A bactericidal composition

Abstract

The application discloses a fungicidal composition, which comprises imazalil and phenoxazine-1-carboxylic acid with a mass ratio of 0.5-5:1, or the fungicidal composition comprises imazalil and phenoxazine-1-carboxylic acid with a mass ratio of 1:3.4-5.5. The fungicidal composition has a synergistic effect on the prevention and treatment of rice sheath blight, strawberry gray mold or pepper anthracnose.

Description

Technical Field

[0001] This invention relates to plant-based bactericidal compositions. Background Technology

[0002] Plants are the material basis for human survival, with rice, strawberries, and peppers being important food and economic crops. In recent years, with the improvement of people's living standards, their requirements for the diversification and quality of agricultural products have also increased, leading to a gradual expansion of the planting area for rice, strawberries, and peppers. These crops are often affected by various factors during their growth. For example, excessive fertilization can lead to root burn, excessive vegetative growth, and lodging. Insufficient fertilization may result in small stems and underdeveloped grains. Therefore, mastering the timing and amount of fertilization is crucial for ensuring yield. Secondly, the looseness of the soil directly affects the vitality of the crop's root system and its ability to absorb nutrients. Thirdly, soil pH affects plant height, tillering number, and the number of microorganisms. Furthermore, different crop varieties exhibit significant differences in photosynthetic characteristics, ecological adaptability, and response to increased CO2 concentrations. Additionally, cultivation practices, planting density, and rotary tillage depth can all influence crop yield. Among all the influencing factors, crop diseases are one of the most important factors leading to a decline in yield and quality. Rice sheath blight is caused by *Rhizoctonia solani*, primarily infecting leaf sheaths and leaves, producing elliptical or cloud-like lesions. Under high humidity, white, net-like mycelium grows on the infected areas, forming sclerotia that easily detach. Infection causes sheath blight, increasing empty grains and reducing thousand-grain weight; in severe cases, it leads to leaf blight and plant collapse, generally reducing yield by 10%–30%, and in severe cases, by more than 50%. Pepper anthracnose is a fungal disease mainly caused by *Anthracnose*. This disease primarily affects leaves and fruits, causing yellowish-brown fruits densely covered with small red spots in concentric rings. Later, a red, gelatinous substance oozes from the surface of the lesions. Severe infection can lead to leaf drop, resulting in losses of over 20%. Strawberry gray mold is prevalent in both greenhouse and open-field cultivation, generally reducing fruit yield by 20%–30%, and in severe cases, by more than 50%, causing significant economic losses. Strawberry gray mold is a fungal disease mainly caused by the genus *Botrytis*. It primarily affects leaves and fruits. Typical leaf symptoms include V-shaped yellowish-brown lesions that eventually lead to scorching and death. In the early stages of infection, mature fruits appear water-soaked, later rotting. Under high humidity, a dense gray mold layer develops on the surface. Based on this, agricultural, biological, and chemical control methods are commonly used in production. Chemical control, with its advantages of economy, efficiency, and convenience, has always been the primary method. However, long-term use of chemical pesticides can easily lead to drug resistance in pathogens, resulting in decreased efficacy. Currently, jinggangmycin has been used to control rice sheath blight for over 30 years, but its efficacy is beginning to decline. Pythium, used to control strawberry gray mold, has reported resistance. Difenoconazole, used to control anthracnose in peppers, has also been discussed for its resistance. Therefore, exploring new methods to control drug resistance in these crop diseases is urgently needed.To address the problem of pesticide resistance in crop pathogens, various methods are available, such as: exploring novel fungicidal compounds, reducing the frequency and dosage of pesticide application, rotating pesticides, and combining pesticides. Among these, combining two pesticides with different mechanisms of action is the most effective and economical method, which can extend the lifespan of the pesticide and improve the control effect.

[0003] Chinese patent CN1961678A uses a powder composed of chlorine dioxide, morpholine guanidine hydrochloride, cytokinin, and microbial products to control rice sheath blight. Chinese patent CN109197890A uses a compound seed dressing agent composed of 0.9-1.8% thiamethoxam, 0.3-1.2% curculigoside, 0.05-0.3% fenvalerate, 0.2-0.5% chlorpyrifos, 6-10% film-forming agent, 4-8% dispersant, 0.1-1% thickener, 0.3-0.5% basic rose extract, 0.01-0.1% salicylic acid, 0.01-0.1% trace elements, and water as the balance to control rice sheath blight. Chinese patent CN107624795A uses a fermented extract mixed with jinggangmycin to control rice sheath blight. Chinese patent CN104106579B uses a compound fungicide of azoxystrobin and pyraclostrobin in a mass ratio of 5:1 to 1:3 to control rice sheath blight. Chinese patent CN104381292A uses a compound fungicide of 2-4 parts acetamiprid, 2-4 parts cypermethrin, 4-5 parts gibberellin, 7-10 parts zinc sulfate, 7-10 parts ammonium molybdate, 10-12 parts abamectin, 12-14 parts chlorpyrifos, 30-35 parts humic acid organic liquid fertilizer, 2-4 parts methyl sterol, 1-2 parts pyrimidine salicylic acid, 4-5 parts oleic acid, 7-10 parts crude oil, 8-22 parts light oil, and 4-6 parts pesticide emulsifier to control rice sheath blight.

[0004] Chinese patent CN1961678A uses a fungicide composed of 5-9 parts Astragalus membranaceus, 5-9 parts Coptis chinensis, 5-9 parts Euphorbia fischeriana, 4-6 parts Ginkgo biloba, 3-5 parts Sophora flavescens, 1-3 parts Artemisia capillaris, 1-3 parts Eupatorium fortunei, 2-4 parts Artemisia argyi, 2-4 parts Galla chinensis, 5-7 parts Lonicera japonica, 5-7 parts Mentha haplocalyx, and 6-10 parts Garlic to control anthracnose in peppers. Chinese patent CN113907083B uses a fungicide made with tetracycline and prochloraz as active ingredients to control anthracnose in peppers. Chinese patent CN107156183A uses a fungicide made with konjac extract and benomyl to control anthracnose in peppers.

[0005] Chinese patent CN105028503A uses Artemisia argyi, Perilla frutescens, Atractylodes lancea, pumpkin seeds, Sophora flavescens, Dryopteris crassirhizoma, Pseudolarix amabilis bark, Ephedra sinica, nettle leaves, Cinnamomum cassia twigs, and pomegranate peel to control gray mold in strawberries. Chinese patent CN106106587A uses 25-30 parts of Rheum palmatum, 15-20 parts of Panax notoginseng leaves, 13-17 parts of tobacco straw, 10-15 parts of Sophora tonkinensis, 10-15 parts of Illicium verum, 8-13 parts of Tripterygium wilfordii, 6-10 parts of Artemisia annua, 5-8 parts of dried ginger, 5-8 parts of Gleditsia sinensis, 4-7 parts of tea oil cake, 3-6 parts of mineral powder, 3-6 parts of leek seeds, 2-4 parts of castor seeds, 0.5-1 part of chromium trichloride, and 0.5-1 part of nano zinc oxide to control gray mold in strawberries. Chinese patent CN103651373A uses pyrimethanil and pyrimethanil to control gray mold in strawberries.

[0006] The overall effects of the above patents are not ideal, therefore there is an urgent need to develop fungicidal compositions that have good control effects on rice sheath blight, pepper anthracnose, or strawberry gray mold.

[0007] Patent CN108041057B discloses a compound fungicide composed of kasugamycin and imidacloprid, which is environmentally compatible, highly efficient, low-toxicity, and low-residue. It is particularly suitable for the control of major fruit tree diseases such as apple ring rot, apple anthracnose, and grape white rot. The weight ratio of the two active ingredients, kasugamycin and imidacloprid, in the compound composition is 60:1 to 1:60, with a synergistic effect of 4:1 to 1:20, and an optimal ratio of 1:4. This invention demonstrates outstanding performance in controlling major fruit tree diseases such as apple ring rot, apple anthracnose, and grape white rot. While this patent only applies to fruit tree diseases, this invention also applies to diseases of gramineous crops. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a bactericidal composition that has a synergistic effect in the prevention and control of rice sheath blight, strawberry gray mold, or pepper anthracnose, in order to address the shortcomings of the existing technology.

[0009] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a bactericidal composition comprising imazalil and phenazine-1-carboxylic acid in a mass ratio of 0.5 to 5:1, or the bactericidal composition comprising imazalil and phenazine-1-carboxylic acid in a mass ratio of 1:3.4 to 5.5.

[0010] Imazalil is an imidazole compound, a moderately toxic fungicide commonly used for fruit preservation during storage. However, it also exhibits high control efficacy against plant pathogenic fungi such as *Lysimachia*, *Fusarium*, and *Syndromea*. Currently, the antifungal mechanism of imazalil is believed to affect fungal cell membrane permeability, physiological functions, and lipid synthesis metabolism, thereby disrupting the fungal cell membrane and preventing normal proliferation, thus impacting the fungus's normal life. Repeated screening experiments have shown that imazalil combined with various antifungal agents does not exhibit synergistic effects.

[0011] Phenazine-1-carboxylic acid is a carboxyl-substituted phenazine compound, a novel fungicidal compound that is highly effective, low in toxicity, and environmentally compatible. Currently, the mechanism of action of phenazine-1-carboxylic acid is believed to involve disrupting the structure and physiological activities of the cell wall and cytoplasmic membrane, thereby exerting its antifungal effect. After repeated screening experiments, phenazine-1-carboxylic acid did not exhibit synergistic effects when combined with various antifungal agents.

[0012] This invention combines two chemical agents and unexpectedly found that a certain mass ratio of imazalil and phenazine-1-carboxylic acid can broaden the antibacterial spectrum, slow down the development of drug resistance, prolong the use time, and improve the control effect.

[0013] When the mass ratio of imazalil to phenazine-1-carboxylic acid is 0.5~5:1 or 1:3.4~5.5, it has a synergistic effect in controlling rice sheath blight, strawberry gray mold, or pepper anthracnose.

[0014] In a preferred embodiment of the present invention, the bactericidal composition comprises imazalil and phenazine-1-carboxylic acid in a mass ratio of 1 to 4:1, or the bactericidal composition comprises imazalil and phenazine-1-carboxylic acid in a mass ratio of 1:3.5 to 5.5, and preferably, the bactericidal composition comprises imazalil and phenazine-1-carboxylic acid in a mass ratio of 2 to 4:1.

[0015] In a preferred embodiment of the present invention, the EC of the bactericidal composition 50 The concentration was 0.08~0.6 μg / mL;

[0016] The pH of the environment during the use of the bactericidal composition is ≤7.

[0017] In a preferred embodiment of the present invention, the total mass concentration of the bactericidal composition is 0.2~0.5 μg / mL, and preferably, the total mass concentration of the bactericidal composition is 0.3~0.4 μg / mL.

[0018] In a preferred embodiment of the present invention, the bactericidal composition comprises imazalil and phenazine-1-carboxylic acid in a mass ratio of 4 to 5:1; or, the bactericidal composition comprises imazalil and phenazine-1-carboxylic acid in a mass ratio of 1:3.4 to 4.5.

[0019] In a preferred embodiment of the present invention, the EC of the bactericidal composition 50 The concentration ranges from 0.08 to 4.1 μg / mL.

[0020] The pH of the environment during the use of the bactericidal composition is ≤7.

[0021] In a preferred embodiment of the present invention, the total mass concentration of the bactericidal composition is 3.6~4.1 μg / mL, and preferably, the total mass concentration of the bactericidal composition is 3.8~4.0 μg / mL.

[0022] In a preferred embodiment of the present invention, the bactericidal composition comprises imazalil and phenazine-1-carboxylic acid in a mass ratio of 0.5 to 3:1. Preferably, the bactericidal composition comprises imazalil and phenazine-1-carboxylic acid in a mass ratio of 0.5 to 1.5:1.

[0023] In a preferred embodiment of the present invention, the EC of the bactericidal composition 50 The concentration was 2.4–2.8 μg / mL.

[0024] The pH of the environment during the use of the bactericidal composition is ≤7.

[0025] In a preferred embodiment of the present invention, the total mass concentration of the bactericidal composition is 8-12 μg / mL, and preferably, the total mass concentration of the bactericidal composition is 9-10 μg / mL.

[0026] The present invention also discloses the application of a fungicide composition in the prevention and control of rice sheath blight, wherein the fungicide composition comprises imazalil and phenazine-1-carboxylic acid in a mass ratio of 1 to 4:1, or, wherein the fungicide composition comprises imazalil and phenazine-1-carboxylic acid in a mass ratio of 1:3.5 to 5.5, preferably, wherein the fungicide composition comprises imazalil and phenazine-1-carboxylic acid in a mass ratio of 2 to 4:1.

[0027] The total mass concentration of the bactericidal composition refers to the sum of the mass concentrations of all components in the bactericidal composition, such as the sum of the mass concentrations of imazalil and phenazine-1-carboxylic acid.

[0028] In a preferred embodiment of the present invention, the EC of the bactericidal composition 50 The concentration was 0.08~0.6 μg / mL;

[0029] The bactericidal composition is used in environments with a pH ≤ 7. This invention is not suitable for alkaline environments, as this will reduce its effectiveness. It is suitable for slightly acidic environments, which can effectively guarantee the effectiveness. The environment includes soil.

[0030] In a preferred embodiment of the present invention, the total mass concentration of the bactericidal composition is 0.2~0.5 μg / mL, and preferably, the total mass concentration of the bactericidal composition is 0.3~0.4 μg / mL.

[0031] The present invention also discloses the application of a fungicide composition in the prevention and control of gray mold in strawberries, wherein the fungicide composition comprises imazalil and phenazine-1-carboxylic acid in a mass ratio of 4 to 5:1; or, the fungicide composition comprises imazalil and phenazine-1-carboxylic acid in a mass ratio of 1:3.4 to 4.5.

[0032] In a preferred embodiment of the present invention, the EC of the bactericidal composition 50 The concentration ranges from 0.08 to 4.1 μg / mL.

[0033] The pH of the environment during the use of the bactericidal composition is ≤7.

[0034] In a preferred embodiment of the present invention, the total mass concentration of the bactericidal composition is 3.6~4.1 μg / mL, and preferably, the total mass concentration of the bactericidal composition is 3.8~4.0 μg / mL.

[0035] The present invention also discloses the application of a bactericidal composition in the prevention and control of anthracnose in peppers, wherein the bactericidal composition comprises imazalil and phenazine-1-carboxylic acid in a mass ratio of 0.5 to 3:1, preferably, the bactericidal composition comprises imazalil and phenazine-1-carboxylic acid in a mass ratio of 0.5 to 1.5:1.

[0036] In a preferred embodiment of the present invention, the EC of the bactericidal composition 50 The concentration was 2.4–2.8 μg / mL.

[0037] The pH of the environment during the use of the bactericidal composition is ≤7.

[0038] In a preferred embodiment of the present invention, the total mass concentration of the bactericidal composition is 8-12 μg / mL, and preferably, the total mass concentration of the bactericidal composition is 9-10 μg / mL.

[0039] Five ratios of imazalil and phenazine-1-carboxylic acid (4:1, 3:1, 2:1, 1:1, and 1:5) showed synergistic effects against rice sheath blight pathogens. The synergistic coefficient was highest at 4:1 (4.4), followed by 2:1, 1:1, and 3:1, with 1:5 showing the lowest. Similarly, ratios of imazalil and phenazine-1-carboxylic acid (5:1, 4:1, and 1:4) showed synergistic effects against strawberry gray mold pathogens. The synergistic coefficient was highest at 4:1 (2.26), followed by 5:1, and then 1:4. Furthermore, ratios of imazalil and phenazine-1-carboxylic acid (1:1, 2:1, and 3:1) showed synergistic effects against pepper anthracnose pathogens. The synergistic coefficient was 1.99 when the mass ratio of imazalil to phenazine-1-carboxylic acid was 1:1. This patented method uses a lower dosage than existing technologies while achieving better synergistic effects.

[0040] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0041] 1. For the first time, it was discovered that the combination of imazalil and phenazine-1-carboxylic acid has a synergistic effect on rice sheath blight, strawberry gray mold, and pepper anthracnose, which provides a theoretical basis for the subsequent preparation of new pesticides.

[0042] 2. Combining imazalil and phenazine-1-carboxylic acid, two agents with different mechanisms of action, can improve the lifespan of the agent and reduce the likelihood of pathogens developing resistance. 3. This invention provides a compound for inhibiting various plant pathogenic fungi, enriching the pool of candidate fungicides for inhibiting diverse plant pathogenic fungi. Attached Figure Description

[0043] Figure 1 This invention illustrates the synergistic effect of imazalil and phenazine-1-carboxylic acid (4:1) on rice sheath blight pathogen in one embodiment of the present invention.

[0044] Note: A: CK; B: 10μg / mL; C: 2μg / mL; D: 0.4μg / mL; E: 0.08μg / mL; F: 0.016μg / mL.

[0045] Figure 2 This illustrates the synergistic effect of imazalil and phenazine-1-carboxylic acid (2:1) on rice sheath blight pathogen in Example 1 of this invention.

[0046] Note: A: CK; B: 10μg / mL; C: 2μg / mL; D: 0.4μg / mL; E: 0.08μg / mL; F: 0.016μg / mL.

[0047] Figure 3 This illustrates the synergistic effect of imazalil and phenazine-1-carboxylic acid (1:1) on rice sheath blight pathogen in Example 1 of this invention.

[0048] Note: A: CK; B: 10μg / mL; C: 2μg / mL; D: 0.4μg / mL; E: 0.08μg / mL; F: 0.016μg / mL.

[0049] Figure 4 This illustrates the synergistic effect of imazalil and phenazine-1-carboxylic acid (3:1) on rice sheath blight pathogen in Example 1 of this invention.

[0050] Note: A: CK; B: 10μg / mL; C: 2μg / mL; D: 0.4μg / mL; E: 0.08μg / mL; F: 0.016μg / mL.

[0051] Figure 5 This illustrates the synergistic effect of imazalil and phenazine-1-carboxylic acid (4:1) on gray mold of strawberries in Example 2 of this invention.

[0052] Note: A: CK; B: 10μg / mL; C: 2μg / mL; D: 0.4μg / mL; E: 0.08μg / mL; F: 0.016μg / mL.

[0053] Figure 6 This illustrates the synergistic effect of imazalil and phenazine-1-carboxylic acid (5:1) against strawberry gray mold in Example 2 of this invention.

[0054] Note: A: CK; B: 10μg / mL; C: 2μg / mL; D: 0.4μg / mL; E: 0.08μg / mL; F: 0.016μg / mL.

[0055] Figure 7 This illustrates the synergistic effect of imazalil and phenazine-1-carboxylic acid (1:4) against strawberry gray mold in Example 2 of this invention.

[0056] Note: A: CK; B: 10μg / mL; C: 2μg / mL; D: 0.4μg / mL; E: 0.08μg / mL; F: 0.016μg / mL.

[0057] Figure 8 This illustrates the synergistic effect of imazalil and phenazine-1-carboxylic acid (1:1) on anthracnose pathogens in pepper in Example 3 of this invention.

[0058] Note: A: CK; B: 18.18μg / mL; C: 9.09μg / mL; D: 4.55μg / mL; E: 2.27μg / mL; F: 1.13μg / mL.

[0059] Figure 9This illustrates the synergistic effect of imazalil and phenazine-1-carboxylic acid (2:1) on anthracnose pathogens in pepper in Example 3 of this invention.

[0060] Note: A: CK; B: 14.29μg / mL; C: 7.15μg / mL; D: 3.58μg / mL; E: 1.79μg / mL; F: 0.90μg / mL.

[0061] Figure 10 This illustrates the synergistic effect of imazalil and phenazine-1-carboxylic acid (3:1) on anthracnose pathogens in pepper in Example 3 of this invention.

[0062] Note: A: CK; B: 12.90μg / mL; C: 6.45μg / mL; D: 3.28μg / mL; E: 1.64μg / mL; F: 0.82μg / mL. Detailed Implementation

[0063] The technical solution of the present invention will be further described below with reference to specific experiments and embodiments. All percentages involved in the experiments and embodiments of the present invention are mass percentages.

[0064] The raw materials used in this invention are readily available and can be obtained through various means.

[0065] Example 1:

[0066] (1) Test materials

[0067] The pathogen used in this experiment was *Rhizoctonia solani* (the rice sheath blight pathogen). Rhizoctonia solani (Provided by the Hunan Provincial Key Laboratory of Plant Disease and Insect Biology and Control, College of Plant Protection, Hunan Agricultural University)

[0068] The reagents used in this experiment were: 97% imazalil, purchased from Sinopharm Chemical Reagent Co., Ltd.; 92% phenazine-1-carboxylic acid, developed by the Microbial Pesticide Laboratory of Hunan Agricultural University; and other analytical reagents such as anhydrous ethanol, Tween-80, acetone, glucose, and agar strips, all purchased from Hunan Wutai Biotechnology Co., Ltd.

[0069] Test culture medium: PDA medium, 200g potato, 20g glucose, 20g agar, natural pH, add water to make up to 1L, sterilize at 121°C for 20min at 1 atmosphere, and set aside.

[0070] (2) Assay for the toxicity of imazalil and phenazine-1-carboxylic acid

[0071] The inhibitory effects of imazalil and phenazine-1-carboxylic acid on rice sheath blight were determined using the mycelial growth rate method. First, appropriate amounts of imazalil and phenazine-1-carboxylic acid were weighed using filter paper on an analytical balance. The weighed technical materials were poured into a dry conical flask, and an appropriate amount of acetone (not exceeding 0.2% of the mother liquor volume) was added to dissolve both technical materials. Then, an appropriate amount of sterile water was added to prepare a 1000 μg / mL mother liquor (if the water solubility of the technical materials is poor, an appropriate amount of Tween-80 can be added). Finally, the flask was ultrasonically cleaned for 25 min or placed in a water bath at a suitable temperature for 30 min. Sterile water was then added to dilute the mother liquor to 500 μg / mL, 250 μg / mL, 125 μg / mL, and 62.5 μg / mL concentrations. Using a pipette, 9 mL of PDA medium melted to 50–60 °C was placed into a 90 mm diameter Petri dish, followed by 1 mL of the drug solution. The mixture was thoroughly mixed to prepare the drug-containing medium. Simultaneously, an equal volume of sterile water was added to the control. Each treatment was replicated in triplicate. A 6 mm diameter hole was punched at the outermost edge of the Petri dish when the mycelium had grown to approximately 2 / 3 of its height. A mycelial cake with the mycelium attached was placed on the solidified Petri dish using an inoculation loop. The dish was inverted and incubated in a 24 °C incubator in the dark. When the mycelium had grown to 2 / 3 of the Petri dish, the mycelial diameter was measured using a cross-hatching method, and the average value was recorded. The optimal concentrations of the two compounds in the combination for *Rhizoctonia solani* were determined based on the inhibition rates observed at concentrations of 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, and 6.25 μg / mL, as shown in Table 1. The inhibition rates of imazalil and phenazine-1-carboxylic acid against rice sheath blight pathogens were calculated using the following formula. Data were processed using DPS9.01 to obtain correlation coefficients and median inhibitory concentrations (EC50). 50 ) and regression equation.

[0072] (1)

[0073] Acetone has high solubility and good inertness. Tween-80 mainly acts as an emulsifier.

[0074] (3) Synergistic effect of imazalil and phenazine-1-carboxylic acid combination on rice sheath blight pathogen

[0075] The toxicity of a mixture of imazalil and phenazine-1-carboxylic acid against rice sheath blight pathogens was determined using the mycelial growth rate method. Imazalil and phenazine-1-carboxylic acid were mixed according to the ratio given in Table 2. The mixed agent was added to sterilized petri dishes, and PDA medium at 50-60℃ was poured in. An equal volume of sterile water was added to the control. Each treatment was designed with 3 replicates. A 6 mm diameter punch was used to punch holes at the outermost edge of the petri dish when the mycelium had grown to about 2 / 3 of its length. The mycelium cake with mycelium attached was placed on the solidified petri dish using an inoculation loop. The petri dishes were inverted and placed in a constant temperature incubator at 24℃ in the dark. When the mycelium grew to 2 / 3 of the petri dish, the mycelial growth diameter was measured using a cross-sectional method, and the average value was taken. The inhibition rate of the mixed agent was calculated according to the method in formula (1). The data were processed using DPS9.01 to obtain the EC 50 The regression equation is then used. The synergistic coefficient of each compound drug is calculated using the Wadley method to evaluate its synergistic effect.

[0076] EC 50 (th)=(a+b) / [a / EC(A) 50 +b / EC(B) 50 (2)

[0077] SR=EC 50 (th) / EC 50 (ob) (3)

[0078] In the formula, A and B represent two compounded drugs, a and b represent the proportions of the two drugs in the mixture, ob is the actual observed value, and th is the theoretical value. SR>1.5 indicates a synergistic effect; 0.5≤SR≤1.5 indicates an additive effect; and SR<0.5 indicates an antagonistic effect.

[0079]

[0080] The first row shows the concentrations obtained by two-fold dilution, and the second row shows the concentrations obtained by five-fold dilution.

[0081]

[0082] (4) Results and Analysis

[0083] 4.1 Toxicity determination of imazalil and phenazine-1-carboxylic acid against rice sheath blight pathogen

[0084] Table 3 shows that both imazalil and phenazine-1-carboxylic acid have inhibitory effects on rice sheath blight pathogens. Imazalil exhibits the strongest virulence against rice sheath blight pathogens, with an EC50 value of [missing information]. 50 It has a concentration of 0.3818 μg / mL, and the toxicity of phenazine-1-carboxylic acid is second only to that of phenazine-1-carboxylic acid, with an EC50 of 0.3818 μg / mL. 50It is 0.8561 μg / mL, which is 0.4460 times that of imazalil.

[0085]

[0086] 4.2 Synergistic effect of imazalil and phenazine-1-carboxylic acid on rice sheath blight pathogen

[0087] As shown in Table 3, among the ratios used, the synergistic effect was lowest (0.65) when imidacloprid and phenazine-1-carboxylic acid were combined at a ratio of 1:2. The synergistic effect was highest (4.41), nearly seven times that of the 1:2 ratio. Figure 1 When compounded in a 2:1 ratio, the synergistic effect coefficient is 4.1, slightly lower than the maximum synergistic effect coefficient. Figure 2 When compounded in a 1:1 ratio, its synergistic effect coefficient is 3.14, which is approximately 5 times the lowest synergistic effect coefficient. Figure 3 When compounded in a 3:1 ratio, the synergistic effect is 2.35, which is only moderate. Figure 4 ).

[0088] Example 2:

[0089] (1) Test materials

[0090] The pathogen used in this experiment was *Gray Mold of Strawberry* (… Botrytis cinerea (Provided by the Hunan Provincial Key Laboratory of Plant Disease and Insect Biology and Control, College of Plant Protection, Hunan Agricultural University)

[0091] The reagents used in this experiment were: 97% imazalil, purchased from Sinopharm Chemical Reagent Co., Ltd.; 92% phenazine-1-carboxylic acid, developed by the Microbial Pesticide Laboratory of Hunan Agricultural University; and other analytical reagents such as anhydrous ethanol, Tween-80, acetone, glucose, and agar strips, all purchased from Hunan Wutai Biotechnology Co., Ltd.

[0092] Test culture medium: PDA medium, 200g potato, 20g glucose, 20g agar, natural pH, add water to make up to 1L, sterilize at 121°C for 20min at 1 atmosphere, and set aside.

[0093] (2) Assay for the toxicity of imazalil and phenazine-1-carboxylic acid

[0094] The inhibitory effects of imazalil and phenazine-1-carboxylic acid on *Botrytis cinerea*, the causal agent of strawberry gray mold, were determined using the mycelial growth rate method. First, appropriate amounts of imazalil and phenazine-1-carboxylic acid were weighed using weighing filter paper on an analytical balance. The weighed technical materials were poured into a dry conical flask, and an appropriate amount of acetone (not exceeding 0.2% of the mother liquor volume) was added to dissolve both technical materials. Then, an appropriate amount of sterile water was added to prepare a 1000 μg / mL mother liquor (if the water solubility of the technical materials is poor, an appropriate amount of Tween-80 can be added). Finally, the flask was ultrasonically cleaned for 25 min or placed in a water bath at a suitable temperature for 30 min. Sterile water was then added to dilute the mother liquor to concentrations of 500 μg / mL, 250 μg / mL, 125 μg / mL, and 62.5 μg / mL. Using a pipette, 9 mL of PDA medium melted to 50–60 °C was placed into a 90 mm diameter Petri dish, followed by 1 mL of the drug solution. The mixture was thoroughly mixed to prepare the drug-containing medium. Simultaneously, an equal volume of sterile water was added to the control. Each treatment was replicated in triplicate. A 6 mm diameter hole was punched at the outermost edge of the Petri dish when the mycelium had grown to approximately 2 / 3 of its height. A mycelial cake with the mycelium side attached was placed on the solidified Petri dish using an inoculation loop. The dish was inverted and incubated in a 24 °C incubator in the dark. When the mycelium had grown to 2 / 3 of the Petri dish, the mycelial diameter was measured using a cross-hatching method, and the average value was recorded. The concentrations of the two compounds in combination for the fungus *Botrytis cinerea* were determined based on the inhibition rates at concentrations of 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, and 6.25 μg / mL, as shown in Table 4. The inhibition rates of imazalil and phenazine-1-carboxylic acid against strawberry gray mold were calculated using the following formula. Data were processed using DPS9.01 to obtain correlation coefficients and median inhibitory concentrations (EC50). 50 ) and regression equation.

[0095] (1)

[0096] (3) Synergistic effect of imazalil and phenazine-1-carboxylic acid combination on strawberry gray mold.

[0097] The toxicity of the mixture of imazalil and phenazine-1-carboxylic acid against *Botrytis cinerea* causal agent of strawberry was determined using the mycelial growth rate method. Imazalil and phenazine-1-carboxylic acid were mixed according to the ratio given in Table 5. The mixed agent was added to sterilized petri dishes, and PDA medium at 50-60℃ was poured in. An equal volume of sterile water was added to the control. Each treatment was designed with 3 replicates. A 6 mm diameter punch was used to punch holes at the outermost edge of the petri dish when the mycelium had grown to about 2 / 3 of its length. The mycelium cake with mycelium attached was placed on the solidified petri dish using an inoculation loop. The petri dishes were inverted and placed in a constant temperature incubator at 24℃ in the dark. When the mycelium grew to 2 / 3 of the petri dish, the mycelial growth diameter was measured using a cross-sectional method, and the average value was taken. The inhibition rate of the mixed agent was obtained according to the calculation method in (1). The data were processed using DPS9.01 to obtain EC 50 The regression equation is then used. The synergistic coefficient of each compound drug is calculated using the Wadley method to evaluate its synergistic effect.

[0098] EC 50 (th)=(a+b) / [a / EC(A) 50 +b / EC(B) 50 (2)

[0099] SR=EC 50 (th) / EC 50 (ob) (3)

[0100] In the formula, A and B represent two compounded drugs, a and b represent the proportions of the two drugs in the mixture, ob is the actual observed value, and th is the theoretical value. SR>1.5 indicates a synergistic effect; 0.5≤SR≤1.5 indicates an additive effect; and SR<0.5 indicates an antagonistic effect.

[0101]

[0102]

[0103] (4) Results and Analysis

[0104] 4.1 Virulence determination of imazalil and phenazine-1-carboxylic acid against gray mold of strawberry

[0105] As shown in Table 6, both imazalil and phenazine-1-carboxylic acid can inhibit the growth of *Botrytis cinerea*, with imazalil exhibiting the strongest virulence against *Botrytis cinerea*, and its EC50 value being [missing information]. 50 It is 1.80 μg / mL, while the EC of phenazine-1-carboxylic acid is... 50 The concentration was 17.0783 μg / mL, indicating that imazalil is more toxic than phenazine-1-carboxylic acid.

[0106]

[0107] 4.2 Synergistic effect of imazalil and phenazine-1-carboxylic acid on gray mold of strawberry

[0108] As shown in Table 6, the synergistic effect of imazalil and phenazine-1-carboxylic acid in ratios of 4:1, 5:1, and 1:4 was high, with the synergistic coefficient being the largest at 4:1, reaching 2.26. Figure 5 Secondly, the ratio is 5:1, with an efficiency improvement coefficient of 1.91. Figure 6 Of the three, the one with the smallest synergistic effect is the one compounded in a 1:4 ratio. Figure 7 Among the compounding ratios used, the lowest synergistic effect coefficient is 0.28 when compounded at a ratio of 1:5.

[0109] Example 3:

[0110] (1) Test materials

[0111] The pathogen used in this experiment was *Anthracnose causal agent* (…). Colletotrichum capsici (Provided by the Hunan Provincial Key Laboratory of Plant Disease and Insect Biology and Control, College of Plant Protection, Hunan Agricultural University)

[0112] The reagents used in this experiment were: 97% imazalil, purchased from Sinopharm Chemical Reagent Co., Ltd.; 92% phenazine-1-carboxylic acid, developed by the Microbial Pesticide Laboratory of Hunan Agricultural University; and other analytical reagents such as anhydrous ethanol, Tween-80, acetone, glucose, and agar strips, all purchased from Hunan Wutai Biotechnology Co., Ltd.

[0113] Test culture medium: PDA medium, 200g potato, 20g glucose, 20g agar, natural pH, add water to make up to 1L, sterilize at 121°C for 20min at 1 atmosphere, and set aside.

[0114] (2) Assay for the toxicity of imazalil and phenazine-1-carboxylic acid

[0115] The inhibitory effects of imazalil and phenazine-1-carboxylic acid on *Anthracnose causal agent* of pepper anthracnose were determined using the mycelial growth rate method. First, appropriate amounts of imazalil and phenazine-1-carboxylic acid were weighed using weighing filter paper on an analytical balance. The weighed technical materials were poured into a dry conical flask, and an appropriate amount of acetone (not exceeding 0.2% of the mother liquor volume) was added to dissolve both technical materials. Then, an appropriate amount of sterile water was added to prepare a 1000 μg / mL mother liquor (if the water solubility of the technical materials is poor, an appropriate amount of Tween-80 can be added). Finally, the flask was ultrasonically cleaned for 25 min or placed in a water bath at a suitable temperature for 30 min. Sterile water was then added to dilute the mother liquor to 500 μg / mL, 250 μg / mL, 125 μg / mL, and 62.5 μg / mL concentrations. Using a pipette, 9 mL of PDA medium melted to 50–60 °C was placed into a 90 mm diameter petri dish, followed by 1 mL of the drug solution. The mixture was thoroughly mixed to prepare the drug-containing medium. Simultaneously, an equal volume of sterile water was added to the control. Each treatment was replicated in triplicate. A 6 mm diameter hole was punched at the outermost edge of the petri dish when the mycelium had grown to approximately 2 / 3 of its height. A mycelial cake with the mycelium side was placed on the solidified petri dish using an inoculation loop. The dish was inverted and incubated in a 24 °C incubator in the dark. When the mycelium had grown to 2 / 3 of the dish's height, the mycelial diameter was measured using a cross-hatching method, and the average value was recorded. The concentrations of the two compounds in the combination of *Anthracnose causal agent* were determined based on the inhibition rates at concentrations of 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, and 6.25 μg / mL, as shown in Table 7. The inhibition rates of imazalil and phenazine-1-carboxylic acid against *Anthracnose capsulatum* were calculated using the following formula. Data were processed using DPS9.01 to obtain correlation coefficients and median inhibitory concentrations (EC50). 50 ) and regression equation.

[0116] (1)

[0117] (3) Synergistic effect of imazalil and phenazine-1-carboxylic acid combination on anthracnose pathogen of pepper

[0118] The toxicity of a mixture of imazalil and phenazine-1-carboxylic acid against *Anthracnose causal agent* was determined using the mycelial growth rate method. Imazalil and phenazine-1-carboxylic acid were mixed according to the ratio given in Table 8. The mixed agent was added to sterilized petri dishes, and PDA medium at 50-60℃ was poured in. An equal volume of sterile water was added to the control. Each treatment was designed with 3 replicates. A 6 mm diameter punch was used to punch holes at the outermost edge of the petri dish when the mycelium had grown to about 2 / 3 of its length. The mycelium cake with mycelium attached was placed on the solidified petri dish using an inoculation loop. The petri dishes were inverted and incubated in a constant temperature incubator at 24℃ in the dark. When the mycelium grew to 2 / 3 of the petri dish, the mycelial growth diameter was measured using a cross-sectional method, and the average value was taken. The inhibition rate of the mixed agent was obtained according to the calculation method in (1). The data were processed using DPS9.01 to obtain EC 50 The regression equation is then used. The synergistic coefficient of each compound drug is calculated using the Wadley method to evaluate its synergistic effect.

[0119] EC 50 (th)=(a+b) / [a / EC(A) 50 +b / EC(B) 50 (2)

[0120] SR=EC 50 (th) / EC 50 (ob) (3)

[0121] In the formula, A and B represent two compounded drugs, a and b represent the proportions of the two drugs in the mixture, ob is the actual observed value, and th is the theoretical value. SR>1.5 indicates a synergistic effect; 0.5≤SR≤1.5 indicates an additive effect; and SR<0.5 indicates an antagonistic effect.

[0122]

[0123]

[0124] (4) Results and Analysis

[0125] 4.1 Virulence determination of imazalil and phenazine-1-carboxylic acid against *Anthracis capsulatum*.

[0126] As shown in Table 9, both imazalil and phenazine-1-carboxylic acid can inhibit the growth of *Anthracis tricuspidata*, the causal agent of anthracis tricuspidata. Imazalil exhibits the strongest virulence against *Anthracis tricuspidata*, with its EC50 being the highest. 50 The EC50 of phenazine-1-carboxylic acid is 2.7375 μg / mL. 50 The EC50 of phenazine-1-carboxylic acid is 24.94 μg / mL. 50 It is about nine times more toxic than imazalil. This indicates that imazalil is more toxic to Phytophthora capsici than phenazine-1-carboxylic acid.

[0127]

[0128] 4.2 Synergistic effect of imazalil and phenazine-1-carboxylic acid on anthracnose pathogens of pepper

[0129] As shown in Table 9, among the used ratios, the synergistic coefficients were relatively large when the ratios were 1:1, 2:1, and 3:1, respectively, with values ​​of 1.99, 1.43, and 1.41. Figures 8-10 The synergistic effect coefficient is the smallest when the compound is made in a 5:1 ratio, which is 0.22, far lower than the synergistic effect coefficient when the compound is made in a 1:1 ratio.

[0130] Example 4:

[0131] (1) Test materials

[0132] The pathogen used in this experiment was *Rhizoctonia solani* (the rice sheath blight pathogen). Rhizoctonia solani (Provided by the Hunan Provincial Key Laboratory of Plant Disease and Insect Biology and Control, College of Plant Protection, Hunan Agricultural University)

[0133] The reagents used in this experiment were: 97% imazalil, purchased from Sinopharm Chemical Reagent Co., Ltd.; 92% phenazine-1-carboxylic acid, developed by the Microbial Pesticide Laboratory of Hunan Agricultural University; and other analytical reagents such as anhydrous ethanol, Tween-80, acetone, glucose, and agar strips, all purchased from Hunan Wutai Biotechnology Co., Ltd.

[0134] Test culture medium: PDA medium, 200g potato, 20g glucose, 20g agar, natural pH, add water to make up to 1 L, sterilize at 121°C for 20 min at 1 atmosphere, and set aside.

[0135] (2) The control efficacy of iramazole combined with phenazine-1-carboxylic acid on detached leaves of rice sheath blight

[0136] The compound of imazalil and phenazine-1-carboxylic acid was prepared into solutions with final concentrations of 0 μg / mL, 0.0974 μg / mL, and 0.3880 μg / mL. A measured amount of the solution was evenly applied to the surface of rice leaves. After drying, rice sheath blight fungal cakes were inoculated for 72 h. Each treatment was repeated 3 times. The leaves were placed in an artificial climate chamber at 26-28℃ and kept moist for 96 h. The diameter of the colony was measured using the cross-cross method. The inhibition rate was calculated according to formula (1) to evaluate the control effect.

[0137] (3) Results and Analysis

[0138] The combination of imazalil and phenazine-1-carboxylic acid (PPA) showed control efficacy against rice sheath blight. Higher concentrations of the PPA combination resulted in better control (Table 10). The best control effect against rice sheath blight was observed at a concentration of 0.3880 μg / mL, which was 2.23 times higher than the lowest concentration. Analysis of variance showed that different treatments significantly controlled rice sheath blight. In conclusion, this study demonstrates that the PPA combination has good control efficacy against rice sheath blight and can be considered a candidate drug for its control.

[0139]

[0140] Example 5:

[0141] (1) Test materials

[0142] The pathogen used in this experiment was *Gyromitra spp.* (Strawberry gray mold). Botrytis cinerea (Provided by the Hunan Provincial Key Laboratory of Plant Disease and Insect Biology and Control, College of Plant Protection, Hunan Agricultural University)

[0143] The reagents used in this experiment were: 97% imazalil, purchased from Sinopharm Chemical Reagent Co., Ltd.; 92% phenazine-1-carboxylic acid, developed by the Microbial Pesticide Laboratory of Hunan Agricultural University; and other analytical reagents such as anhydrous ethanol, Tween-80, acetone, glucose, and agar strips, all purchased from Hunan Wutai Biotechnology Co., Ltd.

[0144] Test culture medium: PDA medium, 200 g potato, 20 g glucose, 20 g agar, natural pH, add water to make up to 1 L, sterilize at 121°C for 20 min at 1 atmosphere, and set aside.

[0145] (2) The control efficacy of thiamethoxam and phenazine-1-carboxylic acid combination on detached fruit of strawberry gray mold

[0146] The compound of imazalil and phenazine-1-carboxylic acid was prepared into solutions with final concentrations of 0 μg / mL, 0.9693 μg / mL, and 3.8772 μg / mL. A measured amount of the solution was evenly applied to the surface of strawberry fruit. After drying, strawberry gray mold fungal cakes were inoculated for 72 hours. Each treatment was repeated 3 times. The fruit was placed in an artificial climate chamber at 26-28℃ and kept moist for 96 hours. The diameter of the colony was measured using the cross-cross method. The inhibition rate was calculated according to formula (1) to evaluate the control effect.

[0147] (3) Results and Analysis

[0148] The results of this experiment show that the compound of imazalil and phenazine-1-carboxylic acid is effective in controlling strawberry gray mold. Higher concentrations of the imazalil-1-carboxylic acid compound resulted in better control of strawberry gray mold (Table 11). The best control effect against strawberry gray mold was observed at a concentration of 3.8772 μg / mL, which was 2.49 times higher than the lowest concentration. Notably, the analysis of variance showed significant differences between different treatments. Therefore, the imazalil-1-carboxylic acid compound can be used as a fungicide for the prevention and control of strawberry gray mold.

[0149]

[0150] Example 6:

[0151] (1) Test materials

[0152] The pathogen used in this experiment was *Anthracnose causal agent* (…). Colletotrichum capsici (Provided by the Hunan Provincial Key Laboratory of Plant Disease and Insect Biology and Control, College of Plant Protection, Hunan Agricultural University)

[0153] The reagents used in this experiment were: 97% imazalil, purchased from Sinopharm Chemical Reagent Co., Ltd.; 92% phenazine-1-carboxylic acid, developed by the Microbial Pesticide Laboratory of Hunan Agricultural University; and other analytical reagents such as anhydrous ethanol, Tween-80, acetone, glucose, and agar strips, all purchased from Hunan Wutai Biotechnology Co., Ltd.

[0154] Test culture medium: PDA medium, 200 g potato, 20 g glucose, 20 g agar, natural pH, add water to make up to 1 L, sterilize at 121°C for 20 min at 1 atmosphere, and set aside.

[0155] (2) The control effect of thiamethoxam and phenazine-1-carboxylic acid combination on detached fruit of pepper anthracnose

[0156] The compound of imazalil and phenazine-1-carboxylic acid was prepared into solutions with final concentrations of 0 μg / mL, 2.4829 μg / mL, and 9.9316 μg / mL. A measured amount of the solution was evenly applied to the surface of the pepper fruit. After drying, the fruit was inoculated with pepper anthracnose fungal cakes for 72 h. Each treatment was repeated 3 times. The fruit was placed in an artificial climate chamber at 26-28℃ and kept moist for 96 h. The diameter of the colony was measured using the cross-cross method. The inhibition rate was calculated according to formula (1) to evaluate the control effect.

[0157] (3) Results and Analysis

[0158] The results of this experiment show that the compound of imazalil and phenazine-1-carboxylic acid has a certain control effect on anthracnose of pepper. The higher the concentration of the imazalil-1-carboxylic acid compound, the better the control effect on anthracnose (Table 12). The best control effect on anthracnose was observed when the concentration of the imazalil-1-carboxylic acid compound was 9.9316 μg / mL, which was 2.64 times that of the lowest concentration. It is noteworthy that the analysis of variance results showed significant differences between different treatments. In conclusion, the imazalil-1-carboxylic acid compound can be used as a backup agent for the control of anthracnose of pepper.

[0159] .

Claims

1. A fungicidal composition, characterized by The fungicidal composition includes imazalil and phenoxazine-1-carboxylic acid in a mass ratio of 1-5:1, or the fungicidal composition includes imazalil and phenoxazine-1-carboxylic acid in a mass ratio of 1:4-5; The pH of the environment during use of the fungicidal composition is ≤7.

2. The bactericidal composition according to claim 1, characterized by, The fungicidal composition includes imazalil and phenoxazine-1-carboxylic acid in a mass ratio of 1-4:

1.

3. The germicidal composition according to claim 1, characterized in that, The fungicidal composition includes imazalil and phenoxazine-1-carboxylic acid in a mass ratio of 2-4:

1.

4. The germicidal composition according to claim 3, characterized in that, EC of the fungicidal composition 50 is 0.08 to 0.6 pg / mL.

5. The germicidal composition according to claim 3, wherein The total mass concentration of the fungicidal composition is 0.2-0.5 μg / mL.

6. The fungicidal composition according to claim 5, characterized in that, The total mass concentration of the fungicidal composition is 0.3-0.4 μg / mL.

7. The germicidal composition according to claim 1, wherein The fungicidal composition includes imazalil and phenoxazine-1-carboxylic acid in a mass ratio of 4-5:

1.

8. The fungicidal composition according to claim 7, characterized in that, EC of the fungicidal composition 50 is 0.08 to 4.1 pg / mL; The pH of the environment during use of the fungicidal composition is ≤7.

9. The fungicidal composition according to claim 7, wherein The total mass concentration of the fungicidal composition is 3.6-4.1 μg / mL.

10. The fungicidal composition according to claim 9, characterized in that, The total mass concentration of the fungicidal composition is 3.8-4.0 μg / mL.

11. The germicidal composition according to claim 1, wherein The fungicidal composition includes imazalil and phenoxazine-1-carboxylic acid in a mass ratio of 1-3:

1.

12. The fungicidal composition according to claim 11, characterized in that, EC of the fungicidal composition 50 was 2.4-2.8 pg / mL; The pH of the environment during use of the fungicidal composition is ≤7.

13. The germicidal composition of claim 11, wherein, The total mass concentration of the fungicidal composition is 8-12 μg / mL.

14. The fungicidal composition according to claim 13, wherein The total mass concentration of the fungicidal composition is 9-10 μg / mL.

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