Application of pyrimethanil in prevention and treatment of pathogenic fungi of crops

Abstract

The invention discloses application of pyrimethanil in prevention and treatment of pathogenic fungi of crops. The pyrimethanil has excellent bacteriostatic activity on pathogenic fungi of crops, and can be used for preventing and treating diseases caused by the pathogenic fungi of important economic crops such as vegetables, tea trees, rice, fruit trees and the like. According to the application disclosed by the invention, the pyrimethanil is found to have a remarkable antibacterial effect on pathogenic fungi such as trichospora fistula, corynespora cassiicola and the like; the bactericidal composition can be compounded with berberine according to a ratio of 1: 2 or 2: 1, or compounded with luteochlorin according to a ratio of 2: 1, and has a synergistic effect in prevention and treatment of corynespora cassiicola. The pyrimethanil disclosed by the invention can delay the generation of drug resistance of germs, expand the bactericidal spectrum of pesticides and reduce the pollution of the pesticides to the environment, conforms to the policy of pesticide reduction and synergism in China, and has a wide application prospect in the field of crop disease control.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of crop pathogenic fungi prevention and treatment, and particularly relates to application of pyrimethanil in crop pathogenic fungi prevention and treatment. BACKGROUND

[0002] Fungal diseases are an important type of crop diseases and insect pests, and the caused gray mold, downy mildew, anthracnose, target spot disease, tea leaf spot disease and the like will have a serious impact on the yield and quality of crops. At present, using fungicides to prevent and treat crop fungal diseases is an important economic and simple measure. However, due to long-term unreasonable use of fungicides, the resistance of fungicides is increasing, and there are risks such as pesticide residues and environmental pollution in crops. Therefore, screening of fungicidal active molecules with novel structure and outstanding activity is an effective way to create fungicides and prevent and treat crop diseases.

[0003] At present, there are many fungicides for preventing and treating fungal diseases, but their action targets are relatively single, mainly concentrated in energy inhibition, inhibition of sterol synthesis, inhibition of biological macromolecule synthesis and the like. Fungicide development based on novel structure molecules is an important means to solve pesticide resistance and create new fungicides. Pyrimethanil is also known as methyl pyrimidine amine and dimethyl pyrimidine amine, which belongs to the benzene amino pyrimidine fungicide. Its chemical name is N-(4,6-dimethyl pyrimidine-2-yl) aniline, the English general name is pyrimethanil, and the chemical formula is C 12 H 13 N3( Figure 1It has the characteristics of high efficiency and low toxicity, and is widely used in agriculture to control various plant diseases. Its main mechanism of action is to inhibit the secretion of hydrolases such as glucanase, cellulase and protease by pathogenic fungi during the infection process, thereby preventing the invasion and spread of the pathogen (Mandrile, L., Giovannozzi, A. M., Durbiano, F., Martra, G., Rossi, A. M. Rapid and sensitive detection of pyrimethanil residues on pome fruits by surface enhanced raman scattering. Food Chemistry, 2018, 244, 16-24.). According to the Fungicide Resistance Action Committee (2020), the main mode of action of pyrimethanil is to inhibit the biosynthesis of methionine (Zhang, Y., Fu, Y., Luo, C., Zhu, F. Pyrimethanil sensitivity and resistance mechanisms in Penicillium digitatum. Plant disease, 2021, 105(6), 1758-1764.). Cystathionine γ-synthase (CGS) and cystathionine β-lyase (CBL) are the two most important enzymes involved in the biosynthesis of methionine. Pyrimethanil can inhibit the activity of both enzymes, reduce the production of methionine, and inhibit fungal growth (Fu, J, Wu, J. B., Jiang, J. H., Wang, Z. Y., Ma, Z. H. Cystathionine gamma-synthase is essential for methionine biosynthesis in Fusarium graminearum. Fungal Biology, 2013, 117, 13-21.).Pyrimethanil has a protective effect and can reduce the death of host cells at the infection site, and is usually used to control fungal diseases (Zhang, Y., Zhou, L., Wang, C., Liu, S. Synergistic antifungal effect and potential mechanism of Dimethomorph combined with Pyrimethanil against Phytophthora capsici. Food Chemistry, 2024, 457, 140158.). Pyrimethanil can synergize with other agents to significantly inhibit the growth of Staphylococcus hominis mycelium and conidial germination, and cause mycelium malformation (Xu, D., Yu, G., Xi, P., Kong, X., Wang, Q., Gao, L., Jiang, Z. Synergistic effects of resveratrol and pyrimethanil against Botrytis cinerea on grape. Molecules, 2018, 23(6), 1455.).

[0004] In addition, studies have found that pyrimethanil can also inhibit the biosynthesis of methionine and other amino acids, further interfering with the growth and reproduction of the pathogen (Buchele, F., Neuwald, D. A., Scheer, C., Wood, R. M., Voegle, R. T., Wunsch, J. N. Assessment of a postharvest treatment with pyrimethanil via thermo-nebulization in controlling storage rots of apples. Agronomy, 2021, 12(1), 34.). In agricultural production, pyrimethanil is also used in post-harvest treatment of citrus fruits to control Penicillium digitatum caused by green mold (Smilanick, J. L., Mansour, M. F., Gabler, F. M., Goodwine, W. R. The effectiveness of pyrimethanil to inhibit germination of Penicillium digitatum and to control citrus green mold after harvest. Postharvest Biology and Technology, 2006, 42(1), 75-85.). It is widely used in the prevention and control of pathogens such as cucumber, tomato, grape, strawberry, etc. For example, Zhang Huiguo et al. showed that at the same dose, 40% pyrimethanil suspension agent had higher control effect on onion botrytis blight than 50% dimethomorph water dispersible granules (Zhang Huiguo, Hu Jiankun, Huang Guodong, et al. Field efficacy test and evaluation of 40% pyrimethanil suspension agent for controlling onion botrytis blight. Yangtze Vegetables, 2022, (20), 71-73.).

[0005] Studies have shown that to improve the efficacy and delay resistance, often with a variety of fungicides used with bupirimate.For example, combined with resveratrol, it can synergistically inhibit the growth of Botrytis cinerea mycelium and spore germination (Xu, D. D., Yu, G., et al. Synergistic effects of resveratrol and pyrimethanil against Botrytis cinerea on grape. Molecules, 2018, 23(6), 1455.); combined with wuyiencin at a mass ratio of 7:3, it has a significant synergistic effect (Xie, J. B., Li, B. Y., et al. Effect of combining wuyiencin and pyrimethanil on controlling grape gray mold and delaying resistance development in Botrytis cinerea. Microorganisms, 2024, 12(7), 1383.); combined with dimethomorph at a mass ratio of 5:1, it has the strongest synergistic effect (synergy coefficient: 1.85), and can destroy the ultrastructure of mycelium and interfere with the metabolic pathway (Zhang, Y., Zhou, L. Y., et al. Synergistic antifungal effect and potential mechanism of dimethomorph combined with pyrimethanil against Phytophthora capsici. Food Chemistry, 2024, 457, 140158.); combined with pyrimethanil and Cryptococcus laurentii, even at low concentrations, the combination can inhibit the rot of Penicillium expansum, and does not affect the activity of the yeast and the quality of the fruit (Yu, C., Zhou, T., et al. Effect of pyrimethanil on Cryptococcus laurentii, Rhodosporidium paludigenum, and Rhodotorula glutinis biocontrol of Penicillium expansum infection in pear fruit. International Journal of Food Microbiology, 2013, 164(2-3), 155-160.).In addition, the study found that pyrimethanil and triazole, dioxime fungicides exist cross resistance, but no cross resistance with fludioxonil, pydiflumetofen, etc. (Zhou, F., Su, H. C., et al. Pyrimethanil resistance detected in gray mold collected from the strawberry fields of Henan province and potential resistance mechanism. Plant Disease, 2025.).

[0006] However, so far there has been no in-depth study on the antibacterial activity of pyrimethanil on pathogenic fungi of other crops, and there is no relevant patent at home and abroad. Therefore, the present patent system evaluates the antibacterial activity of pyrimethanil on pathogenic fungi of cucumber, tomato, grape, tea tree, rice and other crops, and also evaluates the antibacterial activity of its combination with berberine and luteovorin, which lays a foundation for the development of fungicides containing pyrimethanil in the future. SUMMARY

[0007] In view of this, one of the purposes of the present application is to provide the application of pyrimethanil in the prevention and treatment of crop pathogenic fungi.

[0008] Further, the pathogenic fungi include one or more of Lasiodiplodia theobromae, Corynespora cassiicola, Glomerella cingulata f. sp. camelliae, Epicoccum nigrum, Botrytis cinerea, Pseudopestalotiopsis camelliae-sinensis, Alternaria longipes, Rhizoctonia solani and Fusarium graminearum.

[0009] Preferably, the pathogenic fungi are Lasiodiplodia theobromae and / or Corynespora cassiicola.

[0010] Preferably, the pathogenic fungi are Lasiodiplodia theobromae.

[0011] Further, the crops include one or more of vegetables, tea trees, rice, or fruit trees.

[0012] Preferably, the vegetables are cucumbers.

[0013] Preferably, the concentration of the use of the pyrimethanil is greater than or equal to 100 μg / mL.

[0014] The second object of the present application is to provide a pesticide composition for preventing and treating plant pathogenic fungi, which comprises effective component A and effective component B, wherein the effective component A is pyrimethanil, and the effective component B is berberine or luteoxin.

[0015] Preferably, the mass ratio of pyrimethanil to berberine is 2:1 or 1:2.

[0016] Preferably, the mass ratio of pyrimethanil to berberine is 2:1.

[0017] Preferably, the mass ratio of pyrimethanil to luteoxin is 1:1.

[0018] The third object of the present application is to provide the use of any of the above-mentioned compositions in preventing and treating plant pathogenic fungi.

[0019] Further, the plant pathogenic fungi are Corynespora cassiicola.

[0020] Preferably, the concentration of the use of the composition is greater than or equal to 50 μg / mL.

[0021] The pyrimethanil of the present application has excellent bacteriostatic activity on crop pathogenic fungi, and can be used for preventing and treating diseases of important economic crops including cucumbers, tea trees, rice, and fruit trees caused by pathogenic fungi, especially L. theobromae, and has a significant bacteriostatic effect. The pyrimethanil can be compounded with berberine and luteoxin to enhance the bacteriostatic effect on C. cassiicola. The pyrimethanil of the present application can delay the development of drug resistance of pathogenic bacteria, expand the bactericidal spectrum of pesticides, reduce the pollution of pesticides to the environment, and meet the policy of reducing the amount of pesticides and increasing efficiency in China, and has a broad application prospect in the field of crop disease prevention and treatment. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The pyrimethanil of the present application is shown in the following structural formula:

[0023] Figure 2 The berberine of the present application is shown in the following structural formula:

[0024] Figure 3 The luteoxin of the present application is shown in the following structural formula:

[0025] Figure 4 Antifungal activity of pyrimethanil on L. theobromae mycelium in the present application, scale = 1 cm;

[0026] Figure 5 Antifungal activity of pyrimethanil on G. cingulata f. sp. camelliae mycelium in the present application, scale = 1 cm;

[0027] Figure 6 Antifungal activity of pyrimethanil on E. nigrum mycelium in the present application, scale = 1 cm;

[0028] Figure 7 Antifungal activity of pyrimethanil on B. cinerea mycelium in the present application, scale = 1 cm;

[0029] Figure 8 Antifungal activity of pyrimethanil on C. cassiicola mycelium in the present application, scale = 1 cm;

[0030] Figure 9 Antifungal activity of pyrimethanil on Ps. camelliae-sinensis mycelium in the present application, scale = 1 cm;

[0031] Figure 10 Antifungal activity of pyrimethanil on A. longipes mycelium in the present application, scale = 1 cm;

[0032] Figure 11 Antifungal activity of pyrimethanil on R. solani mycelium in the present application, scale = 1 cm;

[0033] Figure 12 Antifungal activity of pyrimethanil on F. graminearum mycelium in the present application, scale = 1 cm;

[0034] Figure 13 Live experiment diagram of C. cassiicola pathogenic cucumber leaves under different concentrations of pyrimethanil in the present application, scale = 1 cm;

[0035] Figure 14 Live antifungal experiment analysis diagram of C. cassiicola pathogenic cucumber leaves under different concentrations of pyrimethanil in the present application;

[0036] Figure 15The myclobutanil and berberine 1:1, 1:2 and 2:1 three kinds of compound proportion (mass ratio) combination on the mycelial inhibition activity of C. cassiicola, scale = 1 cm;

[0037] Figure 16 The myclobutanil and berberine 1:1, 1:2 and 2:1 three kinds of compound proportion (mass ratio) combination on the mycelial inhibition activity of C. cassiicola, scale = 1 cm;

[0038] Figure 17 The myclobutanil and berberine 1:1, 1:2 and 2:1 three kinds of compound proportion (mass ratio) combination on the mycelial inhibition activity of C. cassiicola, scale = 1 cm;

[0039] Figure 18 The myclobutanil and berberine 1:1, 1:2 and 2:1 three kinds of compound proportion (mass ratio) combination on the mycelial inhibition activity of C. cassiicola, scale = 1 cm. DETAILED DESCRIPTION

[0040] The application will be described in detail below with reference to the embodiments, which are only illustrative and not limited to the scope of the application. The application is not limited to the following embodiments or examples, and any modification and transformation made without departing from the spirit of the application shall be included in the scope of the application. The experimental materials used in the following examples are commercially available unless otherwise specified. The percentages mentioned in the following examples are mass percentages unless otherwise specified.

[0041] Example 1: Inhibition activity detection by indoor bioassay

[0042] After analyzing the mechanism of action of pyrimethanil, the bioassay targets were selected as plant pathogenic fungi: L. theobromae strain CGMCC3.20151, G. cingulata f. sp. camelliae strain ACCC35058, E. nigrum strain CGMCC3.20484, B. cinerea strain CGMCC3.20932, C. cassiicola strain GZUGPL2024001, Ps. camelliae-sinensis strain CGMCC3.20199, A. longipes strain CGMCC3.20931, R. solani strain CGMCC3.7376, and F. graminearum strain CGMCC3.4521. All of the above strains were isolated and obtained by the inventors' team and have been deposited and published by preservation institutions such as the China General Microbiological Culture Collection Center and the China Agricultural Microbiological Culture Collection Center; the above strains can also be obtained from the Fine Chemicals Research and Development Center (National Key Laboratory of Green Pesticides) of Guizhou University.

[0043] Preparation method: Use a pipette to take 60 μL of the active ingredient and pyrimethanil at a concentration of 400 g / L, add 4.8 mL of sterile water and mix by pipetting to prepare a stock solution of 5000.0 μg / mL. Dispense the stock solution into 100 mL PDAs according to the concentration gradient and sterilize before use.

[0044] Indoor bioassay methods were used: referring to standard NY / T1156.7-2006, the growth rate method was used to determine the inhibitory effect of the agent on the mycelial growth of pathogenic fungi L. theobromae, G. cingulata f. sp. camelliae, E. nigrum, B. cinerea, C. cassiicola, Ps. camelliae-sinensis, A. longipes, R. solani and F. graminearum.

[0045] The specific experimental steps were as follows: Using water treatment as a blank control, the mycelial growth rate method was employed to determine the inhibitory effect of the agent on the mycelial growth of the pathogens *L. theobromae*, *G. cingulata* f. sp. *camelliae*, *E. nigrum*, *B. cinerea*, *C. cassiicola*, *Ps. camelliae-sinensis*, *A. longipes*, *R. solani*, and *F. graminearum*. Six 250 mL Erlenmeyer flasks were prepared, each containing 100 mL of PDA medium (one flask for water treatment and five for agent treatment). These flasks were sterilized in an autoclave at 121°C for 20 min. The sterilized PDA medium was then removed. The prepared pyrimethanil stock solution had been added to the PDA before sterilization, resulting in the following final concentrations of the agent:

[0046] (1) The concentrations of the agents used to test the antibacterial activity against L. theobromae were 0.0, 7.5, 15.0, 30.0, 60.0 and 120.0 μg / mL;

[0047] (2) The concentrations of the agents used to test the antibacterial activity against the camellia-specific G. cingulata f. sp. camelliae were 0.0, 20.0, 40.0, 80.0, 160.0 and 320.0 μg / mL;

[0048] (3) The concentrations of the agents used to test the antibacterial activity against E. nigrum were 0.0, 5.0, 20.0, 40.0, 60.0 and 80.0 μg / mL;

[0049] (4) The concentrations of the agents used to test the antibacterial activity against Botrytis cinerea were 0.0, 10.0, 30.0, 60.0, 100.0 and 150.0 μg / mL;

[0050] (5) The concentrations of the agents used to test the antibacterial activity against C. cassiicola were 0.0, 10.0, 20.0, 30.0, 60.0 and 120.0 μg / mL;

[0051] (6) The concentrations of the agents used to test the antibacterial activity against Ps. camelliae-sinensis were 0.0, 10.0, 25.0, 45.0, 70.0 and 90.0 μg / mL;

[0052] (7) The concentrations of the agents used to test the antibacterial activity against Alternaria longipes were 0.0, 10.0, 15.0, 30.0, 60.0 and 120.0 μg / mL;

[0053] (8) The concentrations of the agents used to test the antibacterial activity against Rhizoctonia solani were 0.0, 10.0, 20.0, 40.0, 80.0 and 160.0 μg / mL;

[0054] (9) The concentrations of the agents used to test the antibacterial activity against Fusarium graminearum were 0.0, 60.0, 100.0, 130.0, 200.0 and 260.0 μg / mL;

[0055] Then, pour the mixed PDA medium containing the drug into petri dishes, five plates for each concentration. After the medium has cooled and solidified, use a sterilized 4 mm diameter punch to collect mycelial discs from activated fresh colonies of *L. theobromae*, *G. cingulata* f. sp. *camlliae*, *E. nigrum*, *B. cinerea*, *C. cassiicola*, *Ps. camelliae-sinensis*, *A. longipes*, *R. solani*, and *F. graminearum*. Inoculate these mycelial discs onto PDA medium containing the drug solution. After inoculation, seal the petri dishes with sealing film and invert them in a 25°C incubator. When the colonies in the 0.0 μg / mL compound drug medium reach 3 / 4 of their length, measure the colony diameter using the cross-hatching method. Finally, calculate the inhibition rate using the following formula.

[0056] Inhibition rate = (1)

[0057] The regression equation and r were calculated using DPS v 9.01 software (http: / / www.dpsw.cn / dps_eng / ). 2 and EC 50 The regression equation represents the relationship between mycelial growth rate and pesticide dosage; EC 50 This represents the half of the effective concentration.

[0058] Table 1 Evaluation of the antifungal activity of pyrimethanil against multiple plant pathogenic fungi.

[0059] Strains Regression equation EC 50 ( g / mL r 2 <!-- 5 -->]]> L. theobromae strain CGMCC3.20151 y = 0.6473x + 4.4834 6.28 0.9042 G. cingulata strain ACCC 35058 y = 1.7662x + 2.1144 43.04 0.9043 E. nigrum strain CGMCC3.20484 y = 2.0864 x + 2.0528 25.86 0.8502 B. cinerea strain CGMCC3.20932 y = 2.1842x + 1.3525 46.78 0.9761 C. cassiicola strain GZUGPL2024001 y = 2.3959x + 1.6281 25.55 0.9926 Ps. camelliae-sinensis strain CGMCC3.20199 y = 2.3045 x + 1.5744 30.65 0.8338 A. longipes strain CGMCC3.20931 y = 1.0216 x + 3.5608 25.63 0.9614 R. solani strain CGMCC3.7376 y = 1.2034 x + 2.9407 51.42 0.9731 F. graminearum strain CGMCC3.4521 y = 3.6145 x − 2.8033 144.17 0.9781

[0060] Depend on Figure 4-Figure 12 The inhibitory effect of pyrimethanil on different fungal strains increases with increasing concentration.

[0061] Table 1 shows that pyrimethanil has an EC50-95% effect on L. theobromae. 50 Compared to other fungi, it is approximately equal to other strains of EC. 50 Approximately 1 / 4 to 1 / 22 of EC 50 Significantly higher than other groups; followed by C. cassiicola, A. longipes, E. nigrum, Ps. camelliae-sinensis and other EC. 50 The higher number of bacteria showed significant differences, indicating that pyrimethanil has a strong inhibitory effect on L. theobromae, C. cassiicola, A. longipes, E. nigrum, and Ps. camelliae-sinensis.

[0062] Analysis of the above data shows that pyrimethanil exhibits strong antibacterial activity against *L. theobromae*, *G. cingulata* f. sp. *camlliae*, *E. nigrum*, *B. cinerea*, *C. cassiicola*, *Ps. camelliae-sinensis*, *A. longipes*, *R. solani*, and *F. graminearum*, especially showing significant control effects against *L. theobromae*, *C. cassiicola*, *A. longipes*, *E. nigrum*, and *Ps. camelliae-sinensis*. Future development of fungicides targeting these pathogens holds promise.

[0063] Example 2: In vivo antibacterial experiment

[0064] After analyzing the characteristics of pyrimethanil's action and the pathogenesis of the pathogen, EC was selected as the target for research on its antibacterial activity. 50 The results were also significantly lower than those of other groups of *C. cassiicola*, and the protective activity of pyrimethanil against cucumber target spot disease was investigated according to the method described by Zhu et al. (Zhu, J., Zhang, L., Ma, D., Gao, Y., Mu, W., Liu, F. A bioactivity and biochemical analysis of iminoctadine tris (albesilate) as a fungicide against *C. cassiicola*. *Pesticide Biochemistry and Physiology*, 2019, 158, 121.).

[0065] The experimental material preparation steps were as follows: Select healthy and plump cucumber seeds (variety: Changchun Mici), wrap them in moist gauze, place them in a petri dish, and germinate them in a constant temperature incubator at 25℃ for 24 hours. Sow the sprouted seeds into seedling trays and cultivate them in a climate chamber at 25℃ for later use. Transfer uncontaminated pathogens (C. cassiicola) into PDA for later use. Prepare sterile inoculation needles and uncontaminated PDA culture media blocks.

[0066] The specific experimental steps were as follows: The protective activity of pyrimethanil against cucumber target spot disease was verified by selecting cucumbers with similar shape and size, no disease spots, and good growth, showing the third true leaf. Pyrimethanil suspension was applied at doses of 100.0, 200.0, 400.0, 800.0, and 1600 μg / mL as treatments, with a water treatment as a blank control (positive control: *Cyclocarya paliurus* mycelial cake; negative control: sterile PDA mycelial cake). Protective treatment was performed 24 hours before inoculation (randomized distribution). The solution was sprayed evenly onto the cucumber leaves using a handheld sprayer until droplets formed. 24 hours later, the leaf surface was gently punctured with a sterile insect needle (avoiding vascular tissue). Then, a sterile inoculation needle was used to pick up pathogenic mycelial cakes, inoculating them with the hyphae facing down onto the wounds of the treated and positive control plants, and gently pressing to prevent dislodgement. The negative control was inoculated with uncontaminated PDA culture media of the same size. After culturing at 25℃ for 3 days (photoperiod of 12 h) in a climate chamber, the size of the lesions was observed. Each treatment was repeated in 6 replicates, and the experiment was performed 3 times. Significance analysis plots were generated using OriginPro 2024 10.1.0.178, data were processed using DPS V9.50 (mean, standard error, 5% significance level), and lesion area was measured using ImageJ Java 1.8.0_172 software. The formula for calculating the lesion inhibition rate is as follows:

[0067] Inhibition rate = (2)

[0068] Depend on Figure 13 and Figure 14 As shown, within the concentration range of 100-1600 μg / mL, an inhibitory effect begins to appear at a concentration of 100 μg / mL. With increasing concentration, the bacterial plaque becomes smaller, and the inhibition rate against the pathogen shows an upward trend; especially when the pyrimethanil concentration is 800 μg / mL, the inhibition rate is nearly 80%, and at 1600 μg / mL, the inhibition rate exceeds 90%. In conclusion, pyrimethanil has a very promising application prospect in the field of controlling pathogenic fungi in crops.

[0069] Example 3: Antibacterial activity detection of pyrimethanil-based compound formulations

[0070] To determine the synergistic effect of pyrimethanil combined with either berberine or gambogeysin, indoor bioassays were first conducted using these two active ingredients. After analyzing the characteristics of the two active ingredients, the bioassay was performed on the plant pathogenic fungus *C. cassiicola*. The following specific experiments were conducted using the method described in Example 1: Indoor bioassay method: Referring to standard NY / T1156.7-2006, the growth rate method was used to determine the inhibitory effect of the mixed agent on the mycelial growth of *C. cassiicola*.

[0071] (1) Preparation of medicine

[0072] 1) Weigh 0.052 g of pyrimethanil technical grade (96%) (purchased from Shaanxi Yikunte Pharmaceutical Technology Co., Ltd.), dissolve it in 5 mL of DMSO to prepare a stock solution of 10000.0 μg / mL.

[0073] 2) Weigh 0.052 g of berberine technical grade (97%) (purchased from Shandong Keyuan Biochemical Co., Ltd.), dissolve it in 5 mL of DMSO to prepare a stock solution of 10000.0 μg / mL.

[0074] 3) Weigh 0.056 g of gambogeysin technical grade (90%) (purchased from Zhengzhou Lipin Pharmaceutical Technology Co., Ltd.), dissolve it in 5 mL of DMSO to prepare a stock solution of 10000.0 μg / mL.

[0075] 4) Compound formulation: Pyrimethanil is compounded with berberine or gambogeysin respectively, and the compounding ratio is set to 1:1, 1:2 and 2:1. The above mother liquor is prepared according to the ratio to obtain the compound formulations of each ratio.

[0076] (2) Indoor tests of compound preparations of pyrimethanil with berberine or gambogeysin.

[0077] Four concentration gradients were designed for each compound reagent. Each compound reagent was added to PDA medium that had been melted and cooled to room temperature, resulting in final concentrations of 15.0, 25.0, 40.0, 70.0 μg / mL (pyrimethanil and berberine compound) or 10.0, 20.0, 40.0, 60.0 μg / mL (pyrimethanil and gambogeysin compound). After thorough mixing, the mixture was poured into sterile 100mm petri dishes to prepare agar plates. Each treatment was replicated five times, with a treatment containing sterile water serving as a blank control. *C. cassiicola* was aseptically inoculated into the center of the agar plate with the mycelial side down using a sterile punch (4mm). (For details on the strains, compound reagents, and their concentration gradients, see [link to relevant documentation]). Figure 15 and Figure 16The colony was placed upside down in a constant temperature incubator at 25 ℃ and cultured for 6-9 days. The colony diameter was measured by the cross-cross method, and the lesion inhibition rate was calculated according to formula (1) of Example 1.

[0078] The Wadley method was used to evaluate the bioactivity of the mixture of drugs A and B, and SR (synergistic ratio) was calculated according to formulas (3) and (4).

[0079] (3)

[0080] In the formula: a and b are the proportions of single agents A and B in the compound preparation; EC(A) 50 EC(B) 50 ECs for A and B respectively 50 Measured value; EC 50（th） EC as a compounding agent 50 Theoretical value.

[0081] (4)

[0082] SR represents the interaction between A and B after mixing: EC 50（ob） This indicates the EC after compounding. 50 Measured values. SR > 1.5 indicates a synergistic or enhancing effect, 0.5 ≤ SR ≤ 1.5 indicates an additive effect, and SR < 0.5 indicates an antagonistic effect.

[0083] The regression equation and r were calculated using DPS v 9.01 software (http: / / www.dpsw.cn / dps_eng / ). 2 and EC 50 The regression equation represents the relationship between mycelial growth rate and pesticide dosage; EC 50 This represents the half of the effective concentration.

[0084] The results are shown in Tables 2-5 and 15. Figure 16 As shown, the ratio of pyrimethanil to berberine is additive when it is 1:1, and synergistic when the ratio is 1:2 or 2:1, with SR values ​​of 1.91 and 1.99 respectively. However, the EC value of the 2:1 compound is significantly lower. 50 The EC compound is approximately 1:2. 50 The concentrations of pyrimethanil and gamboge were 18.84 and 36.55, respectively. When the ratio was 2:1, the inhibition rate at 70 μg / mL was over 80%, while at ratios of 1:1 and 1:2, the inhibition rates were only 74% and 70%, respectively. When the ratios of pyrimethanil and gamboge were 1:1 and 2:1, the EC50 values ​​were... 50 The value was comparable to the inhibition rate at 60 μg / mL, but the 1:1 ratio of the compound had a synergistic effect, while the 2:1 ratio only had an additive effect.

[0085] Therefore, in the combination of pyrimethanil with berberine and gambogeysin, only the ratios of pyrimethanil to berberine (1:2 and 2:1) and the ratio of pyrimethanil to gambogeysin (1:1) show a synergistic effect. Simultaneously, considering the overall EC... 50 Based on the results of antibacterial rate and other factors, the optimal inhibitory effect on *Trichophyton mentagrophytes* was found when the ratio of pyrimethanil to berberine was 2:1, or when the ratio of pyrimethanil to gambogeysin was 1:1.

[0086] Table 2. Determination of the antibacterial activity of berberine and gambogeysin against *C. cassiicola* GZUGPL2024001.

[0087] Medicament EC 50 (μg / mL) Regression equation r 2 ]]> Berberine 518.2 y = 1.0708 x + 2.0934 0.9855 Luteoviridin 119.71 y = 1.7423 x + 1.3792 0.9880

[0088] Table 3. Determination of antibacterial activity of different ratios of pyrimethanil and berberine against *C. cassiicola* (GZUGPL2024001).

[0089]

[0090] Table 4. Determination of antibacterial activity of different ratios of pyrimethanil and gambogeysin against C. cassiicola (GZUGPL2024001).

[0091]

[0092] Example 4: In vitro in vivo test of pyrimethanil-based compound formulation

[0093] Based on the above indoor SR values, an indoor in vivo test was conducted on the compound composition of pyrimethanil and berberine to control *Corynebacterium multiflorum*, to determine the control effect of the compound composition of pyrimethanil and berberine on *Corynebacterium multiflorum*.

[0094] Following the method described in Example 2, healthy and plump cucumber seeds (Changchun Honeythorn) were sown. The third true leaves of cucumbers with similar shape and size, no disease spots, and good growth were selected, with five replicates for each treatment group. Pyrimethanil and berberine were mixed at ratios of 1:1, 1:2, and 2:1 to prepare solutions with concentrations of 50.0, 100.0, 200.0, 400.0, and 800.0 μg / mL, respectively (preparation method as per Experimental Example 1). These solutions were then sprayed onto the cucumber leaves, with sterile water serving as a positive control. After the solution on the cucumber leaves had air-dried naturally, small triangular holes, approximately 2 mm deep, were punched into the leaves using a syringe. Then, using a sterilized punch with a diameter of 4 mm, mycelial cakes were collected from fresh *C. cassiicola* colonies that had been cultured for 7 days. The collected mycelial cakes were then inoculated, with the inoculated area facing down, onto the punctured part of the leaf. A negative control group was inoculated with sterile PDA medium, and a positive control group was sprayed with sterile water. After 48 hours, the pathogenicity was observed, photographed, and statistically analyzed.

[0095] Disease index = lesion area

[0096] Prevention and control efficacy % = [(Control disease index - Treatment disease index) / Control disease index] * 100

[0097] Table 6. Indoor efficacy test of cucumber leaves against C. cassiicola GZUGPL2024001.

[0098]

[0099] The results are shown in Table 6 and Figure 17 , 18 As shown, after spraying with pyrimethanil and berberine in ratios of 1:1, 1:2, and 2:1, the inhibition rate of *Cercospora multiflora* lesions increased with the increase of the dosage.

[0100] And by Figure 18 It can be seen that within the range of 50-800 μg / mL, an inhibitory effect begins at a concentration of 50 μg / mL, and the inhibition rate increases with the increase of the dosage. When the compound combination with a 2:1 ratio is applied at a dosage of 200 μg / mL, the inhibitory effect is significantly different from that of the 1:1 and 1:2 compound combinations. Therefore, the 2:1 compound combination of pyrimethanil and berberine of this invention exhibits significant antibacterial activity against *Corynebacterium multiflorum*.

[0101] The conventional techniques and solutions not described in detail in the above embodiments are all well known in the art, and therefore will not be elaborated upon here. The above embodiments and / or experimental examples describe the preferred embodiments of the present invention in detail. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. Application of pyrimethanil in the control of pathogenic fungi in crops.

2. The application as described in claim 1, characterized in that, The pathogenic fungi include one or more of the following: *Lasiodiplodia theobromae*, *Corynespora cassiicola*, *Glomerella cingulata f. sp. camelliae*, *Epicoccum nigrum*, *Botrytis cinerea*, *Pseudopestalotiopsis camelliae-sinensis*, *Alternaria longipes*, *Rhizoctonia solani*, and *Fusarium graminearum*.

3. The application as described in claim 2, characterized in that, The pathogenic fungi are *Lasiodiplodia theobromae* and / or *Corynespora cassiicola*.

4. The application as described in claim 3, characterized in that, The crops include one or more of vegetables, tea trees, rice, or fruit trees.

5. The application as described in claim 4, characterized in that, The concentration of pyrimethanil used is greater than or equal to 100 μg / mL.

6. A pesticide composition for controlling plant pathogenic fungi, characterized in that, The composition comprises active ingredient A and active ingredient B, wherein active ingredient A is pyrimethanil and active ingredient B is berberine or gambogeysin.

7. The pesticide composition according to claim 6, characterized in that, The mass ratio of pyrimethanil to berberine is 2:1 or 1:2, or the mass ratio of pyrimethanil to gambogeysin is 1:

1.

8. The use of the pesticide composition according to claim 6 or 7 in the control of plant pathogenic fungi.

9. The application as described in claim 8, characterized in that, The plant pathogenic fungus is Corynespora cassiicola.

10. The application as described in claim 9, characterized in that, The concentration of the composition used is greater than or equal to 50 μg / mL.

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