Compound composition of cumic acid and patchouli ketone, bactericide and application

Abstract

The invention provides a compound composition containing cumic acid and patchouli ketone, a bactericide and application of the compound composition and the bactericide, and the mass ratio of the cumic acid to the patchouli ketone is (8-1): (1-1). Cumic acid and patchoulenone are compounded, so that various melon fusarium wilt can be prevented and treated, and gray mold, powdery mildew and the like of various crops can be prevented and treated. The binary compound agent has a remarkable synergistic effect on preventing and treating the diseases, the use amount of a single agent can be remarkably reduced, and the compound agent is expected to be developed into a new generation of green agricultural botanical fungicide.

Description

Technical Field

[0001] This invention belongs to the field of agricultural fungicide technology, and specifically discloses a synergistic fungicide composition with cuminic acid and patchouli ketone as active ingredients, its preparation method and application. Background Technology

[0002] Crop diseases pose a significant challenge to global food security, causing direct economic losses of tens of billions of dollars annually. Currently, chemical fungicides remain the primary means of plant disease control, but their long-term reliance on them has led to increasingly prominent dual problems: on the one hand, pathogen resistance is continuously increasing, significantly reducing control effectiveness; on the other hand, food safety and environmental issues caused by pesticide residues are receiving considerable attention. Under my country's dual strategic guidance of "food security" and "green development," developing highly efficient, low-toxicity, and environmentally friendly new green fungicides has become an important direction for agricultural science and technology innovation.

[0003] Cuminic acid, also known as p-isopropylbenzoic acid, is the core active ingredient in cumin seed oil and possesses an extremely broad range of antibacterial activity. Under in vitro experimental conditions, cuminic acid exhibits extremely strong inhibitory capabilities against a large number of common plant pathogens, including Sclerotinia sclerotinia, Phytophthora bovis, Fusarium wilt, and rice blast fungus, effectively inhibiting their growth and reproduction. In pot experiments, when plants face disease threats, cuminic acid not only provides excellent preventative protection against various plant diseases before they occur, reducing the chance of infection, but also demonstrates outstanding therapeutic effects after disease onset, significantly alleviating symptoms and helping plants recover healthy growth.

[0004] Pogostone is a natural product with broad-spectrum antibacterial activity, belonging to the 2-pyranone class of natural compounds. As the main component of patchouli essential oil, pogostone has certain preventative effects against common agricultural diseases. Previous studies have shown that pogostone has good inhibitory effects on *Pestalotia eugenae* Pk., *Alternaria solani* (early blight pathogen of tomato), and *Sclerotinia sclorotiorum*, while also exhibiting an inhibitory effect on *Fusarium oxysporum* (MIC value of 8 μg / mL). It also shows good antibacterial activity against the bacterial plant disease *Ralstonia solanacearum*, with a MIC value of 50 μg / mL. Current research on pogostone and its derivatives in agriculture mainly focuses on preliminary studies of their antibacterial activity, with limited research on their antibacterial mechanisms. Medical research has shown that patchouli ketones possess a wide range of biological activities, including anticancer, anti-inflammatory, and antioxidant effects, and have significant development value. Summary of the Invention

[0005] The purpose of this invention is to provide a binary compound of cuminic acid and the natural product patchouli ketone, which can be used to control a variety of crop diseases such as watermelon wilt, powdery mildew and gray mold in various crops.

[0006] To achieve the above objectives, the technical solution adopted by the present invention includes:

[0007] A compound composition of cucurbitacin and patchouli, wherein, by mass ratio, cucurbitacin:patouli = (8-1):1.

[0008] Optionally, by mass ratio, cuminic acid:patchouli ketone = (6-2):1.

[0009] Optional, by mass ratio, cuminic acid:patchool = 4:1.

[0010] The compound composition of cuminic acid and patchouli ketone described in any one of the present invention is used in the preparation of plant fungicides.

[0011] Optionally, the bactericide contains 1% to 30% cucurbitacin and 1% to 30% patchouli ketone by weight percentage.

[0012] Optionally, the plant fungicide is used to control watermelon wilt, grape gray mold, and / or wheat powdery mildew.

[0013] A bactericide composed of cuminic acid and patchouli ketone, wherein the effective component of the bactericide is any of the compound compositions of cuminic acid and patchouli ketone described in this invention;

[0014] The bactericide contains 1% to 30% cucurbitacin and 1% to 30% patchouli ketone by weight percentage.

[0015] Optionally, the bactericide is an emulsion containing water, with a mass percentage of:

[0016] Cuminic acid 1%–30%, patchouli 1%–30%, solvent 1%–10%, emulsifier 1%–10%, dispersant 1%–5%, antifreeze 1%–5%, preservative 0.5%–4%, water to 100%;

[0017] The dispersant is polyvinyl alcohol, MNO, xanthan gum, or magnesium aluminum silicate.

[0018] The solvent is at least one selected from ethyl acetate, cyclohexanone, isoamyl citrate, furfural, 5-methylfurfural, ethanol, and ethyl acetate;

[0019] The antifreeze is sodium nitrite, calcium chloride, ethylene glycol, propylene glycol, glycerin, or urea;

[0020] The preservative is potassium sorbate, benzoic acid, or benzaldehyde;

[0021] The emulsifier is at least one selected from alkyl polyoxyethylene ether, 602, agricultural emulsion 500, NP-10, fatty acid polyoxyethylene ester, TX-10, 4103, OP-10, Well205 and M30.

[0022] Optionally, the bactericide is a soluble liquid with a mass percentage of:

[0023] Cuminic acid 1%–30%, patchouli 1%–30%, emulsifier 5%–25%, antifreeze 1%–5%, solvent to 100%;

[0024] The emulsifier is at least one of OP-10, 2201, Tween 80, agricultural emulsion 500, agricultural emulsion 600, agricultural emulsion BY, agricultural emulsion EL and NP-30;

[0025] The solvent is at least one selected from ethanol, ethyl acetate, N,N-dimethylacetamide, N,N-dimethylformamide, dimethyl sulfoxide, sec-juniper acetate, furfural, and 5-methylfurfural;

[0026] The antifreeze is sodium nitrite, propylene glycol, glycerin, or urea.

[0027] Compared with the prior art, the advantages of the present invention are:

[0028] Systematic formulation studies of this invention have shown that when cucurbitacin and patchouli are combined in a mass ratio of 8:1 to 1:1, the synergistic effect coefficient exceeds the 1.5 threshold, exhibiting significant synergistic properties. This binary compound agent not only has excellent control effects on target diseases but also effectively reduces the amount of single agent used, meeting the needs of green agricultural development and possessing great potential for development into a new generation of plant-derived agricultural fungicides.

[0029] It is noteworthy that the China Pesticide Information Network has registered very few pesticides for the control of watermelon wilt, highlighting the severe shortage of pesticide resources in this field. This compound formulation, however, has demonstrated excellent performance in watermelon wilt control trials, possessing extremely high development value and the potential to fill a market gap, becoming a new option for pesticide registration in the field. Detailed Implementation

[0030] To more intuitively and comprehensively demonstrate the research and development objectives, technical solutions, and significant advantages of this invention, a detailed description is provided below in conjunction with specific embodiments. It should be emphasized that the following embodiments are only some typical applications of the invention's technical solutions, and not all implementation methods. Other implementation methods derived from the embodiments of this invention that do not involve inventive effort are all within the scope of patent protection of this invention.

[0031] This invention is the first to discover that when cumin and patchouli are combined in a specific ratio, they exhibit significant synergistic control effects against various plant diseases, including watermelon wilt (Fusarium oxysporum f.sp. niveum), wheat powdery mildew (Podosphaera xanthii), and gray mold (Botrytis cinerea), providing a novel, highly efficient, low-toxicity, and environmentally friendly fungicide solution for plant disease control.

[0032] This invention provides a compound fungicide of cucurbitacin and patchouli ketone. By scientifically combining cucurbitacin and patchouli ketone at a mass ratio of 8:1 to 1:1, a high-performance formulation is formed. Extensive testing has verified that this formulation not only has a significant control effect on watermelon wilt but is also effective against powdery mildew and gray mold in various crops. In practical applications, this binary compound agent exhibits excellent synergistic effects, significantly reducing the dosage compared to single agents, ensuring control efficacy while aligning with the concept of green agriculture. Based on its superior performance, this compound formulation has great potential to be developed into a new generation of green agricultural plant-derived fungicides, providing a highly efficient and environmentally friendly new solution for crop disease control.

[0033] For example, bactericides can be formulated as water-in-oil emulsions and soluble liquids;

[0034] The components and weight percentages of the water-in-oil emulsion are as follows: cuminic acid 1%–30%, patchouli 1%–30%, solvent 1%–10%, emulsifier 1%–10%, dispersant 1%–5%, antifreeze 1%–5%, preservative 0.5%–4%, water to 100%; polyvinyl alcohol, MNO, xanthan gum, or magnesium aluminum silicate are used as dispersants; N,N-dimethylacetamide, ethyl acetate, cyclohexanone, isoamyl citrate, furfural, 5- At least one or more of methylfurfural, ethanol, and ethyl acetate are used as solvents; sodium nitrite, calcium chloride, ethylene glycol, propylene glycol, glycerin, or urea are used as antifreeze agents; potassium sorbate, benzoic acid, or benzaldehyde are used as preservatives; and one or more of alkyl polyoxyethylene ethers, 602, agricultural emulsion 500, NP-10, fatty acid polyoxyethylene esters, TX-10, 4103, OP-10, Well205, and M30 are used as emulsifiers.

[0035] The components and weight percentages of the soluble liquid are as follows: 1%–30% cuminic acid, 1%–30% patchouli ketone, 5%–25% emulsifier, 1%–5% antifreeze, and solvent to bring the total to 100%; at least one or more of OP-10, 2201, Tween 80, agricultural emulsion 500, agricultural emulsion 600, agricultural emulsion BY, agricultural emulsion EL, and NP-30 are used as emulsifiers; at least one or more of ethanol, ethyl acetate, N,N-dimethylacetamide, N,N-dimethylformamide, dimethyl sulfoxide, sec-juniper acetate, furfural, and 5-methylfurfural are used as solvents; the antifreeze is one or more of sodium nitrite, propylene glycol, glycerol, or urea.

[0036] Example 1: Virulence of cumin and patchouli against Fusarium wilt of watermelon

[0037] To accurately determine the virulence of cucurbitacin and patchouli ketone against pathogens, this study employed the mycelial growth rate method. The specific operational steps are as follows:

[0038] First, using methanol as a solvent, cuminic acid and patchouli technical grade drugs were dissolved separately to prepare a series of stock solutions with varying concentrations. Then, the stock solutions were precisely measured using a pipette and added to PDA medium. After thorough mixing, medium containing different drug concentrations was prepared.

[0039] Next, a 0.5 cm diameter mycelial disc was carefully selected from the edge of the cultured pathogen colony and placed in the center of the drug-containing culture medium. After inoculation, the petri dishes were uniformly placed in a 25°C incubator for cultivation. During the cultivation process, the colony diameter was measured periodically using the cross-sectional method, and the inhibition rate was calculated based on the measurement data. Finally, the experimental data were systematically analyzed using DPS software to calculate the effective inhibitory concentration (EC50). 50 ).

[0040] Table 1. Virulence determination of different ratios of cumin and patchouli against Fusarium wilt of watermelon.

[0041]

[0042] Table 2 shows that cucurbitacin and patchouli have different EC50-like effects on Fusarium wilt pathogens of watermelon. 50 The concentrations were 45.091 μg / mL and 19.176 μg / mL, respectively, indicating that cucurbitacin and patchouli both had good antibacterial activity against Fusarium wilt of watermelon.

[0043] Example 2: Synergistic effect of different ratios of cuminic acid and patchouli ketone

[0044] To systematically determine the virulence of cucurbitacin and patchouli ketone against pathogens at different ratios, this study designed and conducted the following experiments based on the mycelial growth rate method:

[0045] Preparation of stock solutions: Using methanol as a solvent, dissolve cuminic acid and patchouli technical materials separately to prepare stock solutions of the same concentration to ensure consistency of experimental standards.

[0046] Mixing and Proportioning: According to the preset experimental plan, the two mother liquors are precisely mixed in different volume ratios to prepare a series of compound solutions with different proportions, thus constructing a gradient of experimental samples.

[0047] Preparation of drug-containing culture medium: Following the procedure in Example 1, each ratio of compound solution was accurately measured using a pipette and added to the PDA culture medium. After thorough mixing, culture medium containing different concentration gradients of drugs was obtained.

[0048] Inoculation and culture: Select a mycelial disc approximately 0.5 cm in diameter from the edge of the cultured pathogen colony and aseptically transfer it to the center of a PDA plate containing compound agents in different proportions. Place the inoculated plate in a 25°C incubator and incubate for 2 days.

[0049] Data Collection and Analysis: When the colony diameter in the blank control group grew to more than 2 / 3 of the petri dish diameter, the diameter of all colonies was measured using the cross-multiplication method. Three biological replicates were performed for each treatment group to ensure data reliability. Based on the measurement data, the mycelial growth inhibition rate of each treatment group was calculated. Finally, DPS software was used for statistical analysis to obtain the effective inhibition concentration (EC50).

[0050] The synergistic effect coefficient (SYnergY ratio, SR) of compound drugs is calculated using the Wadle Y method according to the following formula. When SR is greater than 1.5, it indicates a synergistic effect; SR between 0.5 and 1.5 indicates an additive effect; and SR less than 0.5 indicates an antagonistic effect (a and b are the proportions of cuminic acid and patchouli in the compound, EC...). 50 (A) and EC 50 (B) is the effective medium concentration of cumin and patchouli ketone, EC 50 (EXp) is the theoretical inhibition concentration, EC 50 (Obs) represents the actual measured concentration of inhibition.

[0051]

[0052] Table 2. Virulence determination of different ratios of cumin and patchouli against Fusarium wilt of watermelon.

[0053]

[0054]

[0055] Based on the data in Table 2, it is clear that the toxicity of cucurbitacin and patchouli against *Fusarium wilt* fungus varies significantly depending on their ratio. The calculated synergistic coefficients range from 0.887 to 1.993. Notably, all the ratios tested exhibited synergistic coefficients higher than 0.5. This strongly suggests that under the various ratios set in this experiment, the combined use of cucurbitacin and patchouli demonstrates an additive effect. In particular, when the ratio of cucurbitacin to patchouli is between 8:1 and 1:1, the synergistic coefficients are all greater than 1.5. This data intuitively illustrates that, under the aforementioned specific ratios, the combined effect of cucurbitacin and patchouli against *Fusarium wilt* fungus is not merely additive, but exhibits a highly significant synergistic effect. Compared to using cucurbitacin or patchouli alone, it more effectively inhibits the growth and reproduction of *Fusarium wilt* fungus, providing a valuable reference for subsequent applications and research.

[0056] Example 3: Production of an aqueous emulsion of a composition of 10% cuminic acid and 10% patchouli ketone

[0057] Accurately weigh 102 kg of 98% cucurbitacin and 102 kg of 98% patchouli ketone. Place the two active ingredients in a 2M container. 3 Mix thoroughly in a mixing tank. Accurately weigh 100 kg of isoamyl citrate and 10 kg of sodium benzoate, add them to the mixing tank, and mix thoroughly again. Then accurately add 95 kg of emulsifier OP-10 to the mixing tank, followed by 10 kg of gum arabic and 15 kg of magnesium aluminum silicate. Add water to bring the total volume to 1000 kg, and mix thoroughly in the mixing tank. Continue stirring for 1 hour. A water-in-oil emulsion product of 1 ton of cuminic acid and patchouli ketone composition is prepared.

[0058] Sampling and analysis revealed that the content of cumin was 10% and the content of patchouli ketone was 10%. The product was dispensed into 500mL double-barrier glass bottles, sealed, and labeled to obtain a 20% composition water-in-oil emulsion product.

[0059] Example 4: Production of a soluble liquid formulation of a composition of 10% cuminic acid and 10% patchouli ketone

[0060] Accurately weigh 102 kg of 98% cucurbitacin and 102 kg of 98% patchouli ketone. Place the two active ingredients in a 2M container. 3The mixture was thoroughly mixed in a mixing tank. The premixed active ingredient mixture was slowly added to 676 kg of ethanol solvent while continuously stirring until the active ingredients were completely dissolved, forming a homogeneous and transparent solution. Then, 100 kg of OP-10 emulsifier was slowly added to the completely dissolved solution, along with 20 kg of sodium nitrite antifreeze. The mixture was continuously stirred to ensure uniform dispersion. After the emulsifier was added, the stirring speed was gradually increased to high speed and emulsification was performed for 30 minutes. High-speed stirring formed a stable emulsion system, uniformly dispersing the active ingredients in the solvent to obtain a soluble liquid with good stability and flowability. One ton of soluble liquid product containing the combination of cuminic acid and patchouli ketone was prepared.

[0061] Sampling and analysis revealed that the content of cumin was 10% and the content of patchouli ketone was 10%. The product was dispensed into 500mL double-barrier glass bottles, sealed, and labeled to obtain a 20% soluble liquid product containing the composition.

[0062] Example 5: The control efficacy of a 20% water-in-oil emulsion and soluble liquid formulation of cumin and patchouli against watermelon wilt and grape gray mold.

[0063] The efficacy of 20% water-in-oil emulsion and soluble liquid formulations against watermelon wilt and grape gray mold was determined using a greenhouse pot experiment. Three concentrations of each test agent were set up. Bacillus amyloliquefaciens water-dispersible granules served as a positive control for watermelon wilt, and 5% calciferic acid aqueous solution served as a positive control for watermelon gray mold. Each treatment was replicated in 5 pots. Watermelon wilt inoculation and disease index surveys were conducted according to the literature reported by Luo Man et al.

[0064] Table 3. Efficacy of the compound preparation of cumin and patchouli against watermelon wilt disease.

[0065]

[0066] Note: According to Fisher's minimum significance test, different letters after the values ​​in the same column indicate that there is a significant difference between the values ​​(P = 0.05).

[0067] Based on the data in Table 3, the results of the greenhouse pot experiment showed that the water-in-oil emulsion and soluble liquid formulation of the compound formulation of cumin and patchouli exhibited good control effects against watermelon wilt. Regarding concentration, when the effective ingredient concentration of the compound formulation was 100 μg / mL, its control effect against watermelon wilt was not significantly different from the control agent. However, when the concentration was increased to 200 μg / mL, the control effect of the compound formulation against watermelon wilt significantly surpassed that of the control agent. From the perspective of formulation, under the same dosage conditions, the soluble liquid formulation of the compound formulation showed slightly better control effects than the water-in-oil emulsion. This result provides an important basis for the selection of concentration and formulation considerations for the subsequent application of this compound formulation in the actual control of watermelon wilt.

[0068] In the study of the efficacy of grape gray mold control, grapevines in the late seedling stage and early flowering stage were selected. A concentration of 5×10⁻⁶ was used. 6 A CFU / mL suspension of *Botrytis cinerea* spores was evenly sprayed onto grape leaves using a backpack sprayer. Forty-eight hours after inoculation, different treatment groups were sprayed with the corresponding fungicide using the same backpack sprayer. Six grapevines were selected for each treatment, with three replicates. Five to seven days after inoculation, ten leaves were collected from each grapevine from top to bottom. The disease incidence on the leaves was investigated and recorded strictly according to the GB / T17980.28-2000 disease classification standard to evaluate the control efficacy of different fungicides against grape gray mold.

[0069] Table 4. The efficacy of the compound of cumin and patchouli against Grape Botrytis cinerea.

[0070]

[0071] Note: According to Fisher's minimum significance test, different letters following the values ​​in the same column indicate significant differences between the values ​​(P = 0.05). Control efficacy (%) = (control lesion diameter - treated lesion diameter) / (control lesion diameter) * 100.

[0072] Based on the data in Table 4, it is clear that the compound preparation of cumin and patchouli is highly effective in controlling grape gray mold. When the dosage of the compound preparation is 50 μg / mL, its control effect exceeds 50%, demonstrating good disease inhibition ability.

[0073] The advantages become more pronounced with increasing concentration. At a concentration of 200 μg / mL, the control effect of the compound formulation is higher than that of the control agent. Regarding formulation differences, under the same dosage conditions, the control effect of the soluble liquid formulation in the compound formulation is also slightly higher than that of the water-in-oil emulsion.

[0074] Example 6: The efficacy of a 20% water-in-oil emulsion and soluble liquid formulation of cuminic acid and patchouli against wheat powdery mildew.

[0075] The efficacy of compound 20% water-in-oil emulsion and soluble liquid formulations against wheat powdery mildew was studied. Three concentrations of the tested agents were set at 100, 200, and 400 μg / mL. 12.5% ​​tebuconazole EC was selected as a positive control for controlling wheat powdery mildew. Each concentration treatment was replicated in quadruplicate, with 50 leaves randomly selected from each replicate, for a total of 200 leaves investigated.

[0076] Table 5. Efficacy of the compound preparation of cumin and patchouli against wheat powdery mildew.

[0077]

[0078] Note: According to Fisher's minimum significance test, different letters after the values ​​in the same column indicate that there is a significant difference between the values ​​(P = 0.05).

[0079] Table 5 clearly shows that the compound formulation of cumin and patchouli exhibits good efficacy in controlling wheat powdery mildew. When the dosage of the compound formulation is 100 μg / mL, the control efficacy of different formulations all exceeds 55%; increasing the dosage to 200 μg / mL further improves the control efficacy, exceeding 70%. When the concentration of the compound formulation reaches 400 μg / mL, its control efficacy is even more significant, exceeding 75%, and at this point, there is no significant difference compared to the control agent. Regarding the comparison of formulations, under the same dosage conditions, the control efficacy of the soluble liquid and water-in-oil formulations of the compound formulation is basically equivalent, without showing any obvious regular differences.

[0080] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A compound composition of cuminic acid and patchouli ketone, characterized in that, By mass ratio, cucurbitacin:patchouli = (8-1):

1.

2. The compound composition of cuminic acid and patchouli ketone according to claim 1, characterized in that, By mass ratio, cucurbitacin:patchouli = (6-2):

1.

3. The compound composition of cuminic acid and patchouli ketone according to claim 1, characterized in that, By mass ratio, cucurbitacin:patchouli = 4:

1.

4. The application of the compound composition of cuminic acid and patchouli as described in any one of claims 1-3 in the preparation of plant fungicides.

5. The application according to claim 4, characterized in that, The bactericide contains 1% to 30% cucurbitacin and 1% to 30% patchouli ketone by weight percentage.

6. The application according to claim 4, characterized in that, The plant fungicide is used to control watermelon wilt, grape gray mold and / or wheat powdery mildew.

7. A bactericide composed of cumin and patchouli, characterized in that, The active ingredient of the bactericide is a compound composition of cuminic acid and patchouli as described in any one of claims 1-3; The bactericide contains 1% to 30% cucurbitacin and 1% to 30% patchouli ketone by weight percentage.

8. The bactericide compounded with cumin and patchouli ketone according to claim 7, characterized in that, The bactericide is an emulsion with a mass percentage of: Cuminic acid 1%–30%, patchouli 1%–30%, solvent 1%–10%, emulsifier 1%–10%, dispersant 1%–5%, antifreeze 1%–5%, preservative 0.5%–4%, water to 100%; The dispersant is polyvinyl alcohol, MNO, xanthan gum, or magnesium aluminum silicate. The solvent is at least one selected from ethyl acetate, cyclohexanone, isoamyl citrate, furfural, 5-methylfurfural, ethanol, and ethyl acetate; The antifreeze is sodium nitrite, calcium chloride, ethylene glycol, propylene glycol, glycerin, or urea; The preservative is potassium sorbate, benzoic acid, or benzaldehyde; The emulsifier is at least one selected from alkyl polyoxyethylene ether, 602, agricultural emulsion 500, NP-10, fatty acid polyoxyethylene ester, TX-10, 4103, OP-10, Well205 and M30.

9. The bactericide compounded with cumin and patchouli ketone according to claim 7, characterized in that, The bactericide is a soluble liquid with a mass percentage of: Cuminic acid 1%–30%, patchouli 1%–30%, emulsifier 5%–25%, antifreeze 1%–5%, solvent to 100%; The emulsifier is at least one of OP-10, 2201, Tween 80, agricultural emulsion 500, agricultural emulsion 600, agricultural emulsion BY, agricultural emulsion EL and NP-30; The solvent is at least one selected from ethanol, ethyl acetate, N,N-dimethylacetamide, N,N-dimethylformamide, dimethyl sulfoxide, sec-juniper acetate, furfural, and 5-methylfurfural; The antifreeze is sodium nitrite, propylene glycol, glycerin, or urea.

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