Use of agaric acid in prevention and treatment of plant rice blast

Abstract

The application discloses an application of pinic acid in preventing and treating rice blast, and belongs to the technical field of plant disease prevention and treatment. The application provides a new use of pinic acid in the prevention and treatment of rice blast. Experiments prove that pinic acid can significantly inhibit the growth, spore germination and appressorium formation of rice blast fungus. Pinic acid can not only effectively inhibit rice blast fungus, but also reduce the occurrence degree of the disease by early application, and has a dual effect of prevention and treatment. The application of pinic acid in a field can effectively control the occurrence and spread of rice blast, and the prevention and treatment effect is equivalent to that of a commonly used chemical fungicide. The application also provides a preparation of pinic acid combined with a chemical fungicide triadimefon or carbendazim. Compared with single drug use, the preparation can not only reduce the application amount, but also has a synergistic effect on the prevention and treatment of rice blast. Safety evaluation shows that pinic acid is safe to the tested crops and has no drug harm in the effective prevention and treatment concentration range, and has a good development and application prospect.

Description

Technical Field

[0001] This invention relates to the field of plant disease control technology, specifically to the application of agaric acid, a natural metabolite, in the control of rice blast. Background Technology

[0002] Plant pathogens causing diseases are a significant factor restricting agricultural production, among which the rice blast fungus (Ascomycota genus) is a major contributor. Magnaporthe oryzae Rice blast, caused by blast fungus, is one of the fungal diseases in rice production. The blast fungus can damage multiple parts of rice at various growth stages. Depending on the location of the infection, it can be classified as seedling blast, leaf blast, node blast, neck blast, grain blast, etc.

[0003] Currently, the control of plant fungal diseases traditionally relies on chemical agents and the breeding of disease-resistant varieties. However, the extensive use of chemical pesticides not only increases planting costs but also easily leads to the evolution of drug resistance in pathogens and environmental pollution. On the other hand, relying solely on disease-resistant varieties often results in the "loss" of resistance due to the rapid variation of physiological races of rice blast fungus, making the control effect difficult to sustain. At the same time, in recent years, with global warming and the increasing intensification of rice cultivation, the pathogenic spectrum of rice blast fungus has continued to expand, and new pathogenic strains have emerged, posing a severe challenge to existing disease-resistant genes.

[0004] Therefore, in-depth analysis of its pathogenic mechanisms, discovery of new targets, and construction of a green, efficient, and sustainable integrated prevention and control system have become urgent needs in current plant disease research, and are of great significance for ensuring food security and sustainable agricultural development.

[0005] Natural products, due to their novel structures, unique mechanisms of action, and good environmental compatibility, have become an important source of lead compounds for novel pesticides. In recent years, phenolic acids, flavonoids, and alkaloids isolated from plants and microorganisms have exhibited good activity against plant pathogens. For example, gallic acid has shown a significant inhibitory effect on rice bacterial leaf streak, with a field control efficacy of up to 64.62%. Its mechanism of action against the pathogen involves interference with respiratory metabolism and inhibition of nucleic acid and protein synthesis (WANG Kaihao, WEI Changying, XIE Huiting, et al Gallic acid isolated from Sedum lineare and its control efficacy on rice bacterial leaf streak [J]. Guihaia, 2018, 38(1): 119-127.). Furthermore, the phenolic compounds abundant in extracts of Brazilian pine and quince have good control effects against anthracnose and gray mold (Pansera MR, Sartori DC, Silveira AB da, 2018, 38(1): 119-127.). et al Araucaria angustifolia and Picrasma crenata extracts as potential agents for the control of phytopathogenic fungi of agricultural interest[J]. Caderno Pedagógico , 2024, 21(8):e6774.).

[0006] Agaric acid is a substance derived from medicinal fungi (such as *Polyporus flocculationii*). Fomitopsis officinalis Magnoliaic acid is a natural tricarboxylic acid compound. Existing research indicates that muscarinic acid, as an inhibitor of adenine nucleotide translocase, can induce mitochondrial permeability transition and affect cellular energy metabolism (García N, et al Agaric acid induces mitochondrial permeability transition through its interaction with the adenine nucleotide translocase. Its dependence on membrane fluidity [J]. Mitochondrion , 2005, 5(4): 272-281.; Chávez E, et al The effect of agaric acid on citrate transport in rat liver mitochondria [J]. Life Sci , 1978, 23(14): 1423-1429.).

[0007] Current research on muscarinic acid mainly focuses on medical microbiology and cell metabolism, and its potential as a plant disease control agent has not yet been systematically explored. Summary of the Invention

[0008] The purpose of this invention is to discover compounds from natural plant metabolites that have fungicidal activity against plant pathogenic fungi, especially rice blast fungus, providing a theoretical basis and candidate compounds for the development of novel, low-toxicity, and drug-resistant plant fungicides, solving problems such as drug resistance, environmental pollution, and residue risks of existing chemical fungicides, and expanding the application fields of these natural products.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides the application of agaric acid in the control of rice blast disease or in the preparation of rice blast disease control agents. The agaric acid is a natural tricarboxylic acid compound with the molecular formula C1. 22 H 40 O7, CAS number 666-99-9.

[0010] This invention systematically evaluated the inhibitory activity and disease control effect of melikonin against rice blast fungus through in vitro tissue experiments, indoor bioassays, and field plot trials, and assessed the safety of melikonin to plants. The results showed that melikonin significantly inhibited the growth and infection of rice blast fungus. Field trials confirmed that melikonin has good control effects against rice blast, comparable to commonly used chemical fungicides. Safety evaluation showed that melikonin is safe and non-toxic to the tested crops within the effective control concentration range.

[0011] The rice blast disease is caused by the fungus *Magnaporphyromonas oryzae* (from the Ascomycota phylum). Magnaporthe oryzae )cause.

[0012] Furthermore, the plant is a grass (Poaceae). Rice blast fungus can infect various grasses such as rice, wheat, and barley. Even more specifically, the plant is either rice or barley.

[0013] Furthermore, the rice blast is leaf blast. This invention demonstrates that muscarinic acid can significantly inhibit the growth and infection of rice blast fungus on crop leaves.

[0014] Furthermore, the application includes applying muscarinic acid to plants during the plant growth stage to prevent or inhibit rice blast fungus infection.

[0015] Specifically, muscarinic acid is dissolved in a solvent such as dimethyl sulfoxide (DMSO) to prepare a stock solution. When using it, it is diluted with water to the appropriate effective concentration and then sprayed on the surface of the plant. This can effectively prevent and inhibit the infection of rice blast fungus on the plant.

[0016] Furthermore, the effective concentration of muscarinic acid is 10 μM to 100 μM. This invention demonstrates that at this effective concentration, it can inhibit rice blast fungus infection in crops and is safe for crops without causing phytotoxicity.

[0017] As a specific embodiment of the present invention, for the control of rice blast in field rice, the effective concentration of pine muscarinic acid is 10 μM~50 μM, and the application rate is 0.5~2.5 μmol / m³. 2 .

[0018] This invention can also be used to prepare formulations for the control of rice blast disease by combining muscarinic acid with pesticide-acceptable adjuvants. The formulation may be, but is not limited to, a liquid or a soluble powder.

[0019] Another object of the present invention is to provide a formulation for controlling rice blast disease, wherein the active ingredients of the formulation include: piracetamic acid and tricyclazole in a molar ratio of 1:200-300; or piracetamic acid and carbendazim in a molar ratio of 1:1000-2000. The present invention has found that the combined use of piracetamic acid with the chemical fungicide tricyclazole or carbendazim has a synergistic effect on the control of rice blast fungus.

[0020] Preferably, the active ingredients of the formulation include: mesona chinensis and tricyclazole in a molar ratio of 1:260; or mesona chinensis and carbendazim in a molar ratio of 1:1400.

[0021] The formulation also includes pesticide-acceptable adjuvants, which may be, but are not limited to, surfactants, solvents, fillers, and antifreeze agents.

[0022] Preferably, gelatin is added to the formulation as an adhesive. The adhesive properties of gelatin allow the muscarinic acid to adhere, spread, and adhere more effectively to the plant surface.

[0023] This invention also provides the application of the aforementioned formulation in the control of rice blast, wherein the effective concentration of melioficin is 5 μM~10 μM. Research in this invention shows that, compared with single application, the combined use of melioficin with chemical fungicides such as tricyclazole or carbendazim not only reduces the application amount but also produces a synergistic effect in the control of rice blast.

[0024] The beneficial effects of this invention are as follows: (1) This invention provides a novel application of muscarinic acid in the control of rice blast disease. Experiments have demonstrated that muscarinic acid can significantly inhibit the growth, spore germination, and appressorium formation of rice blast fungus. Mucilaginic acid not only effectively inhibits rice blast fungus but also reduces the severity of the disease through early application, exhibiting both preventative and curative effects. Field application of muscarinic acid can effectively control the occurrence and spread of rice blast, with control effects comparable to commonly used chemical fungicides.

[0025] (2) The present invention also provides a formulation that combines muscarinic acid with chemical fungicides tricyclazole or carbendazim. Compared with the use of the drug alone, the combination of muscarinic acid with chemical fungicides tricyclazole or carbendazim can not only reduce the application amount, but also produce a synergistic effect on the prevention and control of rice blast.

[0026] (3) Myricetic acid is a natural metabolite with novel mechanisms of action, environmental friendliness, and crop safety. It does not have adverse effects on soil, water, or non-target organisms. Safety evaluations show that myricetic acid is safe and non-toxic to the tested crops within the effective control concentration range. Therefore, it can be prepared into various formulations for the control of plant diseases and has good development and application prospects. Attached Figure Description

[0027] Figure 1 The colony morphology of wild-type rice blast fungus 70-15 after 7 days of growth in CM medium containing 0 μM, 10 μM, and 50 μM muscarinic acid.

[0028] Figure 2 The relative growth rate of wild-type rice blast fungus 70-15 after 7 days of growth in CM medium containing 10 μM and 50 μM muscarinic acid is statistically shown. * indicates a significant difference compared to the 10 μM group, and **** indicates a statistically significant difference. p <0.0001.

[0029] Figure 3 Spore germination was observed under a microscope after inducing rice blast fungus spores with different concentrations of muscarinic acid for 12 h. Scale bar: 100 μm.

[0030] Figure 4 Statistical results of conidial germination rate after 12 h of induction with different concentrations of muscarinic acid for rice blast fungus spores. * in the figure indicates a significant difference compared to the 0 μM group, and **** indicates... p <0.0001.

[0031] Figure 5 Appressor cell formation was observed under a microscope after 24 h of induction of rice blast fungus spores with different concentrations of muscarinic acid. Scale bar: 100 μm.

[0032] Figure 6 Statistical results of appressorium formation rate after 24 h of induction with different concentrations of melatonin from rice blast fungus spores. * in the figure indicates a significant difference compared to the 0 μM group, and **** indicates... p <0.0001.

[0033] Figure 7 The effect of different concentrations of muscarinic acid on the pathogenicity of rice blast fungus spore suspension on detached barley leaves.

[0034] Figure 8 This study investigated the effects of different application methods of muscarinic acid on the pathogenicity of rice blast fungus spore suspensions on rice leaves. Group Aa-A involved the application of a mixture of muscarinic acid and rice blast fungus; Group Aa-B involved pre-application of muscarinic acid followed by inoculation with rice blast fungus.

[0035] Figure 9 The growth of rice after spraying with different concentrations of pine muscarinic acid.

[0036] Figure 10 for Figure 9 Statistical results of plant height of medium-grain rice.

[0037] Figure 11 for Figure 9 Statistical results of stem width of medium-sized rice.

[0038] Figure 12 The growth of barley after spraying with different concentrations of muscarinic acid.

[0039] Figure 13 for Figure 12 Statistical results of plant height of medium-sized barley.

[0040] Figure 14 for Figure 12 Statistical results of stem width of medium-sized barley.

[0041] Figure 15 The inhibitory effects of different pesticides applied in the field on rice blast were investigated.

[0042] Figure 16 This figure shows the statistical results of disease index after different pesticides were applied in the field. * indicates a significant difference compared to the control group, and **** indicates a statistically significant difference. p <0.0001.

[0043] Figure 17 This study investigated the effects of single and combined use of pesticides on the pathogenicity of rice blast fungus spore suspension on rice leaves in an indoor pot experiment.

[0044] Figure 18 The graph shows the statistical results of disease index after applying different agents to indoor potted plants. * indicates a significant difference compared to the control group, and *** indicates a statistically significant difference. p <0.001, **** indicates p <0.0001. Detailed Implementation

[0045] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0047] Specifically: the molecular formula of muscarinic acid is C 22 H 40 O7, CAS number 666-99-9; tricyclazole molecular formula C9H7N3S, CAS number 41814-78-2; carbendazim molecular formula C9H9N3O2, CAS number 10605-21-7.

[0048] Wild-type strain 70-15 of *Oryza sativa*, the blast fungus, was preserved in the laboratory. This strain is internationally recognized as the model strain for rice blast (Dean RA, Talbot NJ, Ebbole DJ). et al The genome sequence of the rice blast fungus *Magnaporthe grisea* (rice blast fungus) Magnaporthe grisea (genomic sequence of the genome) [J]. Nature , 2005, 434(7036): 980-986.; Yang J, et al . An annotated near-completesequence assembly of the Magnaporthe oryzae 70-15 reference genome(Magnaporthe oryzae Magnaporthe oryzae 70-15 (Annotation of near-complete sequence assembly of reference genome) [J]. Scientific Data (2025, 12: 758.), obtained from rice isolate Guy11 through multiple backcrosses. This strain can infect rice and cause typical rice blast symptoms.

[0049] The CM medium formula (1 L) for culturing rice blast fungus is as follows: yeast extract (1 g), casein amino acids (1 g), D-glucose (10 g), potassium dihydrogen phosphate (1.52 g), sodium nitrate (6 g), peptone 140 (2 g), potassium chloride (0.52 g), magnesium sulfate heptahydrate (0.52 g), 0.1% (v / v) vitamin solution, and 0.1% (v / v) trace element solution. Adjust the pH to 6.5 with sodium hydroxide, and add 15 g / L agar to the solid medium. Autoclave at 121°C for 15 min.

[0050] Example 1: Mucilaginic acid inhibits the growth of rice blast fungus. 1. Mucilaginic acid inhibits the vegetative growth of rice blast fungus. (1) Experimental methods Under laboratory conditions, muscarinic acid was dissolved in dimethyl sulfoxide (DMSO) to prepare a 50 mM stock solution, which was then added to CM medium to achieve final concentrations of 10 and 50 μM, respectively. A control group without added muscarinic acid was also included. Rice blast fungus was inoculated onto CM medium containing 0, 10, and 50 μM muscarinic acid and incubated at 25°C for 7 days. Colony morphology was observed, and colony diameter was counted.

[0051] (2) Experimental Results The results are as follows Figure 1 and Figure 2 As shown, the mycelium of rice blast fungus was sparse under the treatment of muscarinic acid, and muscarinic acid at a concentration of 50 μM could significantly inhibit the vegetative growth of rice blast fungus.

[0052] 2. Mucilaginic acid inhibits the germination of rice blast fungus spores. (1) Experimental methods Rice blast fungus spores cultured on CM medium were washed off with sterile water and diluted to a concentration of 5 × 10⁻⁶. 4 Spores / mL. A 50 mM muscarinic acid stock solution was prepared using dimethyl sulfoxide (DMSO) solvent. 50 mM muscarinic acid stock solution was added to achieve final concentrations of 0, 5, 10, and 50 μM in the spore solution. 25 µL of each solution was dropped onto a hydrophobic glass slide to induce spore germination. The spores were then incubated in the dark at 25°C for 12 h. Spore morphology was observed under a microscope. Spores with germ tubes or appressorium structures were considered to have germinated. The germination rate was statistically analyzed and calculated.

[0053] (2) Experimental Results The results are as follows Figure 3 and Figure 4 As shown, muscarinic acid at concentrations above 5 μM can significantly inhibit spore germination.

[0054] 3. Mucilaginic acid inhibits the formation of appressorium spores of rice blast fungus. (1) Experimental methods Appressoria are an essential structure for rice blast fungus to penetrate the host epidermis, and their formation rate directly determines the success rate of invasion.

[0055] Rice blast fungus spores cultured on CM medium were washed off with sterile water and diluted to a concentration of 5 × 10⁻⁶. 4 Spores were cultured at a concentration of 1 / mL using dimethyl sulfoxide (DMSO) to prepare a 50 mM muscarinic acid stock solution. The muscarinic acid stock solution was added to achieve final concentrations of 0, 5, 10, and 50 μM in the spore solution. 25 µL of the solution was dropped onto a hydrophobic glass slide to induce spore germination. The spores were then cultured in the dark at 25°C for 24 h in a humidified chamber. The spore morphology was observed under a microscope, the number of spores with appressorium structures was counted, and the appressorium formation rate was calculated.

[0056] (2) Experimental Results The results are as follows Figure 5 and Figure 6 As shown, muscarinic acid at concentrations above 5 μM can significantly inhibit the formation of appressoria of rice blast fungus.

[0057] Example 2: Mucilaginic acid significantly reduces the infectivity of rice blast fungus. 1. Mucilaginic acid inhibits the infection of barley by rice blast fungus. (1) Experimental methods Rice blast fungus spores cultured on CM medium were washed off with sterile water and diluted to a concentration of 5 × 10⁻⁶. 4 A 50 mM tonic acid stock solution was prepared using dimethyl sulfoxide (DMSO) solvent. The tonic acid stock solution was added to achieve final concentrations of 5, 10, 50, and 100 μM in the spore solution. An equal amount of dimethyl sulfoxide (DMSO) was added to the spore solution of the control group. The prepared spore solution was dropped onto detached barley leaves, 3 drops per leaf, 25 µL per drop. The barley leaves were placed on a tray lined with a moistened paper towel and sealed for 4 days to maintain humidity. The disease incidence on the leaves was then observed.

[0058] (2) Experimental Results The results are as follows Figure 7 As shown in the leaf inoculation experiment, muscarinic acid at concentrations above 10 μM can significantly inhibit the infection of barley by rice blast fungus.

[0059] 2. Mucilaginic acid prevents and inhibits rice blast infection. (1) Experimental methods Tocopheryl acid was dissolved in dimethyl sulfoxide (DMSO) to prepare a 50 mM stock solution. The tocopheryl acid stock solution and an equal amount of dimethyl sulfoxide (DMSO) were diluted separately with sterile water to make the concentration of the tocopheryl acid diluted solution 10 μM. The dimethyl sulfoxide (DMSO) diluted solution was used as the control group treatment reagent.

[0060] Rice blast fungus spores cultured on CM medium were washed off with sterile water and diluted to 5 × 10⁻⁶ concentrations. 4 cells / mL and 1 × 10 5 Seeds / mL. Select plump rice CO39 seeds, place them in petri dishes (with moisturizing paper inside), submerge them in water, and incubate them in a 37°C constant temperature incubator for 36 h. Select seeds with uniform germination and plant them in flowerpots containing rice-specific soil, then incubate them in a 28°C constant temperature and humidity plant culture room with alternating light and dark conditions (12 h each).

[0061] Group Aa-A involved mixing 2 mL of 10 μM muscarinic acid with 2 mL of 1 × 10⁻⁶ μM muscarinic acid. 5Mix rice blast fungus spores at a concentration of 1 spore / mL and spray 4 mL of the mixture onto each pot of rice that has been cultured for 14 days.

[0062] Group Aa-B was pre-treated by spraying each pot of rice with 2 mL of 10 μM melatonin after 10 days of cultivation. After another 4 days of cultivation, each pot of rice previously sprayed with melatonin was sprayed again with 2 mL of 5 × 10⁻⁶ melatonin. 4 Rice blast fungus spore liquid per mL.

[0063] The control group consisted of 2 mL of dimethyl sulfoxide (DMSO) dilution solution and 2 mL of a 1 × 10⁻⁶ solution. 5 The rice blast fungus spore solution was mixed with rice blast fungus spores per mL and sprayed onto rice leaves that had been cultured for 14 days.

[0064] All groups were cultured at 25℃ for 5 days to observe the disease incidence on the leaves.

[0065] (2) Experimental Results The results are as follows Figure 8 As shown in the leaf inoculation experiment, 10 μM muscarinic acid can effectively prevent and inhibit rice blast fungus infection.

[0066] Example 3: Muscarpic acid had no significant inhibitory effect on plant growth. 1. Mucilaginic acid has no significant inhibitory effect on rice growth. (1) Experimental methods Tocopheryl acid was dissolved in dimethyl sulfoxide (DMSO) to prepare a 50 mM stock solution. The tocopheryl acid stock solution and an equal amount of dimethyl sulfoxide (DMSO) were diluted with sterile water to make the tocopheryl acid dilutions 50 μM and 100 μM. The dimethyl sulfoxide (DMSO) dilution was used as the control group treatment reagent.

[0067] Select plump rice CO39 seeds, place them in petri dishes (with moisturizing paper inside), submerge them in water, and incubate them in a 37°C constant temperature incubator for 36 hours. Select seeds with uniform germination and plant them in flowerpots containing rice-specific soil, then incubate them in a 28°C constant temperature and humidity plant culture room with alternating light and dark conditions (12 hours each) for 14 days.

[0068] Spray each pot of rice with 5 mL of dimethyl sulfoxide (DMSO) solution containing 0, 50 μM, or 100 μM muscarinic acid. Culture at 28°C with alternating light and dark conditions (12 h each time) for 4-5 days, and then count the plant height and stem width.

[0069] (2) Experimental Results The results are as follows Figures 9-11 As shown, neither 50 μM nor 100 μM muscarinic acid had a significant inhibitory effect on rice growth.

[0070] 2. Mucilaginic acid has no significant inhibitory effect on barley growth. (1) Experimental methods Tocopheryl acid was dissolved in dimethyl sulfoxide (DMSO) to prepare a 50 mM stock solution. The tocopheryl acid stock solution and an equal amount of dimethyl sulfoxide (DMSO) were diluted with sterile water to make the tocopheryl acid dilutions 50 μM and 100 μM. The dimethyl sulfoxide (DMSO) dilution was used as the control group treatment reagent.

[0071] Select plump barley seeds, soak them in clean water, remove shriveled seeds, and sow them in moist composite substrate soil. Cultivate them at room temperature for 7-9 days.

[0072] Spray each pot with 5 mL of dimethyl sulfoxide (DMSO) solution containing 0, 50 μM, or 100 μM muscarinic acid. Culture at 28°C with alternating light and dark conditions (12 h each) for 4-5 days, and then measure plant height and stem width.

[0073] (2) Experimental Results The results are as follows Figures 12-14 As shown, neither 50 μM nor 100 μM melatonin had a significant inhibitory effect on barley growth.

[0074] Example 4: The effect of pine muscarinic acid in controlling rice blast in the field is comparable to that of commonly used chemical fungicides. (1) Experimental methods Five plots with uniform soil fertility and convenient irrigation and drainage were divided in the field as experimental plots, each with an area of ​​40 m². 2 A 0.5-meter-wide protective row was set up between each plot to prevent interference between pesticides. The test crop was CO39 rice, and the rice varieties in each plot were the same and the growth was uniform.

[0075] The experimental agent was muscarinic acid, and the control agents were tricyclazole and carbendazim. Sterile water treatment was used as a blank control. The concentrations of the agents in each experimental group were set as follows: muscarinic acid 10 μM, muscarinic acid 50 μM, tricyclazole 2.6 mM, and carbendazim 14.0 mM.

[0076] Rice blast fungus spores cultured on CM medium were washed off with sterile water, filtered through three layers of filter paper, and the spore suspension was collected and diluted with sterile water to a concentration of 2×10⁻⁶. 5 Species / mL. A gelatin solution with a mass-to-volume ratio of 0.4% was prepared as an adsorbent and mixed with an equal volume of spore suspension. After mixing, the mass-to-volume ratio of gelatin was 0.2%, and the spore concentration was 1×10⁻⁶. 5 spores / mL. For each experimental group treated with the agent, the agent needs to be added to the spore solution to reach the preset treatment concentration.

[0077] During the susceptible stage of rice, in weather with high air humidity, apply rice blast fungus spore solution evenly to each plot using a sprayer, with 2 L of spore solution sprayed per plot. A survey is conducted 14 days after application. A five-point sampling method is used for each plot, with 100 leaves sampled at each point. The disease level is recorded according to the rice blast grading standard, and the disease index is calculated.

[0078] (2) Experimental Results The results are as follows Figure 15 and Figure 16 As shown, the control group exhibited severe rice leaf blast disease, with a disease index of 52.5. The disease was milder in all pesticide-treated groups. Specifically, the disease index was 9.5 in the tricyclazole treatment group, 4.8 in the carbendazim treatment group, 11.4 in the 10 μM melilotic acid treatment group, and 8.07 in the 50 μM melilotic acid treatment group. This indicates that 10 μM and 50 μM melilotic acid are significantly effective in controlling rice blast, and their control efficacy is comparable to that of traditional chemical fungicides.

[0079] Example 5: The combined use of muscarinic acid and chemical bactericides has a synergistic effect. (1) Experimental methods The experimental agents were tricyclazole, carbendazim, and terbinafine. The experimental groups included single-agent treatment groups and combined-agent treatment groups, with the following concentrations: tricyclazole 1.3 mM, carbendazim 7.0 mM, terbinafine 5.0 μM, terbinafine 5.0 μM + tricyclazole 1.3 mM, and terbinafine 5.0 μM + carbendazim 7.0 mM, respectively. The control group was only sprayed with spore solution and received no agent treatment. Both the control and treatment groups were replicated three times.

[0080] Select plump rice CO39 seeds, place them in petri dishes (with moisturizing paper inside), submerge them in water, and incubate them in a 37°C constant temperature incubator for 36 hours. Select seeds with uniform germination and plant them in flowerpots containing rice-specific soil, then incubate them in a 28°C constant temperature and humidity plant culture room with alternating light and dark conditions (12 hours each).

[0081] Rice blast fungus spores cultured on CM medium were washed off with sterile water, filtered through three layers of filter paper, and the spore suspension was collected and diluted with water to a concentration of 2×10⁻⁶. 5 Species / mL. Prepare a 0.4% (w / v) gelatin solution as an adsorbent and mix it with an equal volume of spore suspension. For each experimental group undergoing treatment, add the agent to the spore solution to achieve the preset treatment concentration.

[0082] Spray the spore suspension evenly onto the leaves of rice seedlings using a sprayer, using 2 mL per pot. Place the rice pots in a 25°C incubator and incubate in the dark for 2 days. Then, incubate for 4-5 days with 16 hours of light / 8 hours of darkness. Analyze the disease index and control effect, and calculate the synergistic effect index using the following formula: Formula 1: Disease index = [∑ (number of diseased leaves at each level × relative level value) / (total number of leaves surveyed × highest level value)] × 100; Formula 2: Prevention and control effect (%) = [(Control disease index - Treatment disease index) / Control disease index] × 100; Formula 3: Theoretical combined control effect (%) = [Control effect of pine sorbic acid alone + Control effect of tricyclazole / carbendazim alone - Control effect of pine sorbic acid alone × Control effect of tricyclazole / carbendazim alone] × 100; Formula 4: Synergistic effect index = Actual combined prevention and control effect / Theoretical combined prevention and control effect; Synergy synergy index > 1 indicates synergy; synergy synergy index = 1 indicates simple addition; synergy synergy index < 1 indicates antagonistic effect.

[0083] (2) Experimental Results The results are as follows Figure 17 , Figure 18 As shown in Table 1, the disease index survey results indicated that the disease index of the blank control group was 36.93, indicating sufficient disease progression. The disease indices of each single-dose treatment group ranged from 20.23 to 21.43, a decrease of 41.97% to 45.22% compared to the control. The disease index of the pine muscarinic acid + tricyclazole combination group was 3.00, and the disease index of the pine muscarinic acid + carbendazim combination group was 2.86, both showing a decrease of more than 85% compared to their respective single-dose groups, indicating a significant improvement in disease control after combination therapy.

[0084] The synergistic effect index of the combination of pine muscarinic acid and tricyclazole was 1.33, and the synergistic effect index of the combination of pine muscarinic acid and carbendazim was 1.37, both greater than 1, indicating that the combination of pine muscarinic acid and tricyclazole, and pine muscarinic acid and carbendazim has a significant synergistic effect on the control of rice blast.

[0085] Table 1. Pot control efficacy and synergistic effect index of combined pesticides against rice blast. deal with Disease index Prevention and control effect (%) Theoretical combined prevention effect (%) Synergistic Efficiency Index Blank control 36.93 -- -- -- pine mushroom acid 20.80 43.68 -- -- Tricyclazole 20.23 45.22 -- -- Carbendazim 21.43 41.97 -- -- pine muscarinic acid + tricyclazole 3.00 91.88 69.15 1.33 Matrine + Carbendazim 2.86 92.26 67.32 1.37

[0086] The above description is merely a specific embodiment of the present invention, intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be construed as limiting the scope of protection of the present invention. All equivalent modifications or substitutions made based on the essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. Application of muscarinic acid in the control of rice blast or in the preparation of rice blast control agents.

2. The application as described in claim 1, characterized in that, The plant in question is a member of the Poaceae family.

3. The application as described in claim 2, characterized in that, The plant in question is either rice or barley.

4. The application as described in claim 1, characterized in that, The rice blast disease mentioned is leaf blast.

5. The application as described in any one of claims 1-4, characterized in that, The application includes applying muscarinic acid to plants during their growth stages to prevent or inhibit rice blast fungus infection.

6. The application as described in claim 5, characterized in that, The effective concentration of muscarinic acid is 10 μM to 100 μM.

7. A preparation for controlling rice blast disease, characterized in that, The active ingredients of the formulation include: meliacin and tricyclazole in a molar ratio of 1:200-300; or meliacin and carbendazim in a molar ratio of 1:1000-2000.

8. The formulation as described in claim 7, characterized in that, The active ingredients of the formulation include: meliacin and tricyclazole in a molar ratio of 1:260; or meliacin and carbendazim in a molar ratio of 1:1400.

9. The formulation as described in claim 7, characterized in that, Gelatin is added as an adhesive in the formulation.

10. The application of the formulation according to any one of claims 7-9 in the control of rice blast, characterized in that, The effective concentration of muscarinic acid is 5 μM to 10 μM.

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