Application of climbazole in preparation of medicine for preventing and treating plant diseases and composition of climbazole

Abstract

The invention discloses application of climbazole as an agricultural bactericide and a synergist in plant diseases, and climbazole has a remarkable inhibition effect on botrytis cinerea and is superior to a positive control drug thiabendazole. In addition, climbazole serving as a synergist is compounded with the tubulin inhibitor, so that the antibacterial activity can be improved. The antibacterial effect is improved by compounding with the thiabendazole, and the compounded EC50 is smaller than that of a contrast drug thiabendazole.

Description

Technical Field

[0001] The present invention relates to the field of plant disease control, and particularly to the application of clotrimazole in the preparation of drugs for controlling plant diseases and its compositions. Background Art

[0002] Plant diseases not only lead to a decrease in crop yields, but also toxins accumulate in grains, affecting food security and further endangering the health of humans and animals. Controlling diseases increases farmers' costs, pollutes the environment, disrupts the ecological balance, affects international trade in agricultural products, and causes economic losses. Diseases caused by plant pathogenic fungi account for about 70-80% of plant diseases. Among them, Botrytis cinerea is a highly influential plant pathogen in the agricultural field, belonging to the genus Botrytis in the order Hyphomycetes of the Deuteromycotina. It has a wide host range and can infect a variety of plants, posing a serious threat to global agricultural production.

[0003] Pesticides play a significant role in the fields of agriculture and public health. They can control pests, diseases, weeds, and rodent pests. Approximately 30% of the global food production depends on their protection, ensuring food security. They can also inhibit the growth of mycotoxins, extend the shelf life of agricultural products, and improve market quality and economic benefits. In public health, they can kill disease vectors such as mosquitoes and flies, reducing the risk of infectious disease transmission. At the same time, pesticides reduce the labor intensity of agriculture and meet the needs of modern intensive agriculture. However, they should be used scientifically and reasonably in combination with green control technologies. According to relevant data, stopping the use of pesticides will lead to a 30% reduction in crop yields and a 50-70% increase in the prices of agricultural products, while the use of pesticides can recover unnecessary losses. However, due to the ban on highly toxic pesticides and drug resistance, etc., we have to continuously develop new green pesticides with high efficiency, low toxicity, and low residues. Clotrimazole has characteristics such as low toxicity, low cost, and a unique mode of action that is not prone to resistance, providing many ideal molecules for drug research and development.

[0004] Clotrimazole, with the chemical name of (RS)-1-(4-chlorophenoxy)-1-(1H-imidazol-1-yl)-3,3-dimethyl-2-butanone, is an organic compound with important application values in the fields of personal care and agriculture. Its molecular formula is C 15 H 17ClN2O2, with a molecular weight of 292.76. It usually appears as a white to off-white crystalline powder. It is slightly soluble in water but dissolves well in organic solvents such as ethanol and acetone. This solubility characteristic makes it have unique usage methods in different application scenarios. It is an anti-dandruff and anti-itching agent developed by Bayer AG in Germany in 1977. This compound has unique antifungal ability and can inhibit Malassezia ovalis or Pityrosporum ovale, Candida albicans, and Trichophyton that cause dandruff. In addition, Climbazole is very suitable for use in formulations. It is not only stable to light and heat but also easily soluble in organic solvents. Climbazole belongs to substances with low toxicity, and adding Climbazole will significantly increase the viscosity of shampoo.

[0005] Climbazole has good antifungal performance and causes the death of fungi by inhibiting the synthesis of ergosterol, an important component of the fungal cell membrane. Climbazole can kill Malassezia ovalis and Pityrosporum ovale, thus inhibiting and eliminating dandruff and achieving the purpose of relieving itching. Due to its stable nature and low solubility in water, it will not cause irritation to the scalp. It can also inhibit Candida albicans that causes gingivitis and periodontitis in the oral cavity. Therefore, it is an important functional additive for daily chemical products and can be widely used in the production of shampoos, conditioners, body washes, soaps, toothpastes, mouthwashes, etc. In addition, Climbazole can also be used in the field of the treatment of skin fungal infections to produce eczema drugs. In this study, we found that commercially sourced Climbazole showed good inhibitory effects on some phytopathogenic fungi and had the potential to be developed into a new type of green pesticide.

[0006] Thiabendazole, chemically named 2-(4-thiazolyl)benzimidazole, inhibits microtubule polymerization by targeting the β-subunit of fungal tubulin and interferes with cell division and hyphal growth. Its mechanism of action is similar to that of carbendazim, but it shows higher inhibitory activity against some pathogens. Thiabendazole is widely used in the agricultural field and can effectively control post-harvest diseases of fruits and vegetables (such as Penicillium digitatum of citrus and Colletotrichum musae of banana) and soil-borne diseases of crops (such as Rhizoctonia solani of potato). At the same time, it is used in the medical field to treat skin fungal infections. This agent has the characteristics of low toxicity and strong systemic absorption and can be applied by spraying, dipping fruits, or seed dressing.

[0007]

[0008] To overcome the problems of insufficient efficacy of a single fungicide and easy generation of drug resistance, in the actual process of agricultural production, different varieties of components are often compounded, and compound agricultural fungicidal compositions with excellent bactericidal effects are found, which are of great significance for reducing the generation of disease drug resistance, environmental protection, and green development. Accordingly, we compounded a low-toxic compound with the commercial drug thiabendazole to reduce the toxicity of the compounded agent and studied the antibacterial activity after compounding to expand the application of Climbazole in the antibacterial aspect. Summary of the Invention

[0009] The object of the present invention is to provide the application of clotrimazole and chemical pesticide synergists as agricultural fungicides in preventing and controlling plant diseases, so as to solve the problems existing in the above-mentioned prior art.

[0010] The present invention provides the application of clotrimazole as an agricultural fungicide and synergist in plant diseases. The agricultural fungicide and synergist contain clotrimazole, and the chemical structure of clotrimazole is shown as follows:

[0011]

[0012] The plant diseases described in the present invention specifically include diseases caused by fungi.

[0013] Furthermore, the plant diseases caused by fungi include diseases caused by Botrytis cinerea.

[0014] A composition for treating plant diseases, comprising a compound of clotrimazole and a medicament with bactericidal effect.

[0015] Furthermore, the medicament with bactericidal effect is thiabendazole.

[0016] Furthermore, the compound mass ratio of thiabendazole to clotrimazole is 1:10 to 10:1. Preferably, the compound mass ratio of thiabendazole to clotrimazole is 1:4, 2:3, 3:2, 4:1.

[0017] A method for preventing and controlling plant diseases caused by fungi, using the compound of myclobutanil and clotrimazole to prevent and control crop diseases.

[0018] The plant diseases caused by fungi include diseases caused by Botrytis cinerea, Colletotrichum gloeosporioides, Colletotrichum graminicola, Colletotrichum capsici, Sclerotinia sclerotiorum, Botryosphaeria dothidea, Gibberella zeae, Verticillium dahliae, Rhizoctonia solani, Botrytis cinerea, Fusarium oxysporum f. sp. capsici, Phytophthora infestans, Colletotrichum coccodes, Alternaria solani, Phytophthora cactorum, Alternaria alternata, Phomopsis longicolla, Sclerotinia sclerotiorum, Cercospora arachidicola, Botrytis cinerea.

[0019] The present invention has the following beneficial effects:

[0020] (1) Clotrimazole shows good biological activity against plant fungi. Experimental data show that: at a concentration of 25 μg / mL, clotrimazole has a significant inhibitory effect on Botrytis cinerea, which is better than the positive control drug thiabendazole. Further findings show that the EC 50 value of clotrimazole against Botrytis cinerea is 0.021 μg / mL, which is better than the positive control drug thiabendazole (EC 50 is 0.790 μg / mL). In summary, clotrimazole has an excellent inhibitory effect on Botrytis cinerea.

[0021] (2) The compounding of clotrimazole and thiabendazole enhances its antibacterial effect. The experimental results against Botrytis cinerea show that the compounding effect is better than that of the control drug, and the drug resistance of the compounding is better than that of the single agent. When the compounding ratio of thiabendazole to clotrimazole is 4:1, the EC 50 value is 0.038 μg / mL, and the synergistic coefficient SR value is 0.684.

[0022] (3) Clotrimazole has many advantages in the environment. It can inhibit harmful microorganisms, reduce crop diseases, and reduce pesticide pollution. In the soil, it has strong adsorption and is easily degraded, which can reduce the pollution risk. In addition, clotrimazole has low residues in the environment, reducing the harm to the ecosystem and food chain and protecting biodiversity. Detailed implementation modes

[0023] The various exemplary implementation modes of the present invention will be described in detail. In the examples, the methods are all conventional methods unless otherwise specified, and the reagents are all conventional commercially available reagents or reagents prepared by conventional methods unless otherwise specified. This detailed description should not be regarded as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation modes of the present invention.

[0024] It should be understood that the terms used in the present invention are only for describing specific implementation modes and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0025] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0026] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation modes of the specification of the present invention, which are obvious to those skilled in the art. Other implementation modes obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are only exemplary.

[0027] For the terms "comprising", "including", "having", "containing", etc. used in this article, they are all open-ended terms, meaning including but not limited to.

[0028] Example 1

[0029] In this invention, clotrimazole (98%) was purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd.

[0030] The natural product clotrimazole has shown good biological activity in anti-plant fungi. Taking the activity against Botrytis cinerea as an example below.

[0031] The mycelial growth rate method, also known as the poisoned medium method, is one of the conventional methods for determining the toxicity of fungicides. The main principle is to mix the test agent with the culture medium, and measure the toxicity of the agent by the growth rate of the colony on the poisoned culture medium. In this example, Botrytis cinerea was used as the test object, and DMSO (dimethyl sulfoxide) was used as the blank control.

[0032] The specific operations are as follows: 1) Weigh an appropriate amount of clotrimazole according to the test concentration, dissolve it with DMSO (the dosage does not exceed 1% of the final poisoned culture medium), then add an aqueous solution containing 0.1% Tween 20 to make up to 10 mL, pour it into 90 mL of melted PDA culture medium, mix well and pour it into 9 petri dishes for standby; 2) Sterilize the puncher (inner diameter 0.5 cm) by burning, and after it cools down, punch the mycelium at the edge of the pre-activated strain, and use an inoculation needle to stick its mycelium surface in the center of the poisoned culture medium. After the treatment is completed, uniformly place it in an incubator at 25°C for cultivation; 3) After the colony diameter of the control group grows to 5.5 - 6.0 cm, use the cross method to measure the colony diameters of the control group and each agent treatment group; 4) Use the following formula to calculate the inhibition rate (%):

[0033] Inhibition rate I% = (C - T) / (C - 0.5) × 100%;

[0034] Wherein, C is the colony diameter of the control group, T is the colony diameter of the agent treatment group, and 0.5 is the diameter of the inoculated agar disc.

[0035] EC 50 (median effective concentration) is an important index for evaluating the sensitivity of plant pathogens to compounds, and is also an important parameter for setting the compound concentration when studying the action mechanism of compounds. In the concentration gradient experiment, the appropriate 5 concentrations are set by the two-fold dilution method. Finally, the inhibition rate of the agent on plant pathogens and the agent concentration are converted into logarithms, and the toxicity curve is obtained through regression analysis by SPSS software, and the EC 50 .

[0036] Embodiments of the present invention are used to illustrate the technical solutions of the present invention, but the content of the embodiments is not limited thereto. The experimental results are shown in Tables 1 and 2.

[0037] Table 1 Inhibitory Activity of Climbazole against Botrytis cinerea (25 μg / mL)

[0038] Compound Inhibition rate % (25 μg / mL) Clotrimazole 99.040±0.960 Thiabendazole 97.400±0.200

[0039] Table 2 EC of Climbazole against Botrytis cinerea 50 value

[0040]

[0041]

[0042] As can be seen from Table 1, at a concentration of 25 μg / mL, climbazole has a significant inhibitory effect on Botrytis cinerea, which is better than the positive control thiabendazole. Further EC 50 testing found that the EC 50 value of climbazole against Botrytis cinerea was 0.021 μg / mL, which is better than the positive control thiabendazole (EC 50 was 0.790 μg / mL); In summary, climbazole has an excellent inhibitory effect on Botrytis cinerea. Thus, climbazole has potential application value as a pesticide and can control fungal diseases such as grape gray mold caused by Botrytis cinerea.

[0043] Example 2

[0044] EC of climbazole as a synergist against the plant pathogenic fungus Botrytis cinerea 50 value.

[0045] The experimental object was Botrytis cinerea, thiabendazole was used as the test control, and DMSO was used as the blank control.

[0046] (1) Using DMSO as the solvent, thiabendazole and climbazole at the same concentration were compounded at a volume ratio of 1:4, 2:3, 3:2, and 4:1.

[0047] (2) The mycelial growth rate method was used to determine the effect of the thiabendazole and climbazole compound on the growth rate of Botrytis cinerea.

[0048] (3) The synergistic coefficient SR was calculated using the Wadley method. When the SR value is less than 0.5, it is an antagonistic effect; when it is between 0.5 and 1.5, it is an additive effect; when it is greater than 1.5, it is a synergistic effect. The calculation formula is:

[0049]

[0050] SR = EC 50 (theory) / EC50 (Actual)

[0051] a and b respectively represent the proportions of thiabendazole and hexachlorophene in the mixture;

[0052] EC(A) 50 and EC(B) 50 represent the EC 50 values of thiabendazole and hexachlorophene.

[0053] The antibacterial activity results of thiabendazole, hexachlorophene and their different ratios against Botrytis cinerea are shown in Tables 3, 4 and 5 respectively.

[0054] Table 3 Joint toxicity of thiabendazole and hexachlorophene against Botrytis cinerea

[0055]

[0056] As can be seen from Table 3, the EC 50 values of thiabendazole and hexachlorophene against Botrytis cinerea are 0.790 and 0.021 μg / mL respectively, showing that the bacteriostatic effect of hexachlorophene is better; the EC 50 values of different compound ratios of the two single agents are between 0.038 and 0.192 μg / mL. When the compound ratios of the two single agents are 1:4, 2:3, 3:2, 4:1, the EC 50 values are all less than that of thiabendazole. Among them, when the compound ratio of thiabendazole and hexachlorophene is 1:4, the additive effect is obvious, the EC 50 value is 0.038 μg / mL, and the synergistic coefficient SR value is 0.684.

[0057] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention..

Claims

1. The application of clotrimazole as an agricultural fungicide and synergist in plant diseases, characterized in that: The agricultural fungicide and synergist contain clotrimazole, and the chemical structure of clotrimazole is shown in the following formula (1):

2. Use of clotrimazole as an agricultural fungicide and synergist in plant diseases according to claim 1, characterized in that: The plant diseases specifically include diseases caused by fungi.

3. The application of clotrimazole as an agricultural fungicide and synergist in plant diseases according to claim 2, characterized in that: The fungi are Botrytis cinerea, Colletotrichum gloeosporioides or Colletotrichum sublineolum.

4. A composition for treating plant diseases, characterized in that: It includes the compounding of clotrimazole and a bactericidal agent with a microtubule inhibitor.

5. The composition for treating plant diseases according to claim 4, characterized in that, The bactericidal agent with a microtubule inhibitor is thiabendazole.

6. The composition for treating plant diseases according to claim 5, characterized in that: The compounding mass ratio of thiabendazole to clotrimazole is 1:10 to 10:

1.

7. The composition for treating plant diseases according to claim 6, characterized in that; The compounding mass ratio of thiabendazole to clotrimazole is 1:4, 2:3, 3:2, 4:

1.

8. A method for controlling plant diseases caused by fungi, characterized in that, Using the drug according to any one of claims 4-7 to control crop diseases.

9. The method for controlling plant diseases caused by fungi according to claim 8, characterized in that: The plant diseases caused by the fungi include diseases caused by Colletotrichum fructicola, Colletotrichum gloeosporioides, Colletotrichum sublineolum, Colletotrichum capsici, Sclerotinia sclerotiorum, Botryosphaeria dothidea, Fusarium graminearum, Verticillium dahliae, Rhizoctonia solani, Botrytis cinerea, Fusarium oxysporum f. sp. capsici, Phytophthora infestans, Colletotrichum coccodes, Alternaria solani, Phytophthora nicotianae, Diaporthe phaseolorum var. caulivora, Fusarium graminearum, Phaeoisariopsis personata, Botrytis cinerea.

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