A pesticide composition containing melatonin and prochloraz and its application in preventing and treating rubber tree anthracnose

Abstract

The application discloses a pesticide composition containing melatonin and prochloraz and application of the pesticide composition in prevention and treatment of rubber tree anthracnose, and belongs to the technical field of rubber tree plant protection. The concentration of the melatonin is 3-18 mmol / L, and the concentration of the prochloraz is 0.005-0.015 mu g / mL, based on the total volume of the pesticide composition. The test proves that the two components have a significant synergistic effect, the combination index is less than 1, the nutrient growth and pathogenicity of the rubber tree anthracnose fungus can be effectively inhibited, and the prevention and treatment effect is significantly improved. The application reduces the dosage of the chemical pesticide prochloraz, reduces environmental residues and pathogenic bacteria resistance risk, simultaneously enhances the disease resistance of the rubber tree by using the melatonin, and provides a new scheme for green and efficient prevention and control of the rubber tree anthracnose.

Description

Technical Field

[0001] This invention belongs to the field of rubber tree plant protection technology, and relates to a pesticide composition containing melatonin and imazalil and its application in the prevention and control of anthracnose in rubber trees. Background Technology

[0002] Anthracnose is one of the most destructive diseases in natural rubber production, caused by the fungus *Anthracnose* (…). Colletotrichum This disease is caused by infection with spp. (rubra). It primarily affects the leaves, young shoots, and fruits of rubber trees, forming typical lesions that lead to premature leaf drop and fruit rot. In severe cases, it can force delays in tapping, significantly impacting the yield and quality of natural rubber. Currently, control of this disease mainly relies on the regular application of chemical fungicides, with broad-spectrum fungicides such as prochloraz and pyraclostrobin being the most widely used.

[0003] However, the long-term or repeated use of chemical fungicides has brought about multiple problems: on the one hand, pathogens are becoming increasingly resistant to commonly used agents, leading to decreased control effectiveness and continuously increasing dosages; on the other hand, the extensive use of chemical agents has exacerbated environmental pollution, negatively impacting non-target organisms and soil microbial communities. Therefore, developing new control products that can reduce the amount of chemical pesticides used while simultaneously achieving highly effective fungicide application and enhancing the plant's own disease resistance has become an important direction for the green control of anthracnose in rubber trees.

[0004] Imazalil is a broad-spectrum imidazole fungicide that effectively inhibits the vegetative growth and reproduction of anthracnose fungi by suppressing the biosynthesis of ergosterol in fungal cell membranes, thereby disrupting cell membrane structure and function. However, long-term reliance on imazalil alone carries the risk of resistance, and its fungicidal spectrum and site of action are relatively limited. Melatonin (N-acetyl-5-methoxytryptamine) is a natural endogenous indoleamine compound. Recent studies have revealed that it has multiple physiological functions in plants: it can act as a broad-spectrum stress-resistance signaling molecule, inducing systemic resistance in plants and enhancing the host's defense response against pathogens; it can also directly inhibit the hyphal growth and spore germination of various pathogens.

[0005] However, there is currently no systematic study on the combined effects of melatonin and imazalil on the anthracnose pathogen of rubber trees. The optimal ratio, synergistic effect, and impact on the pathogenicity of the anthracnose pathogen are all lacking in clear technical solutions. Summary of the Invention

[0006] The purpose of this invention is to provide a synergistic pesticide composition containing melatonin and imazalil, which achieves a significant synergistic control effect through a specific ratio, providing a new technical option for the green control of anthracnose in rubber trees.

[0007] The first aspect of the present invention provides a pesticide composition containing melatonin and imazalil, wherein the pesticide composition comprises two active ingredients, melatonin and imazalil, and the concentration of melatonin is 3-18 mmol / L and the concentration of imazalil is 0.005-0.015 μg / mL by total volume.

[0008] Furthermore, the concentration of melatonin is 9 mmol / L, and the concentration of imazalil is 0.01 μg / mL.

[0009] Furthermore, it also includes agriculturally acceptable auxiliary materials.

[0010] In the embodiments, when the present inventors combined melatonin and imazalil within the aforementioned specific concentration range, they discovered a significant synergistic effect between the two. Combination index (CI) measurements showed that the CI values ​​were all less than 1 for the three tested ratios. Particularly when the melatonin concentration was 9 mmol / L and the imazalil concentration was 0.01 μg / mL, the combination index was as low as 0.00962, exhibiting a very strong synergistic effect. This composition not only effectively inhibits the vegetative growth and conidial germination of anthracnose fungus on rubber trees, but also significantly reduces the pathogenicity of the fungus on rubber tree leaves, inhibiting the formation and spread of lesions.

[0011] A second aspect of the present invention provides the application of the aforementioned pesticide composition in the control of anthracnose in rubber trees.

[0012] Furthermore, the application is to inhibit the mycelial growth of anthrax bacteria.

[0013] Furthermore, the pesticide composition is applied to rubber trees or the rubber tree cultivation environment. After application, the final concentration of melatonin at the site of action on the rubber tree is 3–18 mmol / L, and the final concentration of imazalil is 0.005–0.015 μg / mL.

[0014] Furthermore, the final concentration of melatonin at the application site on the rubber tree was 9 mmol / L, and the final concentration of imazalil was 0.01 μg / mL.

[0015] A third aspect of the present invention provides a method for preventing and controlling anthracnose in rubber trees, comprising applying the pesticide composition to rubber trees or the rubber tree cultivation environment.

[0016] Furthermore, after application of the pesticide composition, the final concentration of melatonin at the site of action on the rubber tree is 3–18 mmol / L, and the final concentration of prochloraz is 0.005–0.015 μg / mL.

[0017] Furthermore, after application of the pesticide composition, the final concentration of melatonin at the site of action on the rubber tree is 9 mmol / L, and the final concentration of imazalil is 0.01 μg / mL.

[0018] The beneficial effects of this invention are: (1) The synergistic use of melatonin and prochloraz can significantly reduce the amount of prochloraz used while ensuring excellent control effect, thereby reducing chemical pesticide residues and environmental risks. (2) Melatonin, as a natural endogenous substance, has the function of inducing systemic resistance in plants, is not prone to developing drug resistance, and is conducive to delaying the evolution of pathogen resistance to prochloraz. (3) The composition can be further prepared into emulsifiable concentrates, suspensions, wettable powders, water emulsions, microemulsions or microcapsule formulations by adding agriculturally acceptable excipients to meet diverse application needs and is suitable for promotion and application in rubber tree planting areas. Attached Figure Description

[0019] Figure 1 The study aimed to achieve a synergistic effect between melatonin and prochloraz in inhibiting the vegetative growth of anthracnose fungus on rubber trees. MT: melatonin; PCZ: prochloraz; MT+PCZ: a mixture of melatonin and prochloraz.

[0020] Figure 2 The inhibition rate of melatonin and prochloraz synergistic inhibition of anthracnose pathogens of rubber trees was determined. MT: melatonin, PCZ: prochloraz; lowercase letters in the bar chart indicate significance at the P<0.05 level.

[0021] Figure 3 To investigate the synergistic inhibition of the pathogenic phenotype of *Trichoderma rubrum* by melatonin and prochloraz. CK: control; MT: melatonin, 9 mM; PCZ: prochloraz, 0.01 μg / mL; MT+PCZ: a mixture of melatonin and prochloraz, with concentrations of 9 mM and 0.01 μg / mL, respectively. Detailed Implementation

[0022] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0023] The anthracnose fungus used in the following examples is *Anthracnose sirenus*, the dominant field species of anthracnose in rubber trees. Colletotrichum siamense The strain is available to the public from the Rubber Research Institute of the Chinese Academy of Tropical Agricultural Sciences. The applicant has committed to distributing the biological material to the public within twenty years from the date of application.

[0024] Example 1: Inhibitory effect of melatonin and prochloraz, alone and in combination, on the vegetative growth of anthracnose fungus on rubber trees. (1) The anthracnose fungus of rubber tree was inoculated in the center of a PDA plate and cultured in a constant temperature incubator at 28 ℃ for 7 days. Subsequently, the edge part of the newly grown hyphae was picked and inoculated into PDB liquid medium and cultured at 28 ℃ and 200 rpm for 4 days. After the culture medium was filtered through a 100 μm cell sieve, a fresh conidia suspension was obtained, and the conidia concentration was determined by microscopic detection. (2) Dilute the freshly prepared conidial suspension to a concentration of 1×10⁻⁶. 5 cells / mL; (3) Prepare drug-containing PDA tablets and set up the following treatment groups: Preparation of melatonin stock solution: Accurately weigh 236.97 mg of melatonin (98%), then add DMSO to dissolve it, making the final volume 1 mL, and then filter it through a 0.22 μm organic phase filter for sterilization.

[0025] Preparation of prochloraz stock solution: Accurately weigh 10 mg of prochloraz technical, then add DMSO to dissolve it completely to make a final volume of 1 mL, which is the 1 mg / mL intermediate stock solution; accurately pipette 100 μL of the above 1 mg / mL intermediate stock solution, add 900 μL of DMSO solution, mix well to obtain 100 μg / mL prochloraz stock solution.

[0026] Control group (CK): PDA plates containing 0.1% DMSO; Melatonin single-dose group (MT): Before PDA medium solidifies, an appropriate amount of melatonin stock solution is added to make the final melatonin concentrations in the plates 3 mM, 9 mM and 18 mM respectively. Prochloraz single-agent group (PCZ): Before the PDA medium solidifies, an appropriate amount of prochloraz stock solution is added to make the final concentrations of prochloraz in the plates 0.005 μg / mL, 0.01 μg / mL, and 0.015 μg / mL, respectively. Mixed group (MT+PCZ): Before the PDA medium solidifies, appropriate amounts of melatonin stock solution and imazalil stock solution are added simultaneously so that the final concentrations of the two agents in the plate correspond one-to-one with the dosages listed in Table 1, namely (3 mM + 0.005 μg / mL), (9 mM + 0.01 μg / mL), and (18 mM + 0.015 μg / mL). After thoroughly mixing the above-prepared culture medium, pour it into a petri dish and air-dry and solidify it in a clean bench. (4) Spot 20 μL of 1×10⁻⁶ solution at the center of each solidified PDA plate. 5Fresh anthrax spores per mL were dried in a sterile laminar flow hood. (5) Seal the dried spore plates with sealing film and incubate them in a 28 ℃ constant temperature incubator for 5 days; (6) After cultivation, the diameter of colonies on each plate was measured using the cross-sectional method. The inhibition rate of mycelial growth by each single agent group and the mixed group was calculated, and the synergistic antibacterial index (combination index, CI) was calculated according to the following formula: Combination Index (CI) = (Actual inhibition rate of the combination group) / (Theoretical additive inhibition rate of the corresponding single-dose group) The effect coefficient is the inhibition fraction in the Chou–Talalay model, calculated by CompuSyn software based on the relative inhibition rate of mycelial growth in each treatment group, and is used to characterize the degree of inhibition of mycelial growth.

[0027] The results are shown in Table 1 and... Figure 1 , Figure 2 As shown.

[0028] Table 1. Synergistic effect of melatonin and imazalil in inhibiting anthracnose pathogens of rubber trees.

[0029] Note: Combination index < 1 indicates that the two drugs have a synergistic effect, combination index = 1 indicates that the two drugs have an additive effect, and combination index > 1 indicates that the two drugs have an antagonistic effect.

[0030] Through systematic screening, the present invention's technical personnel discovered that melatonin and imazalil both exhibited significant synergistic effects (CI < 1) within the tested concentration range. The combination of 9 mmol / L melatonin and 0.01 μg / mL imazalil was determined to be the optimal embodiment of the present invention for the following reasons: (1) The antibacterial effect was equivalent to that of the highest concentration group. Table 1 shows that the effect coefficient of the 9 mM + 0.01 μg / mL group was 0.9999, which was exactly the same as the effect coefficient of the high concentration group of 18 mM + 0.015 μg / mL (0.9999). Both achieved near-complete inhibition at the biological level, and there was no statistically significant difference. In other words, doubling the concentration did not bring any additional therapeutic benefit.

[0031] (2) The synergistic effect was the strongest among the three groups. The combination index (CI) is a core indicator for quantifying the strength of synergistic effects; a lower CI value indicates a stronger synergistic effect. The CI value of the 9 mM + 0.01 μg / mL group was 0.00962, the lowest among the three tested ratios (the CI value of the 18 mM + 0.015 μg / mL group was 0.01462), indicating that the two active ingredients in this ratio had the closest synergistic cooperation and the highest interaction efficiency.

[0032] (3) Significantly better in terms of economic efficiency and environmental friendliness Melatonin, as a natural endogenous substance, has low toxicity. However, the application of any exogenous substance in the field should be minimized while ensuring effectiveness. The melatonin dosage in the 9 mM group was only half that in the 18 mM group, and the dosage of prochloraz was also reduced accordingly. This directly reduces the production cost of the formulation and the cost of pesticides for farmers. On the other hand, it reduces the environmental residual load of chemical agents in the soil and water of rubber tree planting areas, which is more in line with the original research and development intention of this invention: "green prevention and control, and reduced pesticide use".

[0033] (4) It helps delay the development of drug resistance in pathogens. One of the basic principles of pesticide resistance management is to "use the lowest effective dose while ensuring efficacy." Adopting a low-dose, high-efficiency regimen of 9 mM + 0.01 μg / mL can reduce the selective pressure on the pathogen population without affecting control efficacy, thereby delaying the development of resistance to prochloraz by anthracnose fungi and extending the effective lifespan of the pesticide.

[0034] Based on the above reasons, this invention uses 9 mmol / L melatonin and 0.01 μg / mL imazalil as the optimal ratio to ensure the best prevention and control effect. This choice takes into account the comprehensive balance of efficacy, synergistic efficiency, economic cost and environmental safety.

[0035] Example 2: Effects of melatonin and prochloraz alone and in combination on the pathogenicity of anthracnose fungus of rubber trees (1) The anthracnose fungus of rubber tree was inoculated in the center of a PDA plate and cultured in a constant temperature incubator at 28 ℃ for 7 days. Subsequently, the edge part of the newly grown hyphae was picked and inoculated into PDB liquid medium and cultured at 28 ℃ and 200 rpm for 4 days. After the culture medium was filtered through a 100 μm cell sieve, a fresh conidia suspension was obtained, and the conidia concentration was determined by microscopic examination. (2) Set up the following treatment groups for spore dilution: Control group (CK): sterile water containing 0.1% DMSO; Melatonin single-dose group (MT): The conidial concentration was adjusted to 1×10⁻⁶ using sterile water. 5Add an appropriate amount of melatonin stock solution to make the final concentration 9 mM, and shake to mix well; Imazalil single-agent group (PCZ): The conidial concentration was adjusted to 1×10 with sterile water. 5 Add an appropriate amount of prochloraz stock solution to make the final concentration 0.01 μg / mL, and shake to mix well; Mixed group (MT+PCZ): The conidial concentration was adjusted to 1×10⁻⁶ using sterile water. 5 Add appropriate amounts of melatonin stock solution and imazalil stock solution to make final concentrations of 9 mM and 0.01 μg / mL, respectively, and shake to mix. (3) Take leaves of rubber trees in the light green stage with basically the same phenology, and inoculate 10 μL of the conidial liquid of each of the above treatment groups into the wound of the leaf using the puncture inoculation method. Then place the leaves in a constant temperature incubator with a temperature of 28 ℃ and a humidity of 95% for moist culture. (4) Observe and photograph the occurrence of lesions on the leaves of each treatment group 4 days after inoculation (e.g., Figure 3 As shown in the figure, the diameter of the lesions was measured simultaneously. By comparing the differences in lesion phenotype and lesion size between the single-agent group (MT, PCZ) and the mixed group (MT+PCZ), the synergistic inhibitory effect of melatonin and prochloraz combined at the optimal ratio of 9 mmol / L melatonin to 0.01 μg / mL prochloraz on the pathogenicity of anthracnose fungus of rubber trees was directly verified.

[0036] Figure 3 The results showed that after treatment with a mixture of melatonin (9 mM) and imazalil (0.01 μg / mL), the anthracnose lesions on rubber tree leaves were significantly smaller than those in the single-agent treatment groups, which directly verified the synergistic inhibitory effect of the two treatments.

Claims

1. A pesticide composition containing melatonin and prochloraz, characterized in that, The pesticide composition contains two active ingredients, melatonin and imazalil. The concentration of melatonin is 3-18 mmol / L and the concentration of imazalil is 0.005-0.015 μg / mL by total volume.

2. The pesticide composition according to claim 1, characterized in that, The concentration of melatonin was 9 mmol / L, and the concentration of imazalil was 0.01 μg / mL.

3. The pesticide composition according to claim 1, characterized in that, It also includes agriculturally acceptable additives.

4. The use of the pesticide composition according to any one of claims 1 to 3 in the control of anthracnose in rubber trees.

5. The application according to claim 4, characterized in that, The application is to inhibit the mycelial growth of anthrax bacteria.

6. The application according to claim 5, characterized in that, When the pesticide composition is applied to rubber trees or the rubber tree cultivation environment, the final concentration of melatonin at the site of action on the rubber tree after application is 3-18 mmol / L, and the final concentration of imazalil is 0.005-0.015 μg / mL.

7. The application according to claim 6, characterized in that, The final concentration of melatonin at the application site on the rubber tree was 9 mmol / L, and the final concentration of imazalil was 0.01 μg / mL.

8. A method for preventing and controlling anthracnose in rubber trees, characterized in that, This includes applying the pesticide composition of any one of claims 1 to 3 to rubber trees or the rubber tree cultivation environment.

9. The method according to claim 8, characterized in that, After application of the pesticide composition, the final concentration of melatonin at the site of action on the rubber tree is 3–18 mmol / L, and the final concentration of prochloraz is 0.005–0.015 μg / mL.

10. The method according to claim 9, characterized in that, After application of the pesticide composition, the final concentration of melatonin at the site of action on the rubber tree is 9 mmol / L, and the final concentration of prochloraz is 0.01 μg / mL.

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