropriate amount of medium-chain 3-hydroxy fatty acid, add one or more of propylene glycol, ethylene glycol or glycerol, stir thoroughly to form an oily liquid, add water and surfactant, and use an appropriate amount of sulfuric acid or potassium hydroxide to adjust the pH to 4-7. Mix in a high-speed homogenizer at 12000 rpm for 30-60 minutes to obtain a clear and transparent microemulsion.

[0015] The surfactants contained in water-based emulsions and microemulsions are either nonionic or anionic surfactants. Nonionic surfactants are one or more of alkylphenol polyoxyethylene ethers, fatty alcohol polyoxyethylene ethers, alkyl glycosides, sucrose fatty acid esters, sorbitan fatty acid esters, and fatty acid glycerides; anionic surfactants are one or more of alkylbenzene sulfonates, alkyl sulfonates, fatty acid sulfonyl esters, alkyl glycerol ether sulfonates, fatty alcohol polyoxyethylene, higher fatty acid salts, and fatty alcohol sulfate salts.

[0016] The aqueous solution is made from the following raw materials by weight percentage: 1-45% potassium salt of medium-chain 3-hydroxy fatty acids, 0.1-5% adjuvants, and the balance being water and pH adjuster; the adjuvants are one or a mixture of xanthan gum, carboxymethyl cellulose, or soluble starch, which act as thickeners to improve leaf staining; the pH value of the aqueous solution is 5-9, preferably 6-8. The preparation process of the aqueous solution is as follows: first, weigh an appropriate amount of potassium salt of medium-chain 3-hydroxy fatty acids, add water and adjuvants, adjust the pH to 5-9 with an appropriate amount of sulfuric acid or potassium hydroxide, and then add water to make up to the required concentration.

[0017] The soluble powder is made from the following raw materials by weight percentage: 90-95% potassium salt of medium-chain 3-hydroxy fatty acids, with the balance being additives. The additives are one or more of xanthan gum, carboxymethyl cellulose, or soluble starch. The preparation process of the soluble powder is as follows: weigh an appropriate amount of potassium salt of medium-chain 3-hydroxy fatty acids, add the additive powder, pulverize and mix evenly to obtain the soluble powder.

[0018] Fungicides for plant diseases can inhibit or kill fungi that cause plant diseases, including Fusarium graminearum, Pyricularia oryzae, and Fusarium oxysporum.

[0019] The application of fungicides for plant diseases includes:

[0020] 1) Used to control powdery mildew in pumpkins;

[0021] 2) Used to prevent and control rice blast;

[0022] 3) Used to control wheat scab;

[0023] 4) Used to prevent and control watermelon wilt disease.

[0024] Compared with chemically synthesized antifungal pesticides, this plant disease fungicide has the following advantages and novelty: 1) Hydroxy fatty acids are natural products, safe for humans, and can be harvested immediately after application, avoiding health hazards caused by pesticide residues; 2) It has a broad-spectrum antifungal effect against a variety of plant disease fungi; 3) It is easy to apply, requiring only foliar spraying or root irrigation, with fewer applications, saving labor and pesticide costs; 4) It has both preventive and curative effects, spraying before plant infection can reduce the incidence rate, and killing fungi after infection can prevent the spread of the fungus and reduce economic losses.

[0025] The position of the hydroxyl group significantly influences the antibacterial activity of fatty acids. 3-hydroxy fatty acids exhibit higher antibacterial activity than 10-hydroxy fatty acids of the corresponding carbon chain length and fatty acids without hydroxyl groups (see examples for detailed data). Furthermore, medium-chain 3-hydroxy fatty acids demonstrate strong antibacterial activity against various plant pathogenic fungi, but show no significant inhibitory effect on most bacteria. While their bactericidal mechanism is currently unclear, it may be related to the inhibition of fungal cell wall synthesis by 3-hydroxy fatty acids. Molecular simulations show that the binding energy of 3-hydroxydecanoic acid to chitin synthase (a key enzyme in fungal cell wall synthesis) is higher than that of 10-hydroxydecanoic acid and decanoic acid. This indicates that 3-hydroxydecanoic acid can better inhibit fungal cell wall synthesis, thus explaining its higher antibacterial activity.

[0026] The same 3-hydroxy fatty acid compound exhibits better antibacterial effects in its microemulsion formulation compared to other formulations. This may be because the nanoparticle form and lower pH give it a certain degree of hydrophobicity, which helps it to better adhere to fungi and easily penetrate them. Attached Figure Description

[0027] Figure 1 This is a graph showing the inhibitory effect of microemulsion 2 on the mycelial growth of Fusarium oxysporum and rice blast fungus.

[0028] Figure 2 These are photos of field trials of fungicides for inhibiting rice blast. From left to right, they represent the water treatment group, isoprothiolane, tricyclazole, microemulsion 2-500 mg / L, and microemulsion 2-1000 mg / L, respectively.

[0029] Figure 3These are photos from a field trial of a fungicide for inhibiting powdery mildew. The top left image shows pumpkin leaves before the fungicide was applied; the bottom left image shows pumpkin leaves after the fungicide was applied; and the right image shows the overall pumpkin plot after the application. Detailed Implementation

[0030] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. The *Fusarium graminearum* (ATCC46779) and *Fusarium oxysporum* (ATCC 62164) used in the examples were provided by the College of Agriculture, Zhejiang University; the *Pyricularia oryzae* Cav. ATCC 20140 was provided by Hangzhou Normal University. All are conventional standard strains and can be purchased from commercial institutions. The medium-chain hydroxy fatty acids used in this invention can all be purchased from conventional reagent manufacturers, with CAS numbers as follows: 3-hydroxydecanoic acid 5561-87-5; 3-hydroxyoctanoic acid 14292-27-4; 3-hydroxylauric acid 1883-13-2; 3-hydroxytetradecanoic acid 28715-21-1. Their potassium salts can be obtained by reacting commercially available hydroxy fatty acid products with potassium hydroxide. The M-PEDE used in the comparative examples was purchased from Gowan Corporation, USA. Unless otherwise specified in the examples, standard conditions or manufacturer-recommended conditions were followed. Reagents or instruments used in the examples, unless otherwise specified, are commercially available products.

[0031] Example 1: Preparation of a water-in-oil emulsion as a fungicide for plant diseases

[0032] Weigh out appropriate amounts of medium-chain 3-hydroxy fatty acids according to the components and contents in Table 1, add oily solvent, stir thoroughly to form an oily liquid, add water and surfactant, and appropriate amounts of sulfuric acid or potassium hydroxide to adjust the pH. Mix in a high-speed homogenizer at 12000 rpm for 30-60 minutes to obtain uniform milky white water emulsions.

[0033] Table 1. Preparation of fungicides for plant diseases – water-in-oil emulsions

[0034]

[0035] The water-based emulsion will not show significant separation after one month of storage at room temperature. If separation occurs, shaking well before use will not affect its effectiveness.

[0036] Example 2: Preparation of a microemulsion as a fungicide for plant diseases

[0037] Weigh out appropriate amounts of medium-chain 3-hydroxy fatty acids according to the components and contents in Table 2, add polyols, stir thoroughly to form an oily liquid, add water and surfactants, and appropriate amounts of sulfuric acid or potassium hydroxide to adjust the pH. Mix in a high-speed homogenizer at 12000 rpm for 30-60 minutes to obtain clear and transparent microemulsions.

[0038] Table 2 Preparation of microemulsions as fungicides for plant diseases

[0039]

[0040] The microemulsion described above is a clear and transparent solution. Its particle size distribution, measured using a nanoparticle size analyzer, is between 5 and 50 nm. The microemulsion remains clear and transparent after being stored at room temperature for 6 months.

[0041] Example 3: Preparation of a fungicide for plant diseases – aqueous solution

[0042] Weigh out appropriate amounts of potassium medium-chain 3-hydroxy fatty acids according to the components and contents listed in Table 3, add water, adjust the pH to 5-9 with potassium hydroxide or sulfuric acid, and then add water to make up to the required concentration to obtain each aqueous solution.

[0043] Table 3 Preparation of fungicides for plant diseases – aqueous solutions

[0044]

[0045] The aqueous solution is a homogeneous, completely water-soluble liquid that leaves no solid residue after centrifugation. It is stable at room temperature for at least 6 months.

[0046] Example 4: Preparation of a soluble powder fungicide for plant diseases

[0047] Weigh appropriate amounts of potassium medium-chain 3-hydroxy fatty acids according to the components and contents listed in Table 4, add the auxiliary powder, pulverize and mix evenly to obtain each soluble powder.

[0048] Table 4. Preparation of plant disease fungicides – soluble powder

[0049]

[0050]

[0051] The soluble powder is completely soluble in water, yielding a clear and transparent aqueous solution.

[0052] Example 5: Inhibitory effects of 3-hydroxy fatty acids with different carbon chain lengths on different plant pathogens

[0053] Using aqueous formulations 1-4 as examples, and 10-hydroxydecanoic acid, decanoic acid, and M-PEDE (an aqueous formulation containing 49.8% potassium long-chain fatty acids) as comparative examples, the inhibitory effects of fatty acids with different carbon chain lengths and hydroxyl positions on different plant pathogens were investigated. *Fusarium graminearum*, *Pyricularia oryzae* Cav., and *Fusarium oxysporum* were all cultured on PDA medium.

[0054] The agent was diluted with PDA medium at different ratios to achieve potassium hydroxy fatty acid salt (or potassium fatty acid salt) concentrations of 0, 100, 300, 500, 700, 1000, 2000, and 5000 mg / L. Subsequently, mycelia were inoculated onto plates containing the aqueous solution and incubated at 28°C for 3-7 days, with each treatment repeated three times. Based on the inhibition rate of mycelial growth under each concentration treatment, the fatty acid concentration (EC50) at which mycelial growth inhibition was 50% was calculated. 50 The results are shown in Table 5.

[0055] Table 5. Inhibitory effects of 3-hydroxy fatty acids with different carbon chain lengths on different plant pathogens.

[0056]

[0057] It is evident that all four 3-hydroxy fatty acids effectively inhibited the aforementioned common plant pathogenic fungi. Potassium 3-hydroxyoctanoate and potassium 3-hydroxydecanoate showed superior inhibitory effects compared to potassium 3-hydroxylaurate and potassium 3-hydroxytetradecanoate, which have slightly longer carbon chains. As a comparative example, 10-hydroxydecanoic acid exhibited significantly weaker bactericidal activity than 3-hydroxydecanoic acid, while decanoic acid and M-PEDE showed almost no bactericidal activity. This demonstrates that the presence and position of the hydroxyl group have a decisive influence on bactericidal ability. Furthermore, it should be noted that the 10-hydroxydecanoic acid and decanoic acid used in this example are fatty acid compounds that can be mass-produced, while fatty acids with hydroxyl groups in other positions (such as 2-hydroxydecanoic acid) are scarce in nature and have limited product development value; therefore, they were not included in the selection of compounds for this comparative example.

[0058] Example 6: Inhibitory effects of different formulations of 3-hydroxydecanoic acid on different plant pathogens

[0059] Aqueous solutions, soluble powders, emulsions, and microemulsions of 3-hydroxydecanoic acid were selected to investigate their inhibitory effects on different plant pathogens and to evaluate the differences in antibacterial activity among different formulations. The experimental procedures were the same as in Example 5, and the results are shown in Table 6. Figure 2 As shown in Table 6, the results of Example 5 were directly used for aqueous formulation 2 without additional experiments. All other formulations underwent three replicate experiments. Figure 1 The image shows a photograph illustrating the inhibitory effect of microemulsion 2 on the mycelial growth of Fusarium oxysporum and Blastomyces oryzae.

[0060] Table 6. Inhibitory effects of different formulations of 3-hydroxydecanoic acid on different plant pathogens.

[0061]

[0062] As can be seen, the effectiveness of the four formulations is in the order of microemulsion > water-in-oil emulsion > aqueous solution > soluble powder. One reason for this difference is the pH; hydroxydecanoic acid exhibits better antibacterial effects at lower pH levels. Aqueous solutions and soluble powders require higher pH levels to ensure complete dissolution; while in water-in-oil emulsions and microemulsions, hydroxydecanoic acid exists in an oil phase, which may allow for better penetration into hyphae or spores, resulting in a stronger bactericidal effect. Furthermore, microemulsions have smaller particles, often reaching the nanoscale, thus exhibiting better bactericidal effects than water-in-oil emulsions.

[0063] Example 7: Plant tolerance to fungicide concentration test

[0064] To evaluate whether high concentrations of plant disease fungicides cause phytotoxicity to plants, aqueous solutions 1-4, soluble powder 2, water-in-oil emulsion 2, and microemulsion 2 were diluted with water to a hydroxy fatty acid content of 5000 and 10000 mg / L, respectively. These solutions were then applied as foliar sprays to watermelon seedlings (Zaojia 8424) 15 days after transplanting, with 10 ml sprayed per seedling. The control group was sprayed with water. The spraying was repeated after 7 days, followed by another 15 days of cultivation. Each group consisted of 10 seedlings. Normal watering and fertilization were maintained throughout the cultivation period. After cultivation, plant height was measured, and leaf morphology was observed for abnormalities such as leaf curling and yellowing.

[0065] Table 7 Evaluation of whether high concentrations of fungicides cause phytotoxicity in watermelon seedlings

[0066]

[0067] The results are shown in Table 7. Except for the highest concentration (10000 mg / L) of aqueous solution 1, which caused slight seedling damage, all other high-concentration sprays were safe for the plants. Regarding hydroxy fatty acids with different carbon chains, hydroxyoctanoic acid, with the shortest carbon chain, may have stronger acidity, thus causing slight damage to the plants at high concentrations. Generally, its application concentration is between 100-1000 mg / L, far below the concentration that might cause seedling damage, indicating a wide safety window.

[0068] Example 8: In vitro experiment on the control of rice blast disease

[0069] Experimental reagents: Aqueous agent 2 and microemulsion 2 described in the examples were diluted with water to a hydroxydecanoic acid content of 500 mg / L.

[0070] Comparison of pesticides: Isoprothiolane 500 mg / L; Tricyclazole 200 mg / L; Both pesticides were commercially available wettable powders and their active ingredient content was converted.

[0071] Disease control: Shimizu

[0072] Rice variety: Zhejing 27

[0073] Treatment method: For rice in the flowering stage, select uniformly growing panicles, cut them approximately 10cm below the flag leaf node, remove the flag leaf, and insert them into sterile water for spraying. After 24 hours, inoculate with rice blast fungus spores (final concentration 10). 4 The inoculation volume was approximately 1 mL per spikelet (number of spikelets / mL). Each treatment inoculated 20 spikelets, with three replicates per treatment. After inoculation, the spikelets were bagged and kept moist at 25°C for 3 days, followed by hydroponics at 25°C for 7 days. The disease index was classified into four levels: Level 0: Asymptomatic; Level 1: 1 / 4 of spikelets were diseased; Level 2: 2 / 4 of spikelets were diseased; Level 3: 3 / 4 of spikelets were diseased; Level 4: The entire spikelet was diseased or even died. The disease index was calculated using the following formula: Disease Index = Σ(Level Number × Number of Spikes in that Level) / (Total Number of Seedlings × 4) × 100.

[0074] The formula for calculating the incidence rate is as follows: Incidence rate % = Number of infected ears / Total number of ears × 100%

[0075] The formula for calculating the efficacy is as follows: Efficacy % = (1 - Disease index of the treatment group / Disease index of the water group) × 100%

[0076] Table 8. In vitro experiments on the control of rice blast by spraying different pesticides.

[0077]

[0078] The results are shown in Table 8. It can be seen that the aqueous solution 2 and microemulsion 2 of the present invention have higher efficacy against rice blast fungus infection than the commonly used pesticides for rice blast control, such as isoprothiolane and tricyclazole.

[0079] Example 9: Experiment on the control of watermelon wilt disease in plants

[0080] The watermelon variety Zaojia 8424 was used. After conventional soaking and germination, the seeds were sown. After germination, the seedlings were cultivated for another 14 days. Seedlings with uniform growth were selected for transplanting, and the experiment was conducted after a 7-day recovery period. Aqueous agent 2 and microemulsion 2 were diluted to hydroxydecanoic acid concentrations of 500, 1000, 2000, and 5000 mg / L, respectively, and used for root irrigation. 50 ml of this solution was applied to each seedling, with an equal volume of plain water used as a control group. Each group contained 30 watermelon seedlings.

[0081] The mycelia of *Fusarium oxysporum* propagated in PDB medium were filtered out, and spores were collected. The number of spores was counted using a hemocytometer and adjusted to 10⁻⁶. 6Prepare 10 ml of the pathogen per plant. On the second day after root drenching, make a small incision in the damaged root with a knife and inoculate with the pathogen. Maintain high temperature and humidity conditions (28-30℃, humidity >90%) and continue normal fertilization and watering. Symptoms will appear approximately 15 days after inoculation. Record the number of infected plants every two days to calculate the disease incidence rate.

[0082] Table 9. Experimental Study on Plant Control of Watermelon Fusarium Disease

[0083]

[0084]

[0085] The results are shown in Table 9. It can be seen that both aqueous solution 2 and microemulsion 2 can effectively prevent the occurrence of watermelon wilt disease, and the effect of microemulsion is better than that of aqueous solution.

[0086] Example 10: Plant experiment on the control of powdery mildew in pumpkin

[0087] Experimental reagents: The aqueous agent 2 and microemulsion 2 described in the examples were diluted with water to a hydroxydecanoic acid content of 1000 mg / L.

[0088] Drug comparison: Difenoconazole 100mg / L, using a commercially available formulation and converted to the content of active ingredient; M-PEDE, diluted with water to a fatty acid content of 1000mg / L;

[0089] Disease control: Shimizu

[0090] Pumpkin Variety: Beibei Pumpkin Huihe No. 1 Treatment Method: For healthy potted pumpkin plants that have developed to the four-leaf stage, select plants with uniform growth and spray them for control. Three days later, inoculate with fresh powdery mildew spores (collected from diseased pumpkin leaves, centrifuged, counted under a microscope, and adjusted to a final concentration of 10 with physiological saline). 4 The inoculation volume was approximately 5 mL per plant (number of plants per mL). Each treatment was inoculated with 30 plants, and each treatment was replicated 3 times. After inoculation, the plants were kept in a high temperature and high humidity environment (28-30℃, humidity >90%), and watered and fertilized normally for another 15 days.

[0091] The disease index is classified into 5 levels. Level 0: No symptoms; Level 1: Lesions cover less than 1 / 5 of the leaf area; Level 2: Lesions cover 1 / 5 to 1 / 3 of the leaf area; Level 3: Lesions cover 1 / 3 to 1 / 2 of the leaf area; Level 4: Lesions cover 1 / 2 to 2 / 3 of the leaf area; Level 5: Lesions cover more than 2 / 3 of the leaf area, and the leaves turn yellow and withered.

[0092] The disease index is calculated as follows: Disease index = Σ(number of diseased leaves at each level × level) / (total number of leaves × 5) × 100.

[0093] The formula for calculating the incidence rate is as follows: Incidence rate % = Number of infected ears / Total number of ears × 100%

[0094] The formula for calculating the efficacy is as follows: Efficacy % = (1 - Disease index of the treatment group / Disease index of the water group) × 100%

[0095] Table 10 Experiments on the control of powdery mildew in pumpkin by spraying different agents.

[0096]

[0097] The results are shown in Table 10. It is evident that aqueous formulation 2 and microemulsion 2 are more effective against powdery mildew infection in pumpkins than difenoconazole, a commonly used pesticide for powdery mildew control, and microemulsion 2 is more effective than aqueous formulation 2. The comparative formulation M-PEDE, however, showed almost no effect in preventing powdery mildew.

[0098] Example 11: Field trial for the control of rice blast disease

[0099] Experiment location: Fuyang District, Hangzhou City, Zhejiang Province

[0100] Rice variety: Zhejing 27

[0101] Experimental reagent: Microemulsion 2 described in Example 1, diluted with water to a hydroxydecanoic acid content of 500 mg / L and 1000 mg / L.

[0102] Comparison of pesticides: Isoprothiolane 500 mg / L; Tricyclazole 200 mg / L; Both pesticides were commercially available wettable powders and their active ingredient content was converted.

[0103] Disease control: Shimizu

[0104] Processing area: Each treatment plant consists of 3 cells, each cell is 3 square meters. 2 The spacing between the cells is 0.5m.

[0105] Treatment method: For rice in the early flowering stage, spray with a diluted pesticide at a rate of 30L per acre. After 24 hours, inoculate with rice blast fungus spores (final concentration 10). 4 The inoculation amount per ear is approximately 1 mL (each ear is inoculated with one inoculated cell per mL).

[0106] Results: 30 days after inoculation with rice blast fungus spores, photos of rice panicles in each group are shown below. Figure 2 As shown; and 200 spikelets were randomly surveyed in each plot. A 4-level classification was used based on the disease index. Level 0: Asymptomatic; Level 1: 1 / 4 of spikelets were diseased; Level 2: 2 / 4 of spikelets were diseased; Level 3: 3 / 4 of spikelets were diseased; Level 4: The entire spikelet was diseased or even died.

[0107] The disease index is calculated as follows: Disease index = Σ(Grade number × Number of ears in that grade) / (Total number of seedlings × 4) × 100.

[0108] The formula for calculating the incidence rate is as follows: Incidence rate % = Number of infected ears / Total number of ears × 100%

[0109] The formula for calculating the efficacy is as follows: Efficacy % = (1 - Disease index of the treatment group / Disease index of the water group) × 100%

[0110] Table 11 Field trials of spraying different pesticides to control rice blast

[0111]

[0112]

[0113] The results are shown in Table 11. It can be seen that microemulsion 2 has a higher efficacy against rice blast fungus infection than the commonly used pesticides isoprothiolane and tricyclazole for rice blast control, and the effect is better at higher concentrations.

[0114] Example 12: Field trial of treatment for powdery mildew in pumpkin

[0115] Experimental location: Fuyang District, Hangzhou City, Zhejiang Province. Experimental reagents: Aqueous agent 2 and microemulsion 2 as described in the examples, diluted with water to a hydroxydecanoic acid content of 3000 mg / L;

[0116] Drug control: M-PEDE, diluted with water to a fatty acid content of 3000 mg / L;

[0117] Pumpkin variety: Japanese Red Honey. Treatment method: Eight pumpkin plots, each with an area of ​​40m², were selected from the field and found to be infected with powdery mildew. 2 Two plots were sprayed with 2 liters of aqueous solution, two plots with 2 liters of microemulsion, and two plots with M-PEDE (5 L each). The other two plots were sprayed with an equal amount of water. The spraying was repeated once after 7 days. Normal watering and fertilization were maintained after spraying, and the plants were cultivated for another 14 days. One hundred typical leaves were selected from each plot before and after spraying to calculate the disease index.

[0118] The disease index is classified into 5 levels. Level 0: No symptoms; Level 1: Lesions cover less than 1 / 5 of the leaf area; Level 2: Lesions cover 1 / 5 to 1 / 3 of the leaf area; Level 3: Lesions cover 1 / 3 to 1 / 2 of the leaf area; Level 4: Lesions cover 1 / 2 to 2 / 3 of the leaf area; Level 5: Lesions cover more than 2 / 3 of the leaf area, and the leaves turn yellow and withered.

[0119] The disease index is calculated as follows: Disease index = Σ(number of diseased leaves at each level × level) / (total number of leaves × 5) × 100.

[0120] The formula for calculating treatment efficacy is as follows: Treatment efficacy % = (1 - Disease index of the treatment group / Disease index of the water group) × 100%

[0121] Table 12 Field Trial of Spraying Aqueous Agent for Controlling Powdery Mildew of Pumpkin

[0122]

[0123] Figure 3 The table shows photos of field trials of plant fungicide for inhibiting powdery mildew. Table 12 presents the statistical results, showing that aqueous solution 2 and microemulsion 2 effectively inhibited the spread of powdery mildew and significantly reduced the disease index. This plant fungicide not only prevents powdery mildew but also controls existing powdery mildew fungi, reducing damage to plants. While M-PEDE also showed some therapeutic effect, it was less effective.

[0124] Example 13: Field trial of rice blast treatment

[0125] Experiment location: Fuyang District, Hangzhou City, Zhejiang Province

[0126] Rice variety: Zhejing 27

[0127] Experimental reagent: Microemulsion 2 as described in Example 1, diluted with water to a hydroxydecanoic acid content of 1000 mg / L.

[0128] Pesticide comparison: 500 mg / L of isoprothiolane was used, and the content of the active ingredient was converted from the commercially available wettable powder.

[0129] Treatment area: Each treatment area consists of 3 plots, each plot covering 0.3 acres. The plots are spaced 0.5 meters apart.

[0130] Treatment method: Rice plants in the late flowering stage were found to be infected with rice blast in the field, with approximately 5% of the plants showing symptoms. At this time, the control group was sprayed with isoprothiolane, while the experimental group was sprayed with microemulsion 2, using the conventional field application rate (20L per acre). A second application was made 15 days after the first application, for a total of two applications.

[0131] Results calculation: 30 days after the second spraying, 200 spikelets were randomly surveyed from each plot. Disease index was used to classify spikelets into four levels: Level 0: Asymptomatic; Level 1: Diseased spikelets comprise 1 / 4 of the total spikelets; Level 2: Diseased spikelets comprise 2 / 4 of the total spikelets; Level 3: Diseased spikelets comprise 3 / 4 of the total spikelets; Level 4: Entire spikelet is diseased or even dies.

[0132] The disease index is calculated as follows: Disease index = Σ(Grade number × Number of ears in that grade) / (Total number of seedlings × 4) × 100.

[0133] The formula for calculating the incidence rate is as follows: Incidence rate % = Number of infected ears / Total number of ears × 100%

[0134] Table 13 Field trials of spraying different agents to treat rice blast

[0135]

[0136] The results are shown in Table 13. It can be seen that microemulsion 2 is slightly more effective in treating rice blast fungus infection than isoprothiolane, a commonly used pesticide for rice blast control. It effectively inhibits the development of rice blast and avoids losses.

[0137] Example 14: In vitro experiment on the control of wheat scab

[0138] Experimental reagents: Aqueous agent 2 and microemulsion 2 described in the examples were diluted with water to a hydroxydecanoic acid content of 800 mg / L.

[0139] Comparison of agents: Cyazofamid 800 mg / L; Carbendazim 800 mg / L; Both agents were commercially available wettable powders and converted to active ingredient content.

[0140] Disease control: Shimizu

[0141] Wheat variety: Jinchun 6. Treatment method: For wheat in the flowering stage, select uniformly growing ears, cut them about 10cm below the flag leaf node, remove the flag leaf, and insert them into sterile water for spraying. After 24 hours, inoculate with Fusarium graminearum spores (final concentration 10). 4 The inoculation volume was approximately 1 mL per spike (number of spikes per mL). Each treatment was inoculated with 20 spikes, with 3 replicates. After inoculation, the spikes were bagged and kept moist at 25°C for 3 days, followed by hydroponics at 25°C for 7 days.

[0142] The disease index is classified into four levels: Level 0: No symptoms; Level 1: 1 / 4 of the spikelets are affected; Level 2: 2 / 4 of the spikelets are affected; Level 3: 3 / 4 of the spikelets are affected; Level 4: The entire spikelet is affected or even dies. The disease index is calculated as follows: Disease Index = Σ(Level number × Number of spikelets in that level) / (Total number of seedlings × 4) × 100.

[0143] The formula for calculating the incidence rate is as follows: Incidence rate % = Number of infected ears / Total number of ears × 100%

[0144] The formula for calculating the efficacy is as follows: Efficacy % = (1 - Disease index of the treatment group / Disease index of the water group) × 100%

[0145] Table 14 In vitro experiments on the control of wheat scab by spraying different pesticides

[0146]

[0147] The results are shown in Table 14. It can be seen that the aqueous solution 2 and microemulsion 2 of the present invention have higher efficacy against Fusarium head blight infection than the commonly used pesticides cyazofamid and carbendazim.

[0148] Example 15: Field trial for the control of wheat scab

[0149] Experiment location: Fuyang District, Hangzhou City, Zhejiang Province

[0150] Wheat variety: Jinchun No. 6; Experimental agent: Microemulsion 2 as described in Example 1, diluted with water to a hydroxydecanoic acid content of 800 mg / L.

[0151] Comparison of fungicides: Carbendazim 800mg / L, using commercially available wettable powder, and converted to the content of active ingredient.

[0152] Treatment area: Each treatment area consists of 3 plots, each plot covering 0.3 acres. The plots are spaced 0.5 meters apart.

[0153] Treatment method: Wheat in the grain-filling stage was found to be infected with Fusarium head blight in the field, with approximately 3.5% of the plants affected. At this time, the control group was sprayed with carbendazim, while the experimental group was sprayed with microemulsion 2, using the conventional field application rate (20L per acre). A second application was made 20 days after the first application, for a total of two applications.

[0154] Results Calculation: Twenty days after the second spraying, 200 spikelets were randomly surveyed from each plot. Disease index was used to classify spikelets into four levels: Level 0: Asymptomatic; Level 1: Diseased spikelets comprise 1 / 4 of the total spikelets; Level 2: Diseased spikelets comprise 2 / 4 of the total spikelets; Level 3: Diseased spikelets comprise 3 / 4 of the total spikelets; Level 4: Entire spikelet is diseased or even dies.

[0155] The disease index is calculated as follows: Disease index = Σ(Grade number × Number of ears in that grade) / (Total number of seedlings × 4) × 100.

[0156] The formula for calculating the incidence rate is as follows: Incidence rate % = Number of infected ears / Total number of ears × 100%

[0157] Table 15 Field trials of spraying different agents to treat wheat scab.

[0158]

[0159] The results are shown in Table 15. It can be seen that microemulsion 2 has a better therapeutic effect on wheat scab than the commonly used pesticide carbendazim.

[0160] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. The application of a fungicide for plant diseases, characterized in that, Used to prevent and control rice blast fungus ( Pyricularia oryzae Rice blast caused by Fusarium graminearum (Fusarium graminearum) Fusarium graminearum Wheat scab caused by Fusarium oxysporum and Fusarium oxysporum (… Fusarium oxysporum Watermelon wilt caused by ) The plant disease fungicide contains one of medium-chain 3-hydroxy fatty acids or potassium salts of medium-chain 3-hydroxy fatty acids; the medium-chain 3-hydroxy fatty acid is one of 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid, 3-hydroxylauric acid, and 3-hydroxytetradecanoic acid.

2. The application according to claim 1, characterized in that, The medium-chain 3-hydroxy fatty acid is 3-hydroxydecanoic acid.

3. The application according to claim 1, characterized in that, The formulation of the plant disease fungicide is an emulsion, microemulsion, aqueous solution, or soluble powder.

4. The application according to claim 3, characterized in that, The emulsion is made from the following raw materials by weight percentage: 10-45% medium-chain 3-hydroxy fatty acids, 1-10% oily solvent, 1-10% surfactant, and the balance being water and pH adjuster; the oily solvent is one or more of soybean oil, oleic acid, and linoleic acid.

5. The application according to claim 3, characterized in that, The microemulsion is made from the following raw materials by weight percentage: 1-10% medium-chain 3-hydroxy fatty acids, 1-10% polyols, 5-20% surfactants, and the balance being water and pH adjusters; the polyols are one or more of propylene glycol, ethylene glycol, and glycerol.

6. The application according to any one of claims 4-5, characterized in that, The surfactants in the water emulsions and microemulsions are one or more of the following: alkylphenol polyoxyethylene ethers, fatty alcohol polyoxyethylene ethers, alkyl glycosides, sucrose fatty acid esters, sorbitol fatty acid esters and fatty acid glycerides, alkylbenzene sulfonates, alkyl sulfonates, fatty acid sulfonyl esters, alkyl glycerol ether sulfonates, fatty alcohol polyoxyethylene, higher fatty acid salts, and fatty alcohol sulfate salts.

7. The application according to claim 3, characterized in that, The aqueous solution is made from the following raw materials by weight percentage: 1-45% potassium salt of medium-chain 3-hydroxy fatty acids, 0.1-5% thickener, and the balance being water and pH adjuster; the pH value of the aqueous solution is 5-9.

8. The application according to claim 3, characterized in that, The soluble powder is made from the following raw materials by weight percentage: 90-95% potassium salt of medium-chain 3-hydroxy fatty acids, with the remainder being a thickener.

9. The application according to claim 7 or 8, characterized in that, The thickener is one or more of xanthan gum, carboxymethyl cellulose, or soluble starch.

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