A bactericidal composition comprising d-limonene and allicin and uses thereof

Abstract

The application provides a bactericidal composition containing d-limonene and allicin, and active ingredients are composed of d-limonene and allicin, and the mass ratio of d-limonene and allicin is 30:1-1:30. The d-limonene can be used alone to prevent and treat rice sheath blight or tomato gray mold, and the d-limonene and the allicin have a significant synergistic effect when the d-limonene and the allicin are combined, and the field observation is safe to crops.

Description

Technical Field

[0001] This invention belongs to the field of pesticide technology, specifically relating to a bactericidal composition comprising d-limonene and allicin and its application. Background Technology

[0002] Chemical pesticides dominate the pesticide market due to their variety, rapid effectiveness, ease of use, and low price. However, the irrational use of chemical pesticides and their inherent drawbacks have led to environmental pollution, animal poisoning, and pesticide residues. With improved living standards and increased environmental awareness, the development of safe, pollution-free, low-toxicity, and low-residue biological pesticides has become a new research hotspot in the pesticide field. Plant-derived pesticides, as a part of bio-rational pesticides, have consistently been a hot topic in new pesticide research. Plant-derived pesticides originate from nature, degrade naturally, generally do not pollute the environment or agricultural products, and have a low probability of accumulating toxicity in the environment and human body. They are relatively safe for humans and livestock, cause minimal harm to natural enemies of pests, and are characterized by low toxicity and low residues, thus maintaining the high quality of agricultural products. Therefore, plant-derived pesticides have a broad market.

[0003] Limonene, also known as limonene, is a monoterpene compound and a common component of plant essential oils. It is mainly found in citrus fruits such as mandarins, sweet oranges, lemons, tangerines, grapefruits, and kumquats, and is also present in other plants such as peppermint, camphor, cedar oil, pine needles, and spearmint. d-Limonene is the dextrorotatory isomer of limonene. Current research on the agricultural fungicidal activity of d-limonene is relatively limited.

[0004] Allicin is a component extracted from the bulb of garlic. It is known as a natural broad-spectrum antibiotic and has been widely used to inhibit Staphylococcus aureus, Staphylococcus albus, and other pathogenic fungi. Studies have also reported that allicin has a certain inhibitory effect on pathogenic fungi such as Rhizoctonia solani, Fusarium moniliforme, Fusarium oxysporum, rice blast fungus, wheat sheath blight fungus, rapeseed sclerotinia, tomato gray mold, tomato leaf mold, and tomato early blight fungus.

[0005] Plant-derived bactericides are less harmful to natural enemies of pests and have the characteristics of low toxicity and low residue, which can maintain the high quality of agricultural products. However, the bactericidal activity of plant-derived bactericides is also relatively low, and they often need to be compounded to seek synergistic bactericidal compositions to improve bactericidal activity. Summary of the Invention

[0006] To address the problems existing in the prior art, the present invention provides the following technical solution:

[0007] A bactericidal composition comprising d-limonene and allicin, wherein the active ingredients are d-limonene and allicin, and the mass ratio of d-limonene to allicin is 30:1 to 1:30. Preferably, the mass ratio of d-limonene to allicin is 25:1 to 1:5; more preferably, the mass ratio of d-limonene to allicin is 25:1 to 5:1.

[0008] The fungicide composition contains pesticide adjuvant ingredients, with the active ingredient accounting for 1-30% by weight of the fungicide composition. Preferably, the pesticide adjuvant contains known excipients, such as emulsifiers, wetting agents, dispersants, organic solvents, cosolvents, water, preservatives, antifreeze agents, etc.

[0009] The bactericidal composition is formulated as an emulsifiable concentrate, microemulsion, water-in-oil emulsion, or soluble liquid.

[0010] The fungicidal composition described herein is used for the prevention and control of crop diseases, particularly for the prevention and control of rice sheath blight or tomato gray mold.

[0011] This invention also provides the use of d-limonene for the prevention and control of rice sheath blight or tomato gray mold.

[0012] By adopting the above technical solution, the present invention has the following beneficial effects:

[0013] There is limited research on the use of d-limonene for the control of crop pathogens. Research has found that d-limonene can be used to control rice sheath blight or tomato gray mold.

[0014] When the mass ratio of d-limonene to allicin is 25:1 to 1:5, the composition exhibits excellent control effect against rice sheath blight pathogen and has a significant synergistic effect.

[0015] When the mass ratio of d-limonene to allicin is 25:1 to 5:1, the composition exhibits excellent control effects against pathogens and has a significant synergistic effect.

[0016] The emulsifiable concentrate formulation prepared by this invention has excellent field efficacy, and field observations show that it is safe for crops. Detailed Implementation

[0017] The present invention will be further explained below with reference to specific embodiments.

[0018] Example 1: Indoor toxicity determination of d-limonene and allicin against rice sheath blight pathogen

[0019] Test pathogen: Rhizoctonia solani, the pathogen causing rice sheath blight.

[0020] Test method:

[0021] To prepare potato culture medium (PDA), weigh 200g of potatoes and place them in 1000mL of distilled water. Boil for 30 minutes, filter, add 20g of glucose and 20g of agar, melt them, and then add distilled water to make up to 1000mL. Dispense and sterilize for later use.

[0022] The mycelial growth rate method was used for determination. Based on the preliminary experiment, seven concentrations of each agent with an inhibition rate of 10%-90% on the mycelial growth of the pathogen were selected. Stock solutions of the agents were prepared at specific concentrations, and a series of drug-containing culture media with varying concentrations were created. Mycelial discs of the pathogen to be used, cultured on PDA medium, were cut using a punch and inoculated into the center of a solidified drug-containing PDA medium with the mycelial side facing upwards. The media were then incubated at room temperature. When the diameter of the control colonies reached more than 3 / 4 of the plate diameter, the colony diameter (mm) was measured using calipers. The diameter of each colony was measured vertically once using the cross-sectional method, and the average value was taken. Each treatment was repeated four times. The effects of the tested agents on the mycelial growth of the tested pathogens were analyzed and compared. The logarithm of the agent concentration was plotted on the x-axis, and the probability of mycelial growth inhibition rate was plotted on the y-axis. Regression analysis was used to analyze the experimental data, and the EC50 value for each treatment was calculated.

[0023] Growth inhibition rate (%) = (Control colony diameter - Treated colony diameter) / (Control colony diameter - Mycelial cake diameter) × 100

[0024] Synergistic effect evaluation method: The co-toxicity coefficient method is used for evaluation. The co-toxicity coefficient standard is that CTC≥120 indicates synergistic effect, 80≤CTC<120 indicates additive effect, and CTC<80 indicates antagonistic effect.

[0025] Test results:

[0026] The test results in Table 1 show that d-limonene has excellent inhibitory activity against the mycelial growth of *Rhizoctonia solani*, with an EC50 value of 215.60 mg / L. Compared with d-limonene, allicin has higher inhibitory activity against the mycelial growth of *Rhizoctonia solani*, with an EC50 value of 35.48 mg / L. The combined use of d-limonene and allicin can enhance the inhibitory effect of the agent on the mycelial growth of *Rhizoctonia solani*. When the mass ratio of d-limonene to allicin is 25:1 to 1:5, the combination exhibits excellent control effect against the pathogen, showing a superior synergistic effect compared to using either alone.

[0027] Table 1: Indoor toxicity test of d-limonene and allicin against rice sheath blight pathogen

[0028]

[0029]

[0030] Example 2: Indoor toxicity determination of d-limonene and allicin against tomato gray mold pathogen

[0031] Test pathogen: Botrytis cinerea, the pathogen causing gray mold in tomatoes.

[0032] Test method:

[0033] The mycelial growth rate method was used for determination. Based on the preliminary experiment, seven concentrations of each agent with an inhibition rate of 10%-90% on the mycelial growth of the pathogen were selected. Stock solutions of the agents were prepared at specific concentrations, and a series of drug-containing culture media with varying concentrations were created. Mycelial discs of the pathogen to be used, cultured on PDA medium, were cut using a punch and inoculated into the center of a solidified drug-containing PDA medium with the mycelial side facing up. The media were incubated at room temperature. When the diameter of the control colonies reached more than 2 / 3 of the plate diameter, the colony diameter (mm) was measured using calipers. The diameter of each colony was measured vertically once using the cross-sectional method, and the average value was taken. Each treatment was repeated four times. The effects of the tested agents on the mycelial growth of the tested pathogens were analyzed and compared. The logarithm of the agent concentration was plotted on the x-axis, and the probability of mycelial growth inhibition rate was plotted on the y-axis. Regression analysis was used to analyze the experimental data, and the EC50 value of each treatment was calculated.

[0034] Growth inhibition rate (%) = (Control colony diameter - Treated colony diameter) / (Control colony diameter - Mycelium cake diameter) × 100

[0035] Synergistic effect evaluation method: The co-toxicity coefficient method is used for evaluation. The co-toxicity coefficient standard is that CTC≥120 indicates synergistic effect, 80≤CTC<120 indicates additive effect, and CTC<80 indicates antagonistic effect.

[0036] Experimental results:

[0037] The test results in Table 2 show that d-limonene has excellent inhibitory activity against the mycelial growth of *Gycium oryzae*, the causal agent of tomato gray mold, with an EC50 value of 160.33 mg / L. Compared with d-limonene, allicin has higher inhibitory activity against the mycelial growth of *Gycium oryzae*, with an EC50 value of 1.42 mg / L. The combined use of d-limonene and allicin enhances the inhibitory effect of the agent on the mycelial growth of *Gycium oryzae*. When the mass ratio of d-limonene to allicin is 25:1 to 5:1, the combination exhibits excellent control effect against the pathogen, showing a superior synergistic effect compared to using either alone.

[0038] Table 2: Indoor toxicity test of d-limonene and allicin against tomato gray mold pathogen

[0039]

[0040]

[0041] Example 3: Field test of the control efficacy of d-limonene and allicin against rice sheath blight

[0042] Crop diseases: Rice sheath blight

[0043] Test reagents: Self-made reagents 1-5.

[0044] Agent 1: 10% d-limonene·allicin emulsifiable concentrate

[0045] d-Limonene 8%, Allicin 2%, Agricultural Emulsion 600# 6%, Tween 80 4%, Ethylene Glycol 4%, Palm Oil to make up 100%. All of the above are weight percentages.

[0046] Agent 2: 11% d-limonene·allicin emulsifiable concentrate

[0047] d-Limonene 10%, Allicin 1%, Agricultural Emulsion 600# 4%, Tween 80 3%, Ethylene Glycol 3%, Palm Oil to make up 100%. All of the above are weight percentages.

[0048] 3:8% d-limonene·allicin emulsifiable concentrate

[0049] d-Limonene 4%, Allicin 4%, Agricultural Emulsion 600# 5%, Tween 80 2%, Ethylene Glycol 3%, Palm Oil to make up 100%. All of the above are weight percentages.

[0050] 4:4% d-limonene emulsifiable concentrate

[0051] d-Limonene 4%, agricultural emulsion 600# 5%, Tween 80 2%, ethylene glycol 3%, palm oil to make up 100%. All of the above are weight percentages.

[0052] Agent 5: 4% Garlicin Emulsifiable Oil

[0053] Allicin 4%, agricultural emulsion 600# 5%, Tween 80 2%, ethylene glycol 3%, palm oil to make up 100%. All of the above are weight percentages.

[0054] Test method: A pesticide treatment group and a control group were set up, with 4 replicates, randomized block design, and a plot area of ​​45 m2. The pesticide was sprayed once on the stems and leaves during the tillering stage of rice, with a pesticide solution dosage of 450 kg / hm2. The disease incidence was investigated before application, and the disease index was investigated 14 days after application to calculate the control efficacy.

[0055] Rice sheath blight severity grading standards:

[0056] Grade 0: The entire plant is disease-free;

[0057] Grade 1: Disease occurs on the fourth leaf and all leaf sheaths and leaves below it (with the sword leaf as the first leaf);

[0058] Grade 3: Disease occurs on the third leaf and all leaf sheaths and leaves below it;

[0059] Level 5: Disease occurs on the second leaf and all leaf sheaths and leaves below it;

[0060] Level 7: Disease occurs on the sword-shaped leaf blade and all leaf sheaths and leaves below it;

[0061] Level 9: The entire plant is infected and dies prematurely.

[0062] Calculation formula:

[0063] Test results:

[0064] The test results in Table 3 show that the d-limonene·allicin emulsifiable concentrate prepared in this invention has excellent field control efficacy against rice sheath blight. Compared with the use of agents 4 and 5 alone, the agent composition of this invention can significantly improve the control effect against rice sheath blight, exhibiting a significant synergistic effect; and field observations have shown that it is safe for crops.

[0065] Table 3: Field control efficacy tests of d-limonene and allicin against rice sheath blight

[0066]

[0067]

[0068] Example 4: Field test of the control efficacy of d-limonene and allicin against tomato gray mold

[0069] Crop diseases: Tomato gray mold

[0070] Test reagents: Self-made reagents 1-5 from Example 3.

[0071] Experimental Methods: A treatment group and a control group were set up, with four replicates in a randomized block design. Each plot was 30 m². Foliar spraying was used, applying the pesticide solution to both sides of the leaves; two applications were made at 7-day intervals. The disease severity of the leaves was assessed before the first application and again 7 days after the second application. Disease severity grading criteria were as follows:

[0072] Grade 1: No lesions

[0073] Grade 3: Three lesions on a single leaf

[0074] Grade 5: 4-6 lesions on a single leaf

[0075] Grade 7: 7-10 lesions on a single leaf

[0076] Level 9: Dysplasia is densely distributed on a single leaf, covering more than a quarter of the leaf area.

[0077] Formula for calculating drug efficacy:

[0078]

[0079]

[0080] Where: CK o —Critical condition index in the blank control area before drug administration;

[0081] CK1 – Disease index after drug administration in the blank control area;

[0082] PT o —Disease index in the treatment area before application of medication;

[0083] PT1 – Disease index after drug treatment in the treatment area.

[0084] Experimental results:

[0085] The test results in Table 4 show that the d-limonene·allicin emulsifiable concentrate prepared in this invention has excellent field control efficacy against tomato gray mold. Compared with the use of agents 4 and 5 alone, the agent composition of this invention can significantly improve the control effect against tomato gray mold, exhibiting a significant synergistic effect; and field observations have shown that it is safe for crops.

[0086] Table 4: Field control efficacy tests of d-limonene and allicin against tomato gray mold

[0087]

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it.

Claims

1. A bactericidal composition comprising d-limonene and allicin, characterized in that, The active ingredients consist of d-limonene and allicin, with a mass ratio of d-limonene to allicin of 25:1 to 1:

5.

2. The bactericidal composition according to claim 1, characterized in that, The mass ratio of d-limonene to allicin is 25:1 to 5:

1.

3. The bactericidal composition according to any one of claims 1-2, characterized in that, It contains pesticide adjuvants, and the active ingredient accounts for 1-30% by weight of the fungicide composition.

4. The bactericidal composition according to claim 3, characterized in that, The bactericidal composition is formulated as an emulsifiable concentrate, microemulsion, water-in-oil emulsion, or soluble liquid.

5. The use of the bactericidal composition according to any one of claims 1-4 for the prevention and control of crop diseases, characterized in that, The diseases mentioned are rice sheath blight or tomato gray mold.

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