Application of indole-3-propionic acid in the control of rice blast

Abstract

This invention discloses the application of indole-3-propionic acid in the control of rice blast disease, belonging to the technical fields of plant protection and agricultural fungicides. Rice blast disease is caused by *Pyrrosia lingua*. Magnaporthe oryzae Infection-induced disease, this invention is the first to discover that indole-3-propionic acid can inhibit the growth of *Pyrrosia lingua* mycelium, spore germination, and appressorium differentiation. Therefore, using indole-3-propionic acid as the core active ingredient, and adding agricultural adjuvants, a fungicide for controlling rice blast disease was prepared. Indole-3-propionic acid is a natural small molecule, low in toxicity, easily degradable, environmentally friendly, and leaves low pesticide residues in rice. It has a triple effect of inhibiting mycelial infection, spore infection, and inducing plant disease resistance, delaying the development of pathogen resistance. It is simple to use, compatible with existing plant protection equipment, and provides both prevention and treatment, controlling both seedling and neck blast. Furthermore, the raw materials are readily available, the synthesis process is mature, and the cost of large-scale field application is low.

Description

Technical Field

[0001] This invention relates to the fields of plant protection and agricultural fungicides, specifically to the application of natural small molecule indole-3-propionic acid in the control of rice blast disease. Background Technology

[0002] With the development of rice blast disease by *Pyrrosia lingua* Magnaporthe oryza E. blast is a devastating fungal disease affecting rice production, classified into seedling blast, leaf blast, and neck blast. It is characterized by rapid onset, widespread prevalence, and severe yield reduction. Currently, the mainstream field pesticides, tricyclazole and isoprothiolane, have been used alone for a long time, leading to increasing resistance in the pathogen and a significant decline in their effectiveness. Furthermore, chemical pesticide residues can easily cause ecological pollution in farmland and pose risks to rice food safety.

[0003] Indole-3-propionic acid (IPA), with the molecular formula C11H11NO2 and CAS number 830-96-6, is a naturally occurring tryptophan-based indole molecule widely found in plant tissues, soil microorganisms, and animal intestines. Current research focuses on its use in pharmaceuticals for antioxidant and intestinal immune protection. There are no publicly available reports on its inhibition of rice blast fungus or its role in controlling rice blast disease. Developing novel, low-toxicity, naturally derived fungicides has significant production value.

[0004]

[0005] Indole-3-propionic acid (IPA)

[0006] Among the existing reports on patents and papers involving the resistance of indole-3-carboxylic acid derivatives to plant pathogens, the paper (Zeng Hongling, He Kaiwei, He Qinxu Liting, Zhang Weilu Xiang, Tang Yongyan, Zhu Xiaobo, Yin Junjie, He Minchen, Xuewei, Li Weitao. Exogenous Indole-3-Acetic Acid Suppresses Rice Infection of Magnaporthe oryzae by Affecting Plant Resistance and Fungal Growth[J]. Phytopathology, 2024, 114(5): 1050-1056) initially reported that indole-3-acetic acid, 1-naphthaleneacetic acid, and 2,4-dichlorophenoxyacetic acid can inhibit mycelial growth and delay spore germination, thereby playing a role in controlling rice blast. However, in current literature reports and patents, there is no clear evidence that indole-3-propionic acid is used to control rice blast. Summary of the Invention

[0007] In view of this, the present invention explores new agricultural applications for indole-3-propionic acid, providing a highly efficient, low-toxicity, and environmentally friendly compound for the control of rice blast, solving the problems of severe resistance and pesticide residues in existing pesticides, and possessing both direct bactericidal and disease-inducing functions in rice.

[0008] To achieve the above objectives, the present invention first provides the application of indole-3-propionic acid in the control of rice blast disease, wherein the rice blast disease is caused by *Pyrrosia lingua*. Magnaporthe oryzae Infection-induced, indole-3-propionic acid can inhibit the growth of *Pyrrosia lingua* mycelium, spore germination, and pathogenicity.

[0009] Its beneficial effects are as follows: Indole-3-propionic acid can disrupt the cell membrane integrity of rice blast fungus, induce mitochondrial ROS burst and mycelial cell apoptosis, and inhibit conidial germination and infection structure formation; at the same time, it can activate the salicylic acid and jasmonic acid disease resistance signaling pathways in rice plants, enhance the activity of endogenous defense enzymes in rice, induce plant systemic immunity, and bidirectionally block rice blast fungus infection.

[0010] Among a series of indole carboxylic acid derivatives, including indole-3-carboxaldehyde, indole-3-acetonitrile, indole-3-carboxylic acid, indole-3-acetic acid, indole-3-propionic acid, indole-3-butyric acid, and methyl indole-3-acetate, indole-3-propionic acid showed the best control effect against rice blast fungus, and its control effect was far superior to that of indoleacetic acid. The possible mechanism is as follows: the indole parent ring is hydrophobic, while the carboxylic acid group is hydrophilic. Indolecarboxylic acid and indoleacetic acid have short carbon chains and are too hydrophilic, making it difficult to penetrate the hydrophobic hyphae cell membrane of *Fusarium graminearum*, resulting in low intracellular penetration. Indolebutyric acid has a long carbon chain and is too lipid-soluble, easily getting stuck in the cell membrane lipid layer and hindering intracellular entry. Indolepropionic acid (with two methylene groups) has the best hydrophilic / hydrophobic balance, the highest transmembrane efficiency, and the highest intracellular concentration.

[0011] Regarding the mechanism of action, other indole carboxylic acid derivatives mainly act as plant growth regulators, achieving the purpose of antibacterial action by activating the plant's own immunity. However, the indole-3-propionic acid in this invention has a novel mechanism of action: on the one hand, it induces excessive accumulation of intracellular reactive oxygen species, causing oxidative damage, and on the other hand, it destroys cell surface structure and increases membrane permeability, thus jointly inhibiting the growth of pathogens.

[0012] In terms of safety, indole-3-propionic acid is a bioproduct of gut microbiota and is mainly produced by gut symbiotic bacteria (such as Clostridium sporogenes) through the tryptophan metabolism pathway. It is a typical "microbiota-host co-metabolite" and has low harm to humans and other animals. It is easily degraded, environmentally friendly, and conducive to ecological balance and stability.

[0013] In addition, indole-3-propionic acid was tested against various plant diseases, such as rice blast fungus, gray mold, Fusarium graminearum, Fusarium scutellaria, Aspergillus niger, Aspergillus flavus, and Aspergillus fumigatus. Indole-3-propionic acid showed the best control effect against rice blast fungus.

[0014] Preferably, the application specifically refers to: preparing a fungicide for controlling rice blast disease by adding agricultural adjuvants with indole-3-propionic acid as the core active ingredient.

[0015] Preferably, the indole-3-propionic acid has a mass percentage of 0.01 to 50%.

[0016] Preferably, the agricultural adjuvant is an adjuvant that is conventionally acceptable in the agricultural field.

[0017] Preferably, the agricultural adjuvant is a solvent.

[0018] Preferably, the agricultural adjuvant is one or more of the following: dispersant, wetting agent, emulsifier, stabilizer, thickener, disintegrant, antifreeze, and defoamer.

[0019] Preferably, the bactericide is formulated as one of the following: soluble concentrate, emulsion, wettable powder, suspension concentrate, emulsifiable concentrate, granules, or soluble powder.

[0020] More preferably, the bactericide is a soluble formulation, composed of the following raw materials in the indicated mass fractions: 3-10 parts indole-3-propionic acid, 40-60 parts isopropanol, 5-10 parts propylene glycol, 5-10 parts emulsifier, and 20-37 parts water; wherein the emulsifier is either fatty alcohol polyoxyethylene ether or castor oil polyoxyethylene ether.

[0021] Preferably, the fungicide is used for seed dressing, root irrigation during the seedling stage, or foliar spraying during the tillering and heading stages of rice.

[0022] Its beneficial effects are as follows: using the fungicide for seed dressing or root irrigation can control rice blast during the seedling stage, and using it for foliar spraying can play a role in prevention before the disease occurs and treatment in the early stage of the disease.

[0023] Preferably, the effective concentration of indole-3-propionic acid in the application of the bactericide is 64~200 μg / mL.

[0024] In summary, this invention provides the application of indole-3-propionic acid in the control of rice blast disease. Compared with the prior art, this invention has the following significant advantages: (1) This invention discloses for the first time the agricultural use of indole-3-propionic acid to resist rice blast, which is an original innovation.

[0025] (2) Indole-3-propionic acid is a natural small molecule, low in toxicity and easy to degrade, green and environmentally friendly, and has low pesticide residues in rice.

[0026] (3) Indole-3-propionic acid has a triple effect of inhibiting mycelial and spore infection and inducing plant disease resistance, thus delaying the development of pathogen resistance.

[0027] (4) The fungicide of the present invention is simple to use, compatible with existing plant protection equipment, and can prevent and treat both seedling blast and ear blast.

[0028] (5) The raw materials of this invention are readily available, the synthesis process is mature, and the cost of field promotion is low. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0030] Figure 1 A is the mass spectrum of indole-3-propionic acid; B is the nuclear magnetic resonance spectrum of indole-3-propionic acid.

[0031] Figure 2 Plate inhibition effect of different drugs on 7 plant pathogenic fungi at 50 μg / mL.

[0032] Figure 3 Statistical analysis of colony diameter on agar plates.

[0033] Figure 4 IPA anti-rice blast fungus MIC plate diagram.

[0034] Figure 5 MIC plate colony diameter statistics.

[0035] Figure 6 A shows SEM images of hyphae and spores of *Bacillus oryzae* at IPA concentrations of 0, 1 / 2 MIC, and MIC; B shows flow cytometry analysis of ROS content in *Bacillus oryzae* at different IPA concentrations; C shows fluorescence curves of ROS content in *Bacillus oryzae* at different IPA concentrations.

[0036] Figure 7 A shows potted rice plants sprayed with different pesticides; B shows rice blast patches on leaves after spraying with different pesticides; C shows the height of rice plants sprayed with different pesticides; D shows the weight of rice plants sprayed with different pesticides; E shows the percentage of rice blast patches on the entire leaf after spraying with different pesticides.

[0037] Figure 8A shows potted rice plants sprayed with IPA at concentrations of 0.1MIC, 0.5MIC, MIC, 10MIC, and 100MIC; B shows rice blast patches on leaves after spraying with different IPA concentrations; C shows the height of rice plants sprayed with different IPA concentrations; D shows the weight of rice plants sprayed with different IPA concentrations; E shows the percentage of rice blast patches on the entire leaf after spraying with different IPA concentrations. Detailed Implementation

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] Example 1: Determination of the toxicity of indole-3-propionic acid to rice blast 1. Test materials The tested pathogens were: Magnaphalthe oryzae (rice blast fungus), Botrytis cinerea (gray mold), Fusarium graminearum (grass spores), Fusarium oxysporum (sharp spores), Aspergillus niger (black mold), Alternaria alternata (alternaria alternata), Aspergillus flavus (flavus mold), and Aspergillus fumigatus (tobacco mold). Standard strains were identified and provided by the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, and activated in PDA medium for testing.

[0040] Test reagents: indole-3-propionic acid, indole-3-carboxylic acid, indole-3-acetic acid, indole-3-butyric acid, and indole-3-carboxaldehyde, indole-3-acetonitrile, and methyl indole-3-acetate, with a purity ≥98%, purchased from Sigma-Aldrich.

[0041] 2. Test Methods 100 mg of tricyclazole (Tri), indole-3-carboxylic acid (ICA), indole-3-acetic acid (IAA), indole-3-propionic acid (IPA), indole-3-butyric acid (IBA), indole-3-carboxaldehyde (I3A), indole-3-acetonitrile (IAN), and methyl indole-3-acetate (MIA) were dissolved in 1 mL of DMSO to prepare 100 mg / mL drug stock solutions. An appropriate amount of heated liquid PDA solution was mixed with each compound stock solution to prepare a PDA medium containing 50 μg / mL of drug. For the blank control, PDA was mixed with water, and for the negative control, PDA was mixed with DMSO. Magnaphalthe oryzae, Botrytis cinerea, Fusarium graminearum, Fusarium oxysporum, Aspergillus niger, Alternaria alternata, Aspergillus flavus, and Aspergillus fumigatus were collected from rice blast fungi. Agar plates were picked up with a sterile needle and inoculated. The plates were sealed and incubated at 28°C for 6-7 days. The diameters were recorded by photographing. Each experiment was repeated in triplicate.

[0042] For the MIC experiment, prepare 12-well plates. Add 1 mL of PDA medium to each well, mixing different concentrations of indole-3-carboxylic acid (ICA), indole-3-acetic acid (IAA), indole-3-propionic acid (IPA), indole-3-butyric acid (IBA), indole-3-carboxaldehyde (I3A), indole-3-acetonitrile (IAN), and methyl indole-3-acetate (MIA). Cool until solidified. The final concentrations of the reagents are 1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL, 128 μg / mL, and 512 μg / mL, respectively. The blank group (CK) uses water mixed with PDA, and the negative control uses DMSO mixed with PDA. Take rice blast fungus, pick up the inoculum blocks with a sterile needle for inoculation, seal the plate, and incubate at 28℃ for 6-7 days. Take photos and record the diameter. Each experiment is repeated in triplicate.

[0043] 3. Test Results like Figure 2As shown, at a concentration of 50 μg / mL, there were significant differences in the inhibitory effects of IPA and indole carboxylic acid derivatives on the mycelial growth of the seven tested pathogenic fungi. The positive control agent tricyclazole (Tri) showed significant inhibitory effects on multiple fungi, particularly on *M. oryzae*, *B. cinerea*, *F. graminearum*, *F. oxysporum*, and *A. fumigatus*, with a significant reduction in colony diameter on the plates. The IPA treatment group also showed a strong broad-spectrum antifungal effect; its inhibitory effect on *M. oryzae*, *B. cinerea*, *F. graminearum*, *F. oxysporum*, and *A. fumigatus* was similar to that of tricyclazole, with significantly limited colony growth, indicating that IPA has good antifungal activity against plant pathogenic fungi at this concentration.

[0044] from Figure 3 The colony diameter statistics showed that the colony diameters of all tested strains in the CK and DMSO groups remained at a high level, while the colony diameters significantly decreased after Tri and IPA treatments. Particularly in *M. oryzae*, *B. cinerea*, *F. graminearum*, *F. oxysporum*, and *A. fumigatus*, the colony diameters in the IPA-treated groups were close to those in the tricyclazole-treated groups, indicating that IPA has a strong inhibitory effect on these pathogenic fungi. For *A. niger*, both IPA and tricyclazole significantly reduced the colony diameter, but some colony growth was still observed. For *A. flavus*, tricyclazole showed strong inhibition, while IPA showed moderate inhibition, indicating that the sensitivity of IPA to different fungi varies. The test results for resistance to *M. oryzae* showed that IPA was the most effective among a series of indole carboxylic acid derivatives (ICA, IAA, IBA, I3A, IAN, and MIA), and its resistance to *M. oryzae* was far superior to other derivatives (more than 10 times).

[0045] The inhibitory effects of IPA and indole carboxylic acid derivatives on rice blast fungus were further evaluated using a concentration gradient plate MIC experiment. Figure 4 It is evident that as the drug concentration increases, the growth of rice blast fungus colonies is gradually inhibited, with a MIC of 64 μg / mL. Among them, IPA showed the most significant dose-dependent inhibitory effect on rice blast fungus, significantly reducing colony diameter even at lower concentrations; when the concentration continued to increase, colony growth was almost completely inhibited. Figure 5 The colony diameter statistics showed that the colony diameter of the IPA treatment group decreased rapidly with increasing concentration, and its inhibition curve decreased significantly earlier than that of other indole carboxylic acid derivatives, indicating that IPA had the highest sensitivity to rice blast fungus and the strongest antibacterial activity.

[0046] The above results indicate that: ① Among the antagonistic targets of *Oryza sativa*, *Botrytis cinerea*, *Fusarium graminearum*, *Fusarium oxysporum*, *Aspergillus niger*, *Aspergillus flavus*, and *Aspergillus fumigatus*, indole-3-propionic acid exhibits the best toxicity (lowest MIC) against *Oryza sativa*. ② Among a series of indole carboxylic acid derivatives, including ICA, IPA, IAA, IBA, I3A, IAN, and MIA, IPA shows the highest sensitivity and strongest antibacterial activity against *Oryza sativa*.

[0047] Example 2: Indole-3-propionic acid on rice blast fungus ( Magnaporthe oryzae Mechanism of action 1. Method The level of intracellular reactive oxygen species (ROS) in pathogens after sample treatment was detected using the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), and its effect on pathogen morphology was observed using scanning electron microscopy. Specifically: A portion of the washed bacterial cell samples were taken, and 2.5% glutaraldehyde fixative was added to the samples. After fixation at 4℃ for 2 h, the samples were washed three times with PBS buffer. Subsequently, the samples were subjected to gradient dehydration in 30%, 50%, 70%, 90%, and 100% ethanol, with each concentration treated for 20 min. After dehydration, the ethanol was discarded, and the residual liquid on the sample surface was blotted dry with filter paper. The treated samples were then freeze-dried for 12 h and then sputter-coated with gold. Finally, the morphological changes on the bacterial cell surface were observed using scanning electron microscopy, and images were acquired and analyzed.

[0048] Rice blast fungus spores were inoculated into PDB medium containing 1 / 2 MIC concentration of the sample and cultured at 37℃ for 12 h. The control group received the same volume of DMSO as the treatment groups. After culture, the cells from each treatment group were collected by centrifugation and washed three times with sterile PBS buffer to remove residual culture medium components. A portion of the sample was then stained with DCFH-DA at a final concentration of 10 μmol / L and incubated at 37℃ in the dark for 30 min. For flow cytometry analysis, the ROS level of the green fluorescence reaction was measured using the FL1H gate. For fluorescence microplate reader detection, the fluorescence intensity of the sample was measured immediately after staining using a fluorescence microplate reader with an excitation wavelength of 420 nm and an emission wavelength of 460-600 nm, with a step size of 1 nm.

[0049] 2. Test Results The morphological changes of rice blast fungus hyphae and spores after treatment with different concentrations of IPA were investigated, and their intracellular ROS levels were detected. Figure 6In SEM observations, the mycelial morphology of *Magnaporthe oryzae* in group A was intact, with a relatively smooth surface, continuous structure, and plump, clearly defined spores, indicating that the mycelial and spore structures were well maintained under normal culture conditions. After treatment with 1 / 2 MIC IPA, the mycelial surface became rough, and some mycelia showed signs of wrinkling, bending, and local collapse; the spore morphology also changed significantly, exhibiting surface depression, wrinkling, and irregular structure. Further increasing the MIC concentration resulted in more pronounced mycelial damage, with some mycelia showing significant deformation, collapse, and even rupture-like changes, and the spore surface wrinkling worsened, reducing integrity.

[0050] Figure 6 Flow cytometry analysis of ROS in group B showed that, compared with the control group, IPA treatment with 1 / 2 MIC and MIC resulted in enhanced intracellular fluorescence signals and an increased proportion of cells with high fluorescence intensity, indicating that IPA treatment promoted the accumulation of intracellular ROS. The fluorescence signal in the MIC-treated group was stronger than that in the 1 / 2 MIC group, suggesting that IPA-induced ROS production is concentration-dependent.

[0051] Figure 6 The fluorescence spectroscopy results were consistent with the flow cytometry results. All three groups of samples showed a major fluorescence peak around 510-520 nm, with the CK group exhibiting the lowest peak. The fluorescence intensity of the 1 / 2 MIC treated groups increased significantly, while the MIC treated group showed the highest peak, reaching approximately 5 × 10⁻⁶. 5 The fluorescence intensity changes showed that MIC>1 / 2 MIC>CK, further indicating that IPA can significantly induce intracellular ROS accumulation in rice blast fungus, and the ROS level gradually increases with increasing IPA concentration.

[0052] The combined results of SEM and ROS analysis indicate that the inhibitory effect of IPA on rice blast fungus is likely closely related to induced oxidative stress. After IPA treatment, a large amount of ROS accumulated within the rice blast fungus, potentially leading to membrane system damage, cell wall structure disruption, and cellular homeostasis disturbance, ultimately resulting in inhibited hyphal growth, abnormal spore morphology, and decreased pathogen activity. This mechanism corroborates the aforementioned finding that IPA reduces the formation of rice blast lesions on rice leaves, suggesting that IPA may exert its anti-blast effect by disrupting the pathogen's morphology and inducing ROS-mediated oxidative damage.

[0053] Example 33: Preparation of 3% Indole-3-propionic Acid Soluble Solution A solvent was prepared by mixing 40g isopropanol, 10g propylene glycol, 10g fatty alcohol polyoxyethylene ether (AEO-9) and 37g water. 3g indole-3-propionic acid was added and stirred until completely dissolved to obtain a soluble solvent with a mass fraction of 3% indole-3-propionic acid.

[0054] Example 4: Preparation of 10% Indole-3-propionic acid soluble concentrate Disperse 60g isopropanol, 5g castor oil polyoxyethylene ether (EL-40) and 5g propylene glycol, and add deionized water to make up to 90g to prepare a solvent. Weigh 10g of pure indole-3-propionic acid into the solvent and stir until completely dissolved to obtain a 10% indole-3-propionic acid soluble solution.

[0055] Example 5: Pot experiment on the control of rice blast by indole-3-propionic acid 1. Test reagents Example 3 (3% indole-3-propionic acid soluble concentrate) and Example 4 (10% indole-3-propionic acid soluble concentrate), blank control: water, control reagent: tricyclazole (Tri); tricyclazole (Tri), indole-3-carboxylic acid (ICA), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), indole-3-carboxaldehyde (I3A), indole-3-acetonitrile (INA), and methyl indole-3-acetate (MIA) were also prepared as soluble concentrates according to the method of Example 3.

[0056] 2. Test Methods Healthy rice seedlings from the field were transferred to seedling trays 7 cm high, 7 cm in diameter at the top, and 5 cm in diameter at the bottom, and allowed to grow for another 5 days. A working solution was prepared by diluting the tricyclazole (Tri), indole-3-carboxylic acid (ICA), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), indole-3-carboxaldehyde (I3A), indole-3-acetonitrile (INA), and methyl indole-3-acetate (MIA) soluble concentrates prepared according to the method in Example 3 1000 times with water. Then, the seedling leaves were sprayed daily with a solution containing 10... 5 The experimental group was infected with rice blast by spraying 10 mL of an aqueous solution containing 10 mL of different soluble agents. The control group was treated with 10 mL of a 1000-fold diluted solution of the drug solvent, which was sprayed evenly at the early stage of disease occurrence. After 7 days of continuous treatment, the disease index was investigated and the control effect was calculated.

[0057] In addition, the indole-3-propionic acid soluble agent of Example 4 was diluted with water to prepare IPA soluble agents with concentrations of 0.1 MIC, 0.5 MIC, MIC, 10 MIC and 100 MIC. The solvent spray of Example 4 was used as the CK group. Rice seedlings infected with rice blast fungus were sprayed with the same method. After 7 days of continuous treatment, the disease index was investigated and the control effect was calculated.

[0058] 3. Test Results like Figure 7A. Among the various indole compound treatments, the IPA group showed the most significant control effect. Whole-plant phenotypic results showed that rice seedlings treated with IPA maintained good growth, with relatively bright green leaves and no obvious wilting or large-scale yellowing. In vitro leaf results further indicated that only a few scattered lesions were visible on the leaf surface of the IPA-treated group, and the lesion area was significantly lower than that of the ICA, IAA, IBA, I3A, IAN, and MIA treatment groups, with overall performance similar to the tricyclazole treatment group. From the perspective of growth indicators ( Figure 7 C, Figure 7 (D) The seedling height and fresh weight of the Rice Blast group were lower than those of the CK group, indicating that rice blast fungus infection inhibits normal seedling growth. After tricyclazole treatment, seedling length and fresh weight both recovered significantly. The seedling length and fresh weight of the IPA treatment group also remained at a high level, with the fresh weight approaching that of the tricyclazole treatment group, indicating that IPA can not only reduce the occurrence of the disease but also alleviate the adverse effects of rice blast fungus infection on seedling growth to a certain extent. In contrast, although some indicators of the ICA, IAA, IBA, I3A, IAN, and MIA treatment groups improved, the overall recovery was not as stable as that of IPA.

[0059] like Figure 7 In groups B and CK, the plants generally grew well, with bright green leaves and no obvious lesions. In the Rice Blast group, infection with rice blast fungus significantly inhibited plant growth, and numerous brown necrotic spots and yellowing areas appeared on the leaves, indicating successful pathogen infection and significant disease symptoms. Compared to the Rice Blast group, the tricyclazole and IPA treatment groups showed a significant reduction in leaf lesions and better plant recovery, demonstrating typical control effects. The lesion area statistics further validated the above phenotypic observations. Figure 7 The results showed that the proportion of lesion area in the Rice Blast group was significantly increased, while both tricyclazole and IPA treatments significantly reduced the area of ​​rice blast lesions. The lesion area in the IPA group remained at a low level, similar to the tricyclazole group, but significantly lower than other indole compound treatment groups. ICA, IAA, and IBA treatments reduced the lesion area, suggesting some inhibitory effect, but the effect was weaker than IPA; I3A, IAN, and MIA groups still showed many lesions, indicating that their alleviating effect on rice blast was relatively limited. This invention confirms that indole-3-propionic acid soluble concentrate has a good control effect on rice blast.

[0060] To further evaluate the control effect of IPA on rice blast, soluble IPA solutions of 0.1 MIC, 0.5 MIC, MIC, 10 MIC, and 100 MIC were prepared according to the method in Example 4 (diluted with water) and sprayed onto rice seedlings infected with rice blast fungus. Figure 8As shown in Figure A, the rice seedlings in the CK group grew well with bright green leaves. In the Rice Blast group, after infection with rice blast fungus, the overall growth of the plants weakened, and the leaves showed varying degrees of yellowing, drooping, and wilting, indicating that rice blast fungus infection significantly affected the normal growth of rice seedlings. After IPA treatment, the growth of rice plants recovered to varying degrees. The 0.5 MIC and MIC treatment groups showed better growth, with more upright leaves, and their overall condition was significantly better than the Rice Blast group. The plant height statistics showed (…). Figure 8 C), the plant height of the Rice Blast group was significantly lower than that of the CK group, indicating that rice blast fungus infection inhibited rice seedling growth. The plant height recovery was not significant in the 0.1 MIC treatment group, while the plant height increased significantly after treatments with 0.5 MIC and 100 MIC, with the MIC treatment group showing the best recovery effect, even slightly higher than the CK group. Although the 10 MIC and 100 MIC treatment groups maintained a certain growth level, the plant height did not continue to increase with increasing concentration, suggesting that excessively high concentrations of IPA have limited growth-promoting effects on rice. Fresh weight statistics showed ( Figure 8 D) The significant decrease in fresh weight in the Rice Blast group indicates that pathogen infection caused significant biomass loss. After IPA treatment, plant fresh weight gradually recovered, with the MIC treatment group showing the highest fresh weight, significantly better than the Rice Blast and 0.1 MIC groups. This suggests that appropriate concentrations of IPA can not only reduce lesion formation but also alleviate growth inhibition caused by rice blast infection. Notably, the fresh weight of the 10 MIC and 100 MIC treatment groups was lower than that of the MIC group, indicating that while high concentrations of IPA still have good disease control effects, they did not further promote plant growth and may even have some impact on seedling biomass accumulation.

[0061] like Figure 8 The leaf lesion results further showed that the Rice Blast group exhibited numerous typical rice blast lesions on the leaf surface, which were brown and necrotic, accompanied by obvious yellowing areas. After treatment with 0.1 MIC IPA, the number of leaf lesions decreased compared to the Rice Blast group, but still showed relatively obvious lesions, indicating that low concentrations of IPA had a certain alleviating effect on rice blast, but the control effect was limited. As the IPA concentration increased to 0.5 MIC, the number of leaf lesions decreased significantly, with only a few sporadic lesions appearing; in the MIC, 10 MIC, and 100 MIC treatment groups, the leaves remained basically green, and the lesion area further decreased, approaching that of the CK group, indicating that IPA could effectively inhibit the formation of rice blast lesions. (Lesion area percentage statistics...) Figure 8E) This further verified the concentration-dependent control effect of IPA. The Rice Blast group had the highest lesion area, approximately 17%. After treatment with 0.1 MIC, the lesion area decreased to approximately 8%, indicating that low concentrations of IPA already showed some resistance to rice blast. After treatment with 0.5 MIC, the lesion area further decreased to a low level. The lesion areas in the MIC, 10 MIC, and 100 MIC treatment groups were close to those in the CK group, indicating that IPA at MIC and above concentrations can significantly inhibit the expansion of rice blast lesions. Among them, the MIC treatment group showed the best performance in reducing lesion area and maintaining plant growth, and was the treatment concentration with the best overall control effect.

[0062] Based on the combined results of in vitro toxicity assays and pot control efficacy tests, the MIC (micronizable concentration) of indole-3-propionic acid (IPA) against rice blast fungus is 64 μg / mL. Pot concentration gradient experiments showed that while the 0.1 MIC treatment could reduce lesion area to some extent, its control effect was limited; the 0.5 MIC treatment significantly reduced lesions. The MIC treatment showed the best performance in reducing lesion area, restoring plant height, and fresh weight, indicating that IPA can produce a stable control effect against rice blast at the MIC concentration. Although the 10 MIC and 100 MIC treatments still inhibited lesion expansion, they did not further improve plant growth, suggesting that excessively high concentrations are not necessary. Therefore, considering both antibacterial activity and pot control efficacy results, the effective concentration of indole-3-propionic acid for controlling rice blast should be above 64 μg / mL, preferably 64–200 μg / mL.

[0063] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of indole-3-propionic acid in the control of rice blast disease, characterized in that, The rice blast disease is caused by *Pyrethrum oryzae*. Magnaporthe oryzae Infection-induced, indole-3-propionic acid can inhibit the growth of *Pyrrosia lingua* mycelium, spore germination, and pathogenicity.

2. The application according to claim 1, characterized in that, The specific application is as follows: using indole-3-propionic acid as the core active ingredient, and adding agricultural adjuvants to prepare a fungicide for controlling rice blast disease.

3. The application according to claim 2, characterized in that, The bactericide contains indole-3-propionic acid at a mass percentage of 0.01-50%.

4. The application according to claim 2, characterized in that, The agricultural adjuvant is a solvent.

5. The application according to claim 2, characterized in that, The agricultural adjuvant is one or more of the following: dispersant, wetting agent, emulsifier, stabilizer, thickener, disintegrant, antifreeze, and defoamer.

6. The application according to claim 2, characterized in that, The bactericide is a soluble formulation, composed of the following raw materials in the indicated mass fractions: 3-10 parts indole-3-propionic acid, 40-60 parts isopropanol, 5-10 parts propylene glycol, 5-10 parts emulsifier, and 20-37 parts water. The emulsifier is either fatty alcohol polyoxyethylene ether or castor oil polyoxyethylene ether.

7. The application according to claim 2, characterized in that, The fungicide is used for seed dressing, root irrigation during the seedling stage, or foliar spraying during the tillering and heading stages of rice.

8. The application according to claim 7, characterized in that, The effective concentration of indole-3-propionic acid in the application of the bactericide is 64~200 μg / mL.

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