A compound composition and use for preventing and treating pepper fusarium wilt

Abstract

This invention provides compound compositions of two pepper root exudates: 4-methyl-2-oxovalerate and DL-dimethyl malate. The dosage of these compositions for controlling pepper wilt disease is specified, with a specific mass ratio of 6-8:1. The compositions effectively inhibit mycelial growth, spore germination, and sporulation of *Fusarium oxysporum*, the causal agent of pepper wilt, and exhibit a synergistic effect. In pot experiments, the compositions of this invention significantly reduced the incidence of wilt disease. Compared to existing technologies, this invention aims to reduce the use of chemical pesticides through low-cost, safe, and highly effective plant-derived antibacterial compounds.

Description

Technical Field

[0001] This invention relates to the field of crop disease control, specifically to a compound composition for controlling Fusarium wilt in peppers. Background Art

[0002] Fusarium wilt of pepper is a persistent soil-borne disease caused by *Fusarium oxysporum* f.sp. capsici, occurring in tropical, subtropical, and temperate pepper-growing areas, causing severe economic losses to pepper production. This pathogen is a soil-dwelling fungus, highly concealed, and difficult to control. Its spread is closely related to the soil ecosystem, which is directly related to plant growth and development. Roots are an important "window" for nutrient exchange between plants and the soil environment. More than 20% of the carbon source fixed by plant photosynthesis is deposited in the soil in the form of root exudates, playing a crucial role in plant response to environmental stress, regulation of plant adaptation to the microenvironment, shaping of the rhizosphere microbial community, and resistance to or promotion of pathogen infection. On the one hand, root exudates regulate the activity of soil pathogens, altering the soil microecological environment and inducing soil-borne diseases; on the other hand, when subjected to disease stress, plants secrete antimicrobial compounds or recruit beneficial bacteria to achieve biological control of pathogens. Under disease stress, varieties with different resistance exhibit significant differences in the types and quantities of root exudates.

[0003] In actual chili pepper production, Fusarium wilt is quite common. The pathogen's spores overwinter in the soil and diseased plant debris, surviving for several years. Repeated planting of susceptible varieties can lead to a large accumulation of spores in the soil, increasing the difficulty of control. Currently, chemical control is the most widely used method. However, chemical control has increasingly prominent problems such as environmental pollution, disruption of ecological balance, and pesticide residues. Furthermore, long-term, large-scale use of single chemical agents easily leads to drug resistance in pathogens. Chemical-based methods are no longer the most suitable approach. While some biocontrol bacteria, such as *Beauveria bassiana*, *Trichoderma*, *Chaetomium*, bacteria, *Bacillus amyloliquefaciens*, *Bacillus subtilis*, and actinomycetes, show some inhibitory effects in the laboratory, their efficacy is unstable under the complex environmental conditions of the field due to limitations in strain application and the synchronous evolution of pathogens. To date, there is still a lack of commercially available biological agents. Therefore, developing safe and efficient biological control agents (products) for chili pepper Fusarium wilt has become an urgent priority. Summary of the Invention

[0004] This invention applies a compound composition of two pepper root exudates, 4-methyl-2-oxovalerate and DL-malate dimethyl ester, to the control of pepper wilt disease, aiming to reduce the use of chemical pesticides through a low-cost, safe, and efficient plant-derived antibacterial compound control method.

[0005] The present invention provides a compound composition comprising DL-dimethyl malate and 4-methyl-2-oxovalerate in a mass ratio of 6-8:1.

[0006] The present invention also provides the use of the above-described compound composition for the prevention and control of wilt disease in peppers.

[0007] Furthermore, the concentration of the above-mentioned compound composition is 1.0~3.0 mg / mL.

[0008] The present invention also provides the use of the above-described compound composition for the control of Fusarium capsici.

[0009] Furthermore, the above-mentioned compound composition inhibits the mycelial growth, spore germination, and sporulation of Fusarium wilt pathogens in pepper.

[0010] Furthermore, the EC50 of the above compound composition on the growth of Fusarium wilt mycelium of pepper is 2-3 mg / mL.

[0011] Furthermore, the EC50 of the above compound composition on the growth of Fusarium wilt mycelium of pepper is 1-2 mg / mL.

[0012] Dimethyl DL-malate is an esterified derivative of DL-malic acid. In production, it can be obtained through the esterification reaction of malic acid and methanol under an acidic catalyst, or through hydrolysis of maleic acid and fumaric acid, followed by separation from the equilibrium products. This compound has wide applications in food, pharmaceuticals, daily chemical products, environmental protection, and agriculture. In food, it can be used as a food additive to enhance the aroma and taste of food; in pharmaceuticals, it has antioxidant and anti-inflammatory effects; in daily chemical products, it has moisturizing, anti-wrinkle, and whitening effects. However, whether dimethyl DL-malate directly inhibits the growth of pathogenic microorganisms, its antibacterial activity, and its dosage are still unclear.

[0013] 4-Methyl-2-oxovalerate is an organic acid produced through abnormal metabolism in plant roots and is currently mainly used in the chemical, pharmaceutical, pesticide, coating, and dye industries. While it lacks direct structural similarity to known pesticide active ingredients, its chemical structure can be modified to acquire specific biological activities, thereby inhibiting certain pathogenic microorganisms. However, its antibacterial activity, dosage, and the optimal combination ratio with DL-malate dimethyl ester to achieve synergistic disease-fighting effects remain unclear to date.

[0014] This invention provides compound compositions of two pepper root exudates: 4-methyl-2-oxovalerate and DL-dimethyl malate. The dosage of these compositions for controlling Fusarium wilt in peppers was specified, and both compositions were shown to inhibit mycelial growth, spore germination, and sporulation of *Fusarium oxysporum* wilt fungus. In pot experiments, the compositions of this invention significantly reduced the incidence of Fusarium wilt. Compared to existing technologies, this invention aims to reduce the use of chemical pesticides through low-cost, safe, and highly effective plant-derived antibacterial compounds. Attached Figure Description

[0015] Figure 1 The inhibitory effect of DL-dimethyl malate on the mycelial growth of *FOC* and the regression curve. A: Inhibition of mycelial growth; B: Regression curve;

[0016] Figure 2 The inhibitory effect of 4-methyl-2-oxovalerate on the mycelial growth of *FOC* and the regression curve. A: Inhibition of mycelial growth; B: Regression curve;

[0017] Figure 3 The figures show the inhibitory effects of each compound combination on the mycelial growth of *Fusarium oxysporum* var. *fusarium* (FOC) and the regression curves of the 7:1 composition. Note: A represents the inhibitory effect of the 7:1 composition on FOC mycelial growth; B represents the inhibitory effect of the 13:1 composition on FOC mycelial growth; C represents the inhibitory effect of the 19:1 composition on FOC mycelial growth; D represents the regression curve of the 7:1 composition on FOC mycelial growth.

[0018] Figure 4 The regression curve of the 13:1 composition on the growth of FOC mycelia;

[0019] Figure 5 The regression curve of DL-dimethyl malate on FOC spore germination;

[0020] Figure 6 The regression curve for 4-methyl-2-oxovalerate on FOC spore germination;

[0021] Figure 7 Regression curves of the 7:1 composition on FOC spore germination;

[0022] Figure 8 The effect of different concentrations of DL-dimethyl malate and 4-methyl-2-oxovaleric acid on the sporulation rate of FOC;

[0023] Figure 9 The effect of different concentrations of two compositions, DL-dimethyl malate and 4-methyl-2-oxovaleric acid, on the sporulation rate of FOC;

[0024] Figure 10The efficacy of DL-dimethyl malate and 4-methyl-2-oxovalerate and their 7:1 composition at various concentrations against wilt disease in potted peppers was measured. Detailed Implementation

[0025] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to examples. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained in the art without creative effort should fall within the scope of protection of the present invention.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0027] Example 1: Inhibitory effect of the composition on mycelial growth

[0028] 1.1 Effect of DL-dimethyl malate on the mycelial growth of *Fusarium oxysporum* f. sp. *pepifolium*, the causal agent of *Fusarium oxysporum* var. *pepifolium*.

[0029] DL-dimethyl malate was purchased from Haikou Meilan Jinli Instrument Trading Co., Ltd., CAS No.: 617-55-0; 4-methyl-2-oxovalerate was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. The mycelia and spores of *Fusarium oxysporum*, the causal agent of *Fusarium oxysporum* wilt, were isolated, purified, and cultured from pepper plants infected with *Fusarium oxysporum*.

[0030] 20.0 g of DL-dimethyl malate was weighed and dissolved in 40 mL of sterile water to prepare a stock solution with a concentration of 500 mg / mL. PDA agar plates containing this compound were prepared at final concentrations of 10, 20, 40, 80, and 160 mg / mL, with each concentration replicated three times. A sterile water control was included. Subsequently, uniformly grown mycelial discs were ablated from a pre-cultured *Fusarium oxysporum* FOC strain using a 7 mm diameter sterile punch and inoculated into the center of the aforementioned plates. After incubation at 28°C upside down for 7 days, the colony diameters were observed and counted, and the mycelial growth inhibition rate was calculated. The results are shown in Table 1. The average inhibition rate was calculated as follows:

[0031] Table 1. Inhibition of FOC mycelial growth by DL-dimethyl malate after 7 days of treatment.

[0032]

[0033] As shown in the table above, DL-dimethyl malate has a good inhibitory effect on the mycelial growth of *Fusarium oxysporum* var. *capsicum*, the causal agent of *Fusarium oxysporum* var. *capsicum*. Figure 1A) As the concentration continued to rise, the inhibition rate increased significantly. When the concentration was 80.00 mg / mL, the inhibition rate reached 100%.

[0034] Based on the inhibition rate and the logarithm of concentration, a linear regression equation was statistically calculated in the DPS data processing system, and a regression curve was plotted. Figure 1 B). The probability value, EC50, and correlation coefficient r were calculated to determine the concentration of DL-malate required for an inhibition rate exceeding 50%. The results showed that the linear regression equation for the effect of DL-malate on the mycelial growth of *Fusarium oxysporum* causal agent *F. oxysporum* (FOC) was y = 1.8616 + 5.4718x, with a regression intercept of -1.86, a regression coefficient of 5.47, an EC50 value of 17.95 mg / mL, and a correlation coefficient r of 0.9146. Furthermore, the F-test value was 15.35, and the p-value was 0.03, which is less than 0.05, indicating that the established regression model was significant and effective. Therefore, a medium concentration of EC50 of 17.95 mg / mL was found to produce an inhibition rate of over 50% on the mycelial growth of *Fusarium oxysporum* causal agent *F. oxysporum* (FOC).

[0035] 1.2 Effects of 4-methyl-2-oxovalerate on the growth of *Fusarium oxysporum* mycelium (FOC) of *Fusarium oxysporum* var. *capsicum*.

[0036] 0.8 g of 4-methyl-2-oxovalerate was weighed and dissolved in 4 mL of sterile water to prepare a stock solution with a concentration of 200 mg / mL. Based on previous exploratory experiments, PDA medium plates containing this compound were prepared at final concentrations of 0.5, 1, 2, 4, and 8 mg / mL, with each concentration gradient repeated three times, using sterile water as a control. Subsequently, uniformly grown mycelial cakes were punched from pre-cultured *Fusarium oxysporum* FOC strains using a sterile punch with a diameter of 7 mm and inoculated into the center of the above plates. After incubation at 28°C upside down for 7 days, the colony diameter was observed and counted, and the mycelial growth inhibition rate was calculated (Table 2). The statistical and calculation methods were similar to those used for the effect of DL-dimethyl malate on the mycelial growth of *Fusarium oxysporum* FOC strains.

[0037] Table 2. Inhibition of FOC mycelial growth by 4-methyl-2-oxovalerate after 7 days of treatment.

[0038]

[0039] As shown in the table above, 4-methyl-2-oxovalerate has a good inhibitory effect on the mycelial growth of *Fusarium oxysporum* var. *capsicum*, the causal agent of *Fusarium oxysporum* var. *capsicum*. Figure 2 A) As the concentration continued to rise, the inhibition rate increased significantly. At a concentration of 4.00 mg / mL, the inhibition rate was 72.50%; at a concentration of 8.00 mg / mL, the inhibition rate exceeded 90%, reaching 96.67%.

[0040] In DPS software, bioassays were performed, a linear regression equation was established between the logarithmic concentration value and the probability value, and a regression curve was plotted. Figure 2 B). The results showed that the linear regression equation for the effect of 4-methyl-2-oxovalerate on the growth of *Fusarium oxysporum* mycelium (FOC) of *Fusarium oxysporum* was y = 3.5119 + 3.6269x, with a regression intercept of 3.51 and a regression coefficient of 3.63. EC 50 The value was 2.57 mg / mL, and the correlation coefficient r was 0.9914. Furthermore, the F-test value was 171.24, and the p-value was 0.0010, which is less than 0.05, indicating that the established regression model was significant and effective. Therefore, inhibiting medium-concentration EC... 50 At a concentration of 2.57 mg / mL, it exhibited an inhibition rate of over 50% on the mycelial growth of *Fusarium oxysporum* FOC.

[0041] 1.3 Effects of the combination of the two on the mycelial growth of *Fusarium oxysporum* f. sp. *pepifolium*, the causal agent of *Fusarium oxysporum* wilt in peppers (FOC).

[0042] The combination ratios of the two compounds were designed based on their EC50 differences. Regarding their respective EC50 effects on mycelial growth, DL-dimethyl malate had an EC50 of 17.95 mg / mL, while 4-methyl-2-oxovalerate had an EC50 of 2.57 mg / mL, resulting in an EC50 ratio of approximately 7:1. Regarding their respective EC50 effects on spore germination, DL-dimethyl malate had an EC50 of 10.58 mg / mL, while 4-methyl-2-oxovalerate had an EC50 of 0.57 mg / mL, resulting in an EC50 ratio of 19:1. Therefore, the designed composition ratios were 7:1, 19:1, and an intermediate ratio of 13:1. Five final concentration gradients were established for each of the three ratios: 0.8, 1.6, 3.2, 6.4, and 12.8 mg / mL. Each concentration gradient was repeated three times, with sterile water added as a control. The plate culture, survey statistics, and calculation methods are similar to those used for the growth of FOC mycelia of *Fusarium oxysporum* var. *capsicum* using DL-dimethyl malate and 4-methyl-2-oxovalerate. The results are shown in Table 3 below.

[0043] Table 3. Inhibition of FOC mycelial growth by the composition after 7 days of treatment.

[0044]

[0045] The table above shows that the composition 7:1 has the best inhibitory effect on the mycelial growth of *Fusarium oxysporum* var. *citrinum*, the causal agent of *Fusarium oxysporum* var. *citrinum*. Figure 3 As can be seen from A, the inhibition rate was 53.49% at a concentration of 3.2 mg / mL. The inhibition rate increased significantly with increasing concentration, reaching over 90% (99.28%) at a concentration of 12.8 mg / mL. The inhibitory effect of composition 13:1 on the mycelial growth of *Fusarium oxysporum* wilt pathogen (FOC) was second only to composition 7:1. Figure 3 (B, Table 3) At a concentration of 3.2 mg / mL, the inhibition rate was 34.94%; at a concentration of 6.4 mg / mL, the inhibition rate was 44.58%; and at a concentration of 12.8 mg / mL, the inhibition rate also exceeded 85.00%, reaching 87.47%. Figure 3 As shown in C, the 19:1 composition exhibited poor inhibitory effect on mycelial growth, with an inhibition rate of less than 50% at a concentration of 12.8 mg / mL. Therefore, no analysis of variance was performed, and it is not listed in the table.

[0046] Bioassays of the 7:1 and 13:1 compositions were performed in DPS software. A linear regression equation between the logarithmic concentration and the probability value was established, and regression curves were plotted. Figure 3 D and Figure 4The results showed that the linear regression equations for the growth of *Fusarium oxysporum* mycelium (FOC) of the 7:1 and 13:1 compositions on the growth of *Fusarium oxysporum* were y = 3.9523 + 2.6782x and y = 3.5874 + 2.0754x, respectively, with regression intercepts of 3.95 and 3.59, regression coefficients of 2.68 and 2.08, EC50 values ​​of 2.46 and 4.79 mg / mL, and correlation coefficients (r) of 0.9492 and 0.9682, respectively. Furthermore, the F-test values ​​were 27.30 and 44.88, and the p-values ​​were 0.0136 and 0.0068, respectively, all less than 0.05, indicating that the established regression models were significant and effective. The EC50 value of the 7:1 composition was 2.46 mg / mL, corresponding to the concentrations of each active ingredient: DL-dimethyl malate was 2.15 mg / mL, and 4-methyl-2-oxovaleric acid was 0.31 mg / mL. The EC50 values ​​of the 7:1 composition of DL-dimethyl malate and 4-methyl-2-oxovalerate were all lower than their respective EC50 values ​​(DL-dimethyl malate EC50: 17.95 mg / mL, 4-methyl-2-oxovalerate EC50: 2.57 mg / mL), and the differences were significant, indicating that the 7:1 composition of DL-dimethyl malate and 4-methyl-2-oxovalerate has a synergistic effect. The EC50 value of the 13:1 composition was 4.79 mg / mL, and the corresponding concentrations of each active ingredient were 4.45 mg / mL for DL-dimethyl malate and 0.34 mg / mL for 4-methyl-2-oxovalerate. These values ​​were also lower than their respective EC50 values ​​(DL-dimethyl malate EC50: 17.95 mg / mL, 4-methyl-2-oxovalerate EC50: 2.57 mg / mL), and the differences were significant, indicating that the 13:1 composition of DL-dimethyl malate and 4-methyl-2-oxovalerate also has a synergistic effect. However, comparing the EC50 values ​​of the 7:1 and 13:1 compositions and the concentrations of their respective active ingredients, it was found that the EC50 value of the 7:1 composition and the concentration of the active ingredient required to achieve a 50% inhibition rate were both lower than those of the 13:1 composition. From the perspective of mycelial growth inhibition, the 7:1 composition of DL-dimethyl malate and 4-methyl-2-oxovaleric acid had a stronger synergistic effect.

[0047] Example 2: Inhibitory effect of the composition on spore germination

[0048] 2.1 Effect of DL-dimethyl malate on FOC spore germination of Fusarium oxysporum, the causal agent of Fusarium oxysporum var. causal agent of pepper blight

[0049] The *Fusarium oxysporum* FOC strain, the causal agent of *Fusarium oxysporum* var. *pescens*, was removed from a 4°C freezer and inoculated onto PDA plates. It was incubated at 28°C for 7 days in an inverted position. Spores were washed with sterile water, filtered through four layers of sterile gauze, and the filtrate was collected. Centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. The precipitate was resuspended in sterile water, centrifuged (1500 rpm for 5 min), and the supernatant was discarded 2-3 times. Finally, the precipitate was resuspended in 0.5% glucose solution, and the spore suspension concentration was adjusted to 1×10⁵–1×10⁷ spores / mL. Based on previous preliminary experiments, 2.0 mL of DL-dimethyl malate solutions with concentrations of 5.0, 10.0, 20.0, 40.0, and 80.0 mg / mL were prepared using a 0.1% Tween 40 aqueous solution. Transfer 0.5 mL of the above-mentioned concentration solution to a 1.5 mL sterile centrifuge tube, add 0.5 mL of spore suspension, mix thoroughly, and then take 30 μL of the above mixed solution and place it in the center of the concave glass slide. Incubate at 28°C with humidity. Repeat each treatment three times, with sterile water treatment as a control. Observe the germination of control spores every 2 hours. When the spore germination rate reaches 90% or higher, examine the germination of spores at each concentration under a microscope. Calculate the spore germination rate and relative inhibition rate based on the germination of more than 200 spores in three randomly selected fields of view.

[0050] Spore germination rate = (Number of germinated spores in treatment / Total number of spores in treatment) * 100%

[0051] Corrected spore germination rate for each treatment = (Spore germination rate of treated spores / Spore germination rate of control spores) * 100%

[0052] Relative inhibition rate = (control corrected spore germination rate - treatment corrected spore germination rate) / control corrected spore germination rate * 100%.

[0053] The results showed that after 16 hours, the spore germination rate of the control group reached over 90%, and all treatments with DL-dimethyl malate had a certain inhibitory effect on spore germination (Table 4). At a concentration of 5.00 mg / mL, the average inhibition rate was 20.70%; at a concentration of 20.00 mg / mL, the inhibition rate far exceeded 50%, reaching 80.90%; as the concentration continued to increase, the inhibition rate increased significantly, reaching 94.02% at a concentration of 40.00 mg / mL.

[0054] Table 4. Inhibitory effect of DL-dimethyl malate on FOC spore germination

[0055]

[0056] In DPS software, bioassays were performed, a linear regression equation was established between the logarithmic concentration value and the probability value, and a regression curve was plotted. Figure 5The results showed that the linear regression equation of DL-dimethyl malate on the germination of FOC spores of *Fusarium oxysporum* sp. was y = 2.2739 + 2.6605x, with a regression intercept of 2.27 and a regression coefficient of 2.66. EC 50 The concentration was 10.58 mg / mL, and the correlation coefficient r was 0.9960. Furthermore, the F-test value was 374.03, and the p-value was 0.0003, which is less than 0.05, indicating that the established regression model was significant and effective. Therefore, inhibiting medium-concentration EC... 50 At a concentration of 10.58 mg / mL, it exhibited an inhibition rate of over 50% on the germination of FOC spores of *Fusarium oxysporum*. This was compared to the effect on mycelial growth by EC. 50 At a concentration of 17.95 mg / mL, the concentration required to inhibit spore germination is lower, and the inhibitory effect is more significant.

[0057] 2.2 Effect of 4-methyl-2-oxovalerate on the germination of FOC spores of *Fusarium oxysporum*, the causal agent of *Fusarium oxysporum* var. *capsicum*.

[0058] Referring to the inhibition test of DL-dimethyl malate on the germination of FOC spores of *Fusarium oxysporum*, the causal agent of *Fusarium oxysporum* wilt, based on previous preliminary experiments, 2.0 mL of each of 0.1% Tween 40 aqueous solution and 4-methyl-2-oxovalerate solutions at concentrations of 0.2, 0.4, 0.8, 1.6, and 3.2 mg / mL were prepared. Subsequently, following the above-mentioned procedure for the spore germination inhibition test, when the germination rate of the control spores reached over 90%, the germination status of spores at each concentration was examined under a microscope, and the germination rate and relative inhibition rate were calculated.

[0059] The results showed that after 16 hours, the spore germination rate of the control group reached over 90%, and all treatments with 4-methyl-2-oxovalerate had a certain inhibitory effect on spore germination (Table 5). At a concentration of 0.2 mg / mL, the average inhibition rate was only 0.34%; at 0.4 mg / mL, the inhibition rate increased to 34.24%; at 0.8 mg / mL, the inhibition rate far exceeded 50%, reaching 66.75%; with further increases in concentration, the inhibition rate increased significantly, reaching 97.38% at 1.6 mg / mL; and reaching 100% at 3.2 mg / mL.

[0060] Table 5. Inhibitory effect of 4-methyl-2-oxovalerate on FOC spore germination

[0061]

[0062] In DPS software, bioassays were performed, a linear regression equation was established between the logarithmic concentration value and the probability value, and a regression curve was plotted. Figure 6The results showed that the linear regression equation for the germination of *Fusarium oxysporum* FOC spores by 4-methyl-2-oxovalerate was y = 6.4238 + 5.8993x, with a regression intercept of 6.42, a regression coefficient of 5.90, an EC50 of 0.57 mg / mL, and a correlation coefficient r of 0.9804. Furthermore, the F-test value was 74.33, and the p-value was 0.003, which is less than 0.05, indicating that the established regression model was significant and effective. Therefore, when the EC50 concentration was 0.57 mg / mL, it produced an inhibition rate of over 50% on the germination of *Fusarium oxysporum* FOC spores. Compared to the EC50 value of 2.57 mg / mL for mycelial growth, the concentration required to inhibit spore germination was lower, and the inhibitory effect was more significant.

[0063] 2.3 Effects of the combination of DL-dimethyl malate and 4-methyl-2-oxovalerate on the germination of FOC spores of *Fusarium oxysporum*, the causal agent of *Fusarium oxysporum* in pepper.

[0064] Based on previous studies of EC50 differences in mycelial growth and spore germination, compositions containing both were designed. Three composition ratios were designed: 7:1, 19:1, and 13:1. Five final concentration gradients were established for all three ratios. Except for the 7:1 composition, which had concentration gradients of 0.4, 0.8, 1.6, 3.2, and 6.4 mg / mL, the other two ratios had concentration gradients of 0.8, 1.6, 3.2, 6.4, and 12.8 mg / mL. The procedure was the same as the method used in the above-mentioned experiments on the inhibition of spore germination by the monomeric compounds. When the germination rate of the control spores reached over 90%, the spore germination at each concentration of the different composition ratios was examined under a microscope, and the spore germination rate and relative inhibition rate were calculated.

[0065] Table 6. Inhibitory effect of three ratios of the composition on FOC spore germination

[0066]

[0067] The results showed that after 16 hours, the spore germination rate of the control group reached over 90%, and all concentrations of the three ratios of the composition had a certain inhibitory effect on spore germination (Table 6). When the concentration of the 7:1 composition was 0.8 mg / mL, the average inhibition rate was 10.11%; at 3.2 mg / mL, the average inhibition rate was well over 50%, at 92.28%; and at 6.4 mg / mL, the average inhibition rate was close to 100%, at 99.65%. For the 13:1 composition, the average inhibition rate was 2.43% at 0.8 mg / mL; at 3.2 mg / mL, the average inhibition rate was 50.46%; and at 6.4 mg / mL, the average inhibition rate was 82.30%. For the 19:1 composition, the average inhibition rate was 1.12% at 0.8 mg / mL; at 3.2 mg / mL, the average inhibition rate was 32.69%; and at 6.4 mg / mL, the average inhibition rate was 65.62%. A comprehensive comparison of the inhibitory effects of the three ratios of the composition at various concentrations on spore germination reveals that the 7:1 composition requires the lowest concentration to achieve the same inhibition rate. Furthermore, under the same concentration conditions, the 7:1 composition exhibits the most significant inhibitory effect on spore germination. Therefore, the 7:1 composition is the optimal formulation.

[0068] Bioassays were performed in DPS software, and linear regression equations were established between the logarithmic values ​​of the concentrations and probability values ​​of the three ratio compositions (Table 7). The results showed that the linear regression equation for the 7:1 composition on the germination of FOC spores of *Fusarium oxysporum* var. *capsicum* was y = 4.3659 + 3.9081x, with a regression intercept of 4.37 and a regression coefficient of 3.91. EC50... 50 The concentration was 1.45 mg / mL, and the correlation coefficient r was 0.9873. Furthermore, the F-test value was 115.43, and the p-value was 0.0017, less than 0.05, indicating that the established regression model was significant and effective. The linear regression equation for the germination of FOC spores of *Fusarium oxysporum* 'Fusarium oxysporum' in the 13:1 composition was y = 3.5036 + 3.0196x, with a regression intercept of 3.50 and a regression coefficient of 3.02. EC50 was [missing value]. 50 The concentration was 3.13 mg / mL, and the correlation coefficient r was 0.9938. Furthermore, the F-test value was 239.54, and the p-value was 0.0006, less than 0.05, indicating that the established regression model was significant and effective. The linear regression equation for the germination of FOC spores of *Fusarium oxysporum* 'F. causal agent' by the 19:1 composition was y = 3.0799 + 2.9483x, with a regression intercept of 3.08 and a regression coefficient of 2.95. EC50 was [missing value]. 50 The concentration was 4.48 mg / mL, and the correlation coefficient r was 0.9983. Furthermore, the F-test value was 859.36, and the p-value was 0.00009, less than 0.05, indicating that the established regression model was also significant and effective. The EC50 of the three values ​​was compared. 50The concentrations of the active ingredients in the 7:1 composition were 1.45 mg / mL, equivalent to 1.27 mg / mL of DL-malate and 0.18 mg / mL of 4-methyl-2-oxovalerate. The concentrations in the 13:1 composition were 3.13 mg / mL, equivalent to 2.91 mg / mL of DL-malate and 0.22 mg / mL of 4-methyl-2-oxovalerate. The concentrations in the 19:1 composition were 4.48 mg / mL, equivalent to 4.26 mg / mL of DL-malate and 0.22 mg / mL of 4-methyl-2-oxovalerate. All these values ​​were lower than the EC50 values ​​of the individual compounds, indicating that DL-malate and 4-methyl-2-oxovalerate have a synergistic effect on spore germination. Furthermore, the 7:1 composition has the lowest EC50, which translates to the least amount of each active ingredient required, indicating a more significant synergistic effect. This mixture represents the optimal formulation and is suitable for further research and application. Figure 7 ).

[0069] Table 7 Linear regression analysis of the three ratios of the composition on FOC spore germination

[0070] Composition Name Linear equation (y=a+bx) Regression intercept (a) Regression coefficient (b) Correlation coefficient (r) <![CDATA[EC 50 (mg / mL)]]> 7:1 composition y = 4.3659 + 3.9081x 4.37 3.91 0.9873 1.45 13:1 composition y = 3.5036 + 3.0196x 3.50 3.02 0.9938 3.13 19:1 composition y = 3.0799 + 2.9483x 3.08 2.95 0.9983 4.48

[0071] Example 3 Effect of the composition on FOC sporulation

[0072] After measuring the colony growth diameter of each drug solution and its 7:1 composition cultured in a 28℃ constant temperature incubator for 7 days, 10-20 mL of sterile water was added to each plate. Hyphae and spores were scraped off using a sterile spatula or small sterile medicine spoon, ensuring the spores were dispersed in the liquid phase as much as possible. The solution was then filtered through double-layered sterile gauze to remove the mycelia on the filter screen. The collected filtrate was concentrated and centrifuged multiple times (1500 rpm, 5 min), and washed 2-3 times with sterile water before resuspending in 1 mL of sterile water. Finally, 20 μL of each spore suspension was transferred to a hemocytometer (16*25 type), and the number of spores in the four corners and the center was observed under a microscope. The spore concentration, i.e., the sporulation yield per plate, was calculated using the following formula. Each treatment was repeated 3 times, with the colony sporulation yield from culture plates without drug solution cultured for 7 days serving as a blank control. Spore concentration = (Number of spores in the four corner squares + Number of spores in the center square) ÷ 100 × 400 × 10 4 cfu / mL.

[0073] Microscopic observation revealed a negative correlation between the sporulation rate of each compound and its combinations at different ratios and the mycelial growth inhibition rate (Table 8). Higher inhibition rates resulted in lower sporulation rates. Compared to the control, after 7 days of culture on plates, the concentrations of DL-dimethyl malate showed significant differences (p≤0.05). At a concentration of 10 mg / mL, the sporulation rate was (5.93±0.67)*10. 5 The cfu / mL concentration was only half that of the control; at a concentration of 20 mg / mL, the sporulation rate was (3.13 ± 0.35) * 10⁻¹⁰. 5 The cfu / mL concentration was less than one-third of the control; at a concentration of 40 mg / mL, the sporulation rate was (1.17 ± 0.4) * 10⁻⁶. 5 The concentration of cfu / mL was only 1 / 9 of the control; at a concentration of 80 mg / mL, colony growth was completely inhibited, and sporulation was 0. This shows that as the concentration increases, sporulation gradually decreases, and may even be completely inhibited. Figure 8 A). When the concentration of 4-methyl-2-oxovalerate is 0.5 mg / mL, the sporulation rate is (10.03 ± 1.21) * 10⁻⁶. 5 The cfu / mL concentration was not significantly different from the control (p≥0.05); at a concentration of 2 mg / mL, the sporulation yield was (5.07±0.21)*10 5 The cfu / mL concentration was only half that of the control; at a concentration of 4 mg / mL, the sporulation rate was (1.53 ± 0.35) * 10⁻¹⁰. 5 The concentration of cfu / mL was only 1 / 9 of the control; at a concentration of 8 mg / mL, almost no sporulation occurred. Figure 8 B).

[0074] Table 8. Effect of concentrations of compounds and their compositions on FOC sporulation.

[0075]

[0076] The 7:1 and 13:1 compositions of DL-dimethyl malate and 4-methyl-2-oxovaleric acid showed better inhibitory effects on sporulation of FOC (Table 8). Figure 9At a concentration of 0.8 mg / mL, the sporulation rates of the 7:1 and 13:1 compositions were (6.77±0.50)*10⁵ CFU / mL and (7.77±0.55)*10⁵ CFU / mL, respectively, with no significant difference between them (p≥0.05), but significant differences compared to the control (p≤0.05). At a concentration of 3.2 mg / mL, the sporulation rates of the 7:1 and 13:1 compositions were (2.43±0.21)*10⁵ CFU / mL and (4.83±0.35)*10⁵ CFU / mL, respectively, with significant differences between them (p≤0.05), and both were much lower than the control sporulation rate (p≤0.05). This indicates that the inhibitory effect on FOC sporulation continuously increases with increasing composition concentration. Interestingly, the 7:1 composition at a concentration of 3.2 mg / mL exhibited a stronger inhibitory effect on FOC sporulation than the 13:1 composition at a concentration of 6.4 mg / mL, while the sporulation rate was slightly lower (3.00 ± 0.30) * 10⁵ CFU / mL. The individual monomer concentrations of the two compositions were 2.8 and 0.4 mg / mL, and 5.94 and 0.46 mg / mL, respectively. This demonstrates that the 7:1 composition exhibits a better synergistic effect, consistent with its inhibitory effect on mycelial growth and spore germination.

[0077] Example 4: The effect of the compound on potted plant control of wilt disease in peppers.

[0078] Based on the EC50 values ​​of DL-malate (17.95 mg / mL for inhibiting mycelial growth and 10.58 mg / mL for inhibiting spore germination), two concentrations, 10.0 and 20.0 mg / mL, were designed for potted plant efficacy evaluation. Similarly, based on the EC50 values ​​of 4-methyl-2-oxovalerate (2.57 mg / mL for inhibiting mycelial growth and 0.57 mg / mL for inhibiting spore germination), two concentrations, 0.5 and 3.0 mg / mL, were designed for potted plant efficacy evaluation. Finally, based on the optimal ratio of DL-malate and 4-methyl-2-oxovalerate (7:1), three concentrations, 1.0, 2.0, and 3.0 mg / mL, were designed for potted plant efficacy evaluation, indicating an EC50 of 2.46 mg / mL for inhibiting mycelial growth and 1.45 mg / mL for inhibiting spore germination. Each concentration was replicated three times, with 15 pots per replicate, and 2 seedlings per pot, resulting in a total of 90 uniformly growing chili seedlings per concentration. When the chili seedlings had 6 leaves and a bud, they were first inoculated using the method of "needle pricking the base of the stem + 30 ml of each concentration of the compound and a 7:1 mixture per pot for irrigation". The blank control was replaced with sterile water. 24 hours later, each pot was inoculated with 30 ml of spore suspension (107 spores / ml) by irrigation. After 10 days, the number of diseased plants was investigated, and the incidence rate and control effect were statistically analyzed.

[0079] Incidence rate (%) = Number of diseased plants treated / Total number of plants * 100

[0080] Prevention and control efficacy (%) = (Control incidence rate - Treatment incidence rate) / Control incidence rate * 100

[0081] Table 9. Effective control efficacy of compounds and their 7:1 compositions against Fusarium wilt in potted plants.

[0082]

[0083] The results showed that all treatments had good control effects against Fusarium wilt of pepper (Table 9). Figure 10 Among them, DL-dimethyl malate at a concentration of 10.0 mg / mL showed a control efficacy of 48.59% against Fusarium wilt, while at a concentration of 20.0 mg / mL, the control efficacy exceeded 60%, reaching 62.26%. 4-Methyl-2-oxovalerate at a concentration of 0.5 mg / mL showed a control efficacy of 42.04% against Fusarium wilt; the control efficacy increased rapidly with increasing concentration, and at a concentration of 3.0 mg / mL, the control efficacy exceeded 60%, reaching 62.10%. This indicates that both concentrations exceeded their respective EC50 limits for mycelial growth and spore germination. 50After applying DL-dimethyl malate at concentrations of 17.95 and 10.58 mg / mL, and 4-methyl-2-oxovalerate at concentrations of 2.57 and 0.57 mg / mL, the control efficacy can rapidly increase to over 60%. Therefore, DL-dimethyl malate concentrations between 10.0 and 20 mg / mL have good control effects against Fusarium wilt, and 4-methyl-2-oxovalerate concentrations between 0.5 and 3.0 mg / mL also have good control effects against Fusarium wilt, providing a reference for field application. When the concentration of the 7:1 combination of the two compounds was 1.0 mg / mL, the control efficacy was 60.18%, comparable to that of 20.0 mg / mL of DL-malate dimethyl ester and 3.0 mg / mL of 4-methyl-2-oxovalerate, with no significant difference (p≥0.05). However, it was significantly higher than that of 10.0 mg / mL of DL-malate dimethyl ester and 0.5 mg / mL of 4-methyl-2-oxovalerate (p≤0.05). At a concentration of 2.0 mg / mL, the control efficacy exceeded 70%, reaching 71.17%. At this concentration, compared to the individual compound concentrations of 1.75 and 0.25 mg / mL, respectively, it was far lower than the EC50 of each individual compound on mycelial growth and spore germination. When the concentration was further increased to 3.0 mg / mL, the control efficacy exceeded 80%, reaching 82.32%, higher than the control efficacy of other treatments (p≤0.05). This indicates that the 7:1 composition of DL-dimethyl malate and 4-methyl-2-oxovalerate has a strong synergistic effect, and its concentration of 1.0~3.0 mg / mL has a good control effect on Fusarium wilt.

Claims

1. A compound composition, characterized in that: The composition comprises DL-dimethyl malate and 4-methyl-2-oxovalerate in a mass ratio of 6-8:

1.

2. Use of the compound composition as described in claim 1 for the prevention and control of wilt disease in peppers.

3. The use as described in claim 2, characterized in that: The concentration of the compound composition is 1.0~3.0 mg / mL.

4. Use of the compound composition according to claim 1 for the control of Fusarium capsici.

5. The use as described in claim 4, characterized in that: The compound composition inhibits mycelial growth, spore germination, and sporulation of *Fusarium oxysporum*, the causal agent of *Fusarium oxysporum* var. *capsicum*.

6. The use as described in claim 5, characterized in that: The EC50 of the compound composition inhibiting the mycelial growth of *Fusarium oxysporum* var. *capsicum* was 2-3 mg / mL.

7. The use as described in claim 5, characterized in that: The EC50 of the compound composition on the inhibition of spore germination by *Fusarium oxysporum* spores was 1-2 mg / mL.

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