Use of the compound pan-ras-in-1 for the preparation of a formulation for the control of magnaporthe oryzae

Abstract

The application belongs to the technical field of plant protection, and relates to application of a compound Pan-RAS-IN-1 in preparation of a preparation for preventing and treating Magnaporthe oryzae. Research finds that the compound Pan-RAS-IN-1 or an agriculturally acceptable salt thereof can inhibit the formation of appressorium in the infection process of Magnaporthe oryzae under the condition of agricultural application, so as to reduce the infection ability of the pathogenic bacteria and achieve the prevention and treatment effect on rice blast. The safety evaluation result shows that the compound does not have obvious adverse effects on the growth of rice in the effective prevention and treatment concentration range. The application provides a new technical scheme for developing a novel rice blast prevention and treatment agent in the action stage.

Description

Technical Field

[0001] This invention belongs to the field of pesticide and plant disease control technology, specifically relating to the agricultural use of an active compound that controls rice blast disease by inhibiting the formation of key structures for rice blast fungus infection. Background Technology

[0002] Rice ( Oryza sativa Rice (L.) is one of the world's most important food crops, and its stable and high yields are crucial for ensuring food security. However, during rice production, the rice blast fungus (L.) can cause serious damage. Magnaporthe oryzae Rice blast, caused by blast disease, has long been a serious threat to rice yield and quality, and is one of the most destructive fungal diseases in the world.

[0003] Currently, the control of rice blast mainly relies on chemical fungicides, including sterol biosynthesis inhibitors, mitochondrial respiration inhibitors, and melanin synthesis inhibitors. While these agents have some efficacy in production, long-term, single-use application can easily lead to the accumulation of drug resistance in rice blast fungi, resulting in decreased control efficacy. Furthermore, their targets are relatively concentrated, making it difficult to meet the needs of sustainable rice blast control.

[0004] During the infection process of rice blast fungus, the pathogen undergoes multiple stages, including spore germination, mycelial growth, and differentiation of infection structures. Among these, the appressorium is a highly specialized structure essential for the fungus to invade the host plant; its formation and maturation directly determine whether the pathogen can successfully infect the host. Studies have shown that once the appressorium formation process is inhibited, the rice blast fungus will struggle to effectively invade, and its pathogenicity will be significantly reduced.

[0005] Therefore, intervening in the formation process of key structures involved in rice blast fungus infection is considered an effective control strategy that differs from traditional fungicide methods and can reduce the risk of disease occurrence from the source of infection. However, existing rice blast control agents mainly work by inhibiting the basic growth or metabolic processes of the pathogen, such as the invention patents with publication numbers CN117717073A and CN117204431A. Control technologies targeting the appressorium formation process are still relatively lacking. Summary of the Invention

[0006] The purpose of this invention is to provide a novel mechanism for the prevention and control of rice blast disease. By inhibiting the formation of appressorium during the infection process of rice blast fungus, the infectivity of the pathogen is reduced, thereby achieving effective prevention and control of rice blast disease.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention provides the compound Pan-RAS-IN-1 or an agriculturally acceptable salt thereof for the preparation of a fungicide for the control of rice blast fungus. Magnaporthe oryzaeApplication in formulations of [compound name missing]. Studies have found that, within agriculturally acceptable application concentrations, the compound significantly inhibits the formation of appressoriums during rice blast fungus infection, while having a relatively weak inhibitory effect on basal mycelial growth, thus effectively reducing the pathogenicity of the pathogen without relying on strong antifungal agents.

[0008] The molecular formula of the compound Pan-RAS-IN-1 is C 36 H 41 Cl2F3N6O2 has the following chemical structural formula: .

[0009] This invention also provides the use of compound Pan-RAS-IN-1 or an agriculturally acceptable salt thereof in the preparation of formulations that inhibit appressorium formation during infection by rice blast fungus, wherein the structural formula of compound Pan-RAS-IN-1 is as follows: .

[0010] As a preferred technical solution, when applying the compound Pan-RAS-IN-1, the effective application concentration is 25-100 μM.

[0011] As a preferred technical solution, the preparation is applied to the plant by foliar spraying, and is applied before or in the early stage of infection by rice blast fungus (in this embodiment of the invention, the preparation is applied 24 hours after inoculation with rice blast fungus).

[0012] Preferably, the formulation is a pesticide, but it is not limited thereto.

[0013] The present invention also provides a method for controlling rice blast fungus, comprising spraying compound Pan-RAS-IN-1 or its agriculturally acceptable salt onto the leaves of plants, wherein the structural formula of compound Pan-RAS-IN-1 is as follows: .

[0014] As a preferred technical solution, the effective application concentration of the compound Pan-RAS-IN-1 is 25-100 μM.

[0015] As a preferred technical solution, the plant is rice.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention achieves the control effect by inhibiting the formation of appressorium, a key structure for rice blast fungus infection. The action stage is clear, which is different from traditional fungicides that mainly inhibit basic growth. (2) Within the effective control concentration range, this compound shows good safety for rice growth and has potential for agricultural application; (3) It provides a new technical path for developing rice blast control technologies with novel targets and low risk of drug resistance. Attached Figure Description

[0017] Figure 1 These are morphological observation photographs showing the effects of different concentrations of the compound Pan-RAS-IN-1 on the colony growth of rice blast fungus.

[0018] Figure 2 This is a bar chart showing the colony diameter of different concentrations of the compound Pan-RAS-IN-1 inhibiting the growth of rice blast fungus.

[0019] Figure 3 These are photomicrographs of appressorium formation of rice blast fungus after treatment with different concentrations of the compound Pan-RAS-IN-1.

[0020] Figure 4 This is a statistical bar chart showing the effect of different concentrations of compound Pan-RAS-IN-1 on the appressorium formation rate of rice blast fungus; different letters indicate statistically significant differences (P<0.05).

[0021] Figure 5 These are photographs of the disease development on detached barley leaves 96 hours after inoculation with rice blast fungus spore suspensions treated with different concentrations of the compound Pan-RAS-IN-1.

[0022] Figure 6 These are photos showing the development of diseases on living rice leaves 6 days after treatment with different concentrations of the compound Pan-RAS-IN-1.

[0023] Figure 7 This is a statistical bar chart showing the effect of different concentrations of the compound Pan-RAS-IN-1 on the area of ​​lesions on living rice leaves; different letters indicate statistically significant differences (P<0.05).

[0024] Figure 8 These are photographs showing the effects of compound Pan-RAS-IN-1 applied 24 hours before (prevention) and 24 hours after (treatment) inoculation on the development of diseases in living rice leaves.

[0025] Figure 9 This is a statistical bar chart showing the effect of applying compound Pan-RAS-IN-1 at different times before and after inoculation on the area of ​​lesions on living rice leaves; different letters indicate statistically significant differences (P<0.05).

[0026] Figure 10 These are photos of a safety test observation of the effects of different concentrations of the compound Pan-RAS-IN-1 on rice seed germination.

[0027] Figure 11 These are phenotypic photographs showing the effects of different concentrations of the compound Pan-RAS-IN-1 on the growth and development of potted rice plants.

[0028] Figure 12 This is a statistical bar chart showing the effect of different concentrations of the compound Pan-RAS-IN-1 on the plant height of rice; where ns indicates P>0.05.

[0029] The discrete small circles in the figure represent different samples. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention.

[0031] 1. Materials and Sources 1.1 General Description Unless otherwise stated, the raw materials, reagents and equipment used in the embodiments of the present invention can be purchased commercially; the experimental procedures involved, unless otherwise specified, are conventional technical methods in the field.

[0032] 1.2 Test strains The rice blast fungus used in this experiment ( Magnaporthe oryzae This is the wild-type strain Guy11 preserved in the laboratory. This strain originated from the American Type Culture Collection (ATCC), product number: ATCC 201236.

[0033] 1.3 Culture medium formulation This experiment used complete medium (CM) for the culture of rice blast fungus. The specific formula of CM medium (per 1L) is as follows: Main ingredients: 10g glucose, 14g peptone, 1g casein amino acids, 1g yeast extract; macroelements: 6g NaNO3, 1.52g KH2PO4, 0.52g KCl, 0.52g MgSO4·7H2O; Trace elements and vitamins: Biotin 0.1mg, Vitamin B1 0.1mg, Thiamine 0.1mg, Riboflavin 0.1mg, Niacin 0.1mg, Para-aminobenzoic acid 0.1mg; The trace salt solution contains: Na₂MoO₄·5H₂O 1.5 mg, CuSO₄·5H₂O 1.6 mg, CoCl₂·6H₂O 1.7 mg, MnCl₂·4H₂O 5 mg, FeSO₄·7H₂O 5 mg, H₃BO₃ 11 mg, ZnSO₄·7H₂O 22 mg, and Na₄EDTA·2H₂O 50 mg. Coagulant: 15g agar powder.

[0034] Preparation method: Dissolve the above ingredients in water, adjust the pH to 6.5 with NaOH solution, and finally bring the volume to 1L with distilled water. Autoclave at 121℃ for 30 min, and set aside.

[0035] 1.4 Test Compounds Name: Pan-RAS-IN-1, CAS No.: 1835283-94-7, Source: Purchased from MedChemExpress (MCE), Item No.: HY-101295.

[0036] Preparation of stock solution: Weigh an appropriate amount (e.g., 5 mg) of compound Pan-RAS-IN-1 powder, dissolve it thoroughly in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution, dispense it into individual containers, and store it at -20°C for later use. Dilute with sterile water or culture medium to the required working concentration as needed during experiments.

[0037] Example 1: Determination of the antibacterial activity of compound Pan-RAS-IN-1 1. Experimental Materials and Methods 1.1 Experimental materials: Rice blast fungus ( Magnaporthe oryzae The strain Guy11 was inoculated onto CM plates and incubated in a 25°C incubator for 3-5 days. Once the colonies had grown well, it was used as the test strain.

[0038] 1.2 Preparation of drug-containing plates: The stock solution of compound Pan-RAS-IN-1 was added to sterilized MM medium cooled to 45-50℃ to prepare plates with final concentrations of 12.5, 25, 50, 100, and 150 μM. Plates containing an equal amount of DMSO (<0.1%) served as blank controls.

[0039] 1.3 Inoculation and Cultivation: Use a 4.5 mm diameter punch to create mycelial cakes, and inoculate them in the center of a plate with the mycelial side facing down. Each treatment is repeated 3 times. Incubate at a constant temperature of 25℃ in the dark.

[0040] 1.4 Data Investigation and Statistical Analysis: When the control group colonies grew to near the edge of the dish, the colony diameter was measured using the cross-sectional method. The calculation formula is as follows: Mycelial growth inhibition rate (%) = [(Control group colony diameter - Mycelial cake diameter) - (Treatment group colony diameter - Mycelial cake diameter)] / (Control group colony diameter - Mycelial cake diameter) × 100%. Statistical software was used to establish a virulence regression equation based on the inhibition rate data, and the half-maximum effect concentration (EC50) was calculated. 50 ).

[0041] 2. Results Analysis The test results are as follows Figure 1 , Figure 2 As shown in Table 1.

[0042] like Figure 1 As shown, all concentration treatments in the experiment exhibited significant inhibitory effects on the mycelial growth of rice blast fungus.

[0043] like Figure 2 As shown, the mycelial growth inhibition rate increased significantly with increasing drug concentration, exhibiting a significant dose-dependent effect.

[0044] As shown in Table 1, the toxicity regression analysis revealed that a toxicity regression equation was established based on the inhibition rate data, and the half-maximum effective concentration (EC50) of compound Pan-RAS-IN-1 for inhibiting the mycelial growth of rice blast fungus was calculated. 50 The value was 139.54 μM. It should be noted that this invention is not aimed at reducing EC. 50 Instead of targeting specific pathogens, the control effect is achieved by interfering with key stages of infection. Therefore, the toxicity parameters that are traditionally based on mycelial growth cannot fully reflect its control potential.

[0045] In particular, at the highest tested concentration of 150 μM, compound Pan-RAS-IN-1 exhibited excellent antibacterial activity, with a relative inhibition rate of 63.70%. These results confirm that compound Pan-RAS-IN-1 can significantly inhibit the in vitro growth of rice blast fungus mycelia and has the potential to be developed into a novel anti-rice blast fungus agent.

[0046] Table 1. Indoor toxicity of different concentrations of compound Pan-RAS-IN-1 against rice blast fungus. Example 2: Determination of the inhibitory activity of compound Pan-RAS-IN-1 on appressorium formation of rice blast fungus 1. Test Methods The inhibitory effect of compound Pan-RAS-IN-1 on appressorium formation of *Magnapordia oryzae* was determined using a hydrophobic surface induction method. The specific procedures were as follows: *Magnapordia oryzae* was inoculated onto CM medium and cultured at 25°C for 7-9 days. Mycelia and spores were washed away, mycelia were removed by filtration, and the spore suspension concentration was adjusted to 1×10⁻⁶. 5Spores / mL. Pan-RAS-IN-1 was dissolved in DMSO to prepare a stock solution, which was then diluted with sterile water and mixed with an equal volume of the spore suspension to achieve final concentrations of 12.5, 25, 50, and 100 μM (DMSO final concentration ≤0.1%). Sterile water containing an equal volume of DMSO served as a blank control. A drop of the mixture was placed on a glass slide, placed in a humidified petri dish, and incubated in the dark at 25°C for 24 hours. Appressorium formation was then observed under a microscope. At least 300 spores were counted for each treatment. The formula for calculating the appressorium formation rate (%) was: (Number of spores forming appressoriums / Total number of spores surveyed) × 100%.

[0047] 2. Results Analysis The test results are as follows Figure 3 and Figure 4 As shown.

[0048] like Figure 3 As shown, microscopic observation results indicate that the compound Pan-RAS-IN-1 has a significant inhibitory effect on the formation of appressorium of rice blast fungus, and this effect is dose-dependent.

[0049] like Figure 4 Quantitative analysis showed that the compound exhibited good inhibitory activity at low concentrations. In particular, at concentrations of 50 μM and above, normal appressorium formation was difficult to observe in the field of view, indicating a high relative inhibition rate, suggesting that the compound can effectively block the appressorium differentiation process at this concentration.

[0050] The above results indicate that the compound Pan-RAS-IN-1 can effectively inhibit the formation of rice blast fungus appressoria and has the potential to be used as a preventative fungicide.

[0051] Example 3: Inhibitory effect of compound Pan-RAS-IN-1 on the pathogenicity of rice blast fungus This embodiment combines an in vitro barley leaf inoculation model and a live rice plant inoculation model to comprehensively evaluate the antibacterial activity of compound Pan-RAS-IN-1, and further determines its control effect at different time points before and after inoculation.

[0052] 1. Experimental Materials and Methods (1) Preparation of plant materials.

[0053] In vitro barley: Select healthy 7-day-old barley seedlings grown in greenhouse, cut leaf segments with uniform growth, and place them in petri dishes lined with moisturizing filter paper.

[0054] Live rice: The susceptible variety CO39 was selected and cultured in a greenhouse until it reached 2 weeks of age (3-4 leaf stage). Healthy seedlings with uniform growth were selected for testing.

[0055] (2) Preparation of strain and reagent. Wild-type strains of rice blast fungus were cultured on CM medium for 7-9 days, then washed and filtered to prepare a spore suspension, the concentration of which was adjusted to 1×10⁻⁶. 5 Spores / mL. Dissolve the stock solution of compound Pan-RAS-IN-1 in a system containing 0.2% gelatin (for in vivo spraying) or sterile water (for in vitro droplet addition).

[0056] (3) Experimental design Experiment 1: Pathogenicity inhibition determination at different concentrations. Different concentrations of Pan-RAS-IN-1 (final concentrations of 12.5, 25, 50, and 100 μM) were mixed with equal volumes of spore suspension to simulate the scenario where the agent and the pathogen come into contact simultaneously.

[0057] Barley in vitro inoculation: Using the micro-drop method, 20 μL of the mixed solution was dropped onto the leaf surface.

[0058] Live inoculation of rice: The mixed solution was sprayed evenly onto the surface of the plants using a spraying method. After inoculation, the plants were incubated at 25℃ with a moist environment. Disease spots on barley leaves were assessed 4 days later; the affected area of ​​rice was assessed 6 days later.

[0059] Experiment 2: Determination of control efficacy at different application times. A 25 μM Pan-RAS-IN-1 solution was selected for testing.

[0060] Preventive application (-24h): First spray the drug solution, then incubate in a moist environment for 24 hours before spraying the drug-free spore suspension.

[0061] Therapeutic application (+24h): First, spray with a drug-free spore suspension, then incubate in a moist environment for 24 hours (until pathogens invade), and then spray with the drug solution. Observe and photograph 7 days after inoculation. Using the solvent control group as a baseline, count the lesion area and calculate the control effect under different drug application time windows.

[0062] 2. Results Analysis (1) In detached barley leaves ( Figure 5 ) and on living rice plants ( Figures 6 to 7 The compound Pan-RAS-IN-1 exhibited consistent and significant dose-dependent inhibitory effects on pathogenicity. With increasing drug concentration, the lesion area decreased significantly. Specifically, in treatments at 25 μM and above, detached barley leaves and living rice leaves remained green, with no obvious brown spot or spindle-shaped lesions observed. This result confirms that this compound can effectively control rice blast in different hosts even at relatively low concentrations.

[0063] (2) Compound Pan-RAS-IN-1 showed positive disease inhibition at different application times. Figures 8 to 9Pretreatment 24 hours before inoculation significantly reduced the number and area of ​​lesions in the treated group compared to the control group, indicating that the compound has good protective activity when applied before pathogen infection. Application 24 hours after inoculation resulted in significantly smaller lesion expansion rates and final lesion areas in the treated group compared to the control group. This result suggests that even after pathogen inoculation, the compound can still inhibit disease development to some extent.

[0064] Conclusion: The above results indicate that the compound Pan-RAS-IN-1 can exert antibacterial efficacy when applied before and after inoculation, suggesting that it has a flexible application window in agricultural applications and has the potential to be developed as both a protectant and a therapeutic agent.

[0065] Example 4: Preliminary evaluation of the safety of compound Pan-RAS-IN-1 for rice growth In this embodiment, the effects of compound Pan-RAS-IN-1 on rice seed germination and seedling growth were determined within the effective antibacterial concentration range to preliminarily assess its safety to the host crop.

[0066] 1. Test Methods (1) Rice seed germination test. Plump and uniformly sized rice seeds were selected, soaked in 75% ethanol for 1 min and surface disinfected with 1% sodium hypochlorite solution for 10 min, and then rinsed 3-5 times with sterile distilled water for later use. The paper bed germination method was used. Different concentrations of Pan-RAS-IN-1 treatment solution were added to petri dishes lined with sterile filter paper, with final concentrations of 12.5, 25, 50 and 100 μM (containing 0.1% DMSO). Sterile distilled water containing 0.1% DMSO was used as a blank control. 30 seeds were evenly sown in each dish, and each treatment was replicated 3 times. The seeds were placed in a 30℃ artificial climate chamber for dark and humidified culture. The seed germination status was investigated and the germination rate was calculated after 7 days.

[0067] (2) Rice plant growth experiment. Seedlings with uniform growth from the above germination experiment were selected and transplanted into plastic pots (30 plants / pot) filled with standardized nutrient soil. They were placed under natural light and managed routinely. After 4 weeks of continued cultivation, the plant phenotype was observed and the plant height (vertical height from the soil surface at the base of the plant to the highest leaf tip) was measured. One-way ANOVA was performed on the data using DPS software.

[0068] 2. Results Analysis The test results are as follows Figures 10 to 12 As shown.

[0069] (1) Effects on seed germination. For example... Figure 10As shown, statistical results indicate that within the concentration range of 12.5-100 μM, the germination rate of rice seeds treated with compound Pan-RAS-IN-1 was not significantly different from that of the blank control group. This suggests that the compound did not significantly inhibit the normal germination of rice seeds at the tested concentrations.

[0070] (2) Effects on seedling growth and phenotype. For example... Figure 11 As shown, at the highest test concentration (100 μM), the rice plants in the treatment group showed basically normal growth, with green leaves and no obvious symptoms of typical phytotoxicity such as leaf curling, yellowing, or necrotic spots. The plant type was not significantly different from the control group. Figure 12 As shown, the final plant height of rice in each concentration treatment group (12.5-100μM) ranged from 26.25±5.25cm to 26.63±7.13cm. Compared with the average plant height of the control group (26.06±3.94cm), there was no significant difference in statistical analysis (P>0.05).

[0071] Conclusion: The above results indicate that under the experimental conditions, compound Pan-RAS-IN-1 exhibits good safety in rice seed germination and seedling growth within the effective antibacterial concentration range, suggesting its potential for application in agricultural production.

Claims

1. Compound Pan-RAS-IN-1 or its agriculturally acceptable salt in the preparation of a fungicide for the control of rice blast fungus ( Magnaportheoryzae The application of Pan-RAS-IN-1 in formulations, the chemical structural formula of which is as follows: 。 2. The use of compound Pan-RAS-IN-1 or an agriculturally acceptable salt thereof in the preparation of formulations that inhibit appressorium formation during infection by rice blast fungus, wherein the structural formula of compound Pan-RAS-IN-1 is as follows: 。 3. The application as described in claim 1 or 2, characterized in that, When applied, the effective concentration of the compound Pan-RAS-IN-1 is 25-100 μM.

4. The application as described in claim 3, characterized in that, When applied, the formulation is applied to the plant by foliar spraying.

5. The application as described in claim 4, characterized in that, When applied, the formulation is administered before infection by rice blast fungus.

6. The application as described in claim 1 or 2, characterized in that, The formulation is a pesticide.

7. A method for controlling rice blast fungus, characterized in that, The compound Pan-RAS-IN-1 or its agriculturally acceptable salt was sprayed onto the leaves of the plant. The structural formula of the compound Pan-RAS-IN-1 is as follows: 。 8. The method for controlling rice blast fungus as described in claim 7, characterized in that, The effective application concentration of the compound Pan-RAS-IN-1 is 25-100 μM.

9. The method for controlling rice blast fungus as described in claim 7, characterized in that, The plant in question is rice.

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