Application of compound HLF1-11 in preparation of medicine for preventing and treating magnaporthe oryzae

Abstract

The invention provides application of a compound HLF1-11 in preparation of a medicine for preventing and controlling magnaporthe oryzae and a medicine for inhibiting spore germination of the magnaporthe oryzae, and further provides a pesticide and a method for preventing and controlling the magnaporthe oryzae. The compound HLF1-11 has an obvious inhibiting effect on development of magnaporthe oryzae appressorium, can effectively inhibit pathogenicity of magnaporthe oryzae spores and can prevent and treat magnaporthe oryzae, so that a foundation is laid for research and development of related prevention and control drugs.

Description

Technical Field

[0001] This invention belongs to the field of plant disease control, specifically relating to the application of compound HLF1-11 in the preparation of a drug for controlling rice blast fungus. Background Technology

[0003] The rice blast fungus attaches to the rice epidermis via conidia, secreting chitinase and lipase to degrade the waxy layer. Invasive hyphae penetrate stomata or epidermal cells, forming infectious hyphae that spread to the vascular bundles. Its secreted effector proteins (such as MoT) can inhibit the expression of rice resistance genes (such as Pi-ta and Pik) while activating pathogenic virulence genes (such as MC69), leading to rice cell necrosis and triggering typical symptoms such as leaf blast and panicle blast.

[0004] There are various methods to control rice blast fungus, such as chemical control, biological control, agricultural management, and disease-resistant breeding.

[0005] 1) Chemical control. For example, traditional fungicides such as tricyclazole can quickly block the germination of pathogen spores. In 2023, the national control area exceeded 3.2 million hectares, with a control efficacy of over 80% in a short period of time. Patent application CN112273028A discloses a method for controlling rice blast, including the following steps: Step 1, planting site treatment; Step 2, seedling raising: from the 4-leaf stage of the seedlings in the seedbed until transplanting, spray the seedlings twice with a 0.3%~0.4% potassium dihydrogen phosphate solution, with a spraying amount of 50~60 kg per mu (approximately 0.067 hectares). During the second application, add 50~60 g of a 35%~40% tricyclazole suspension to the potassium dihydrogen phosphate solution; Step 3, planting site treatment before transplanting; Step 4, seedling transplanting; Step 5, seedling treatment after transplanting: 15~40 days after transplanting, mix 50~60 kg of a 0.3% potassium dihydrogen phosphate solution and 50~60 g of a 40% tricyclazole suspension per mu (approximately 0.067 hectares), add water, and spray once. Repeat the same method when the rice is in the heading stage. Rice blast can be controlled by promoting seedling growth and using tricyclazole suspension to inhibit rice blast fungal spores. However, long-term use can lead to resistance, and excessive application can pollute the soil and water sources.

[0006] 2) Biological control. For example, Bacillus subtilis inoculants inhibit pathogens through competition and antimicrobial peptides, exhibiting high environmental compatibility and a control efficacy of approximately 60%-70%. Patent application CN120384015A discloses a strain of Bacillus subtilis and its applications. This strain, designated WR4, is accessed at the China General Microbiological Culture Collection Center (CGMCC No. 29148) and isolated from rice rhizosphere soil. It coexists harmoniously with the rice rhizosphere soil microecology. Furthermore, this strain possesses a certain degree of salt tolerance, allowing for a wider range of soil applications. It can broadly control pathogenic fungi such as Fusarium, Verticillium dahliae, Rice blast fungus, and Fusarium graminearum, as well as pathogenic bacteria such as potato scab pathogen. However, it exhibits poor field stability; rainy weather reduces the activity of Bacillus subtilis; and some strains have a narrow antimicrobial spectrum.

[0007] 3) Agricultural management. For example, crop rotation can reduce the pathogen population by 25%-40%; precision irrigation reduces field humidity and inhibits spore spread. However, this relies on large-scale farmland and cooperation among farmers, making it difficult to promote in scattered planting areas.

[0008] 4) Disease-resistant breeding. For example, gene editing (such as CRISPR-Cas9 targeted editing of OsSWEET11) can breed broad-spectrum disease-resistant varieties, and the coverage rate of its promotion exceeded 65% in 2023. However, pathogens mutate rapidly ("escape-type" physiological races have been found in Yunnan and other places), and single disease-resistant genes are prone to failure.

[0009] Therefore, the development of drugs to control rice blast fungus remains of great significance. Summary of the Invention

[0010] To address the aforementioned technical problems in the prior art, this invention provides the application of compound HLF1-11 in the preparation of drugs for controlling rice blast fungus and drugs for inhibiting the germination of rice blast fungus spores, and also provides a pesticide and method for controlling rice blast fungus.

[0011] This invention provides compound HLF1-11 in the preparation of a fungicide for controlling rice blast fungus (… Magnaporthe oryzae Its application in drugs.

[0012] Preferably, the concentration of the compound HLF1-11 is 300~1000 μM.

[0013] More preferably, the concentration of compound HLF1-11 is 800~1000 μM, which has a more significant inhibitory effect on rice blast fungus.

[0014] More preferably, the concentration of compound HLF1-11 is 1000 μM, which has a better effect on preventing and controlling rice blast fungus.

[0015] Preferably, the rice blast fungus infects grass plants, and the drug is sprayed onto the leaves of the grass plants during application.

[0016] The present invention also provides the use of compound HLF1-11 in the preparation of a drug for inhibiting the germination of spores of rice blast fungus.

[0017] Preferably, the concentration of the compound HLF1-11 is 300~1000 μM.

[0018] More preferably, the concentration of compound HLF1-11 is 800~1000 μM, which can more effectively inhibit the pathogenicity of rice blast fungus spores.

[0019] More preferably, the concentration of compound HLF1-11 is 1000 μM, which has a better effect on inhibiting the spore germination of rice blast fungus.

[0020] The present invention also provides a pesticide for controlling rice blast fungus, wherein the active substance is compound HLF1-11.

[0021] The present invention also provides a method for controlling rice blast fungus, wherein the above-mentioned pesticide for controlling rice blast fungus is sprayed on the leaves of gramineous plants.

[0022] Preferably, the concentration of compound HLF1-11 in the pesticide used to control rice blast fungus is 300~1000 μM.

[0023] More preferably, the concentration of compound HLF1-11 in the pesticide used to control rice blast fungus is 800~1000 μM, which has a significant inhibitory effect on rice blast fungus.

[0024] More preferably, the concentration of compound HLF1-11 in the pesticide used to control rice blast fungus is 1000 μM, which has a more significant inhibitory effect on rice blast fungus.

[0025] Compared with the prior art, the present invention has the following beneficial effects: The compound HLF1-11 in this invention has a significant inhibitory effect on the development of appressorium of rice blast fungus, which can effectively inhibit the pathogenicity of rice blast fungus spores and prevent and control rice blast, thus laying the foundation for the development of related control drugs. Attached Figure Description

[0026] Figure 1 The inhibitory effect of compound HLF1-11 (1 mM) on appressorium formation of rice blast fungus.

[0027] Figure 2The disease incidence in detached barley leaves was observed when different concentrations of the compound HLF1-11 were added to the spore liquid of rice blast fungus.

[0028] Figure 3 This study describes the disease incidence 12 days after spraying 1 mM compound HLF1-11 simultaneously with inoculation with rice blast fungus spores in a net-house experiment.

[0029] Figure 4 The percentage of each level of disease in the net-house experiment.

[0030] Figure 5 This refers to the disease index in the net room experiment.

[0031] Figure 6 This study describes the disease incidence 8 days after spraying 1 mM compound HLF1-11 simultaneously with inoculation with rice blast fungus spore liquid in a field experiment.

[0032] Figure 7 The percentages of leaf blight at each level in the field experiment.

[0033] Figure 8 The disease index of leaf blight in the field experiment.

[0034] Figure 9 This study investigated the incidence of neck blast 14 days after a second application of 1 mM compound HLF1-11 one week later during a field experiment at the beginning of heading.

[0035] Figure 10 The proportion of each level of neck blast disease in field experiments.

[0036] Figure 11 The disease index of rice neck blast in field experiments.

[0037] Note: In the figure, *** indicates p < 0.001, and **** indicates p < 0.0001. Detailed Implementation

[0038] In the following embodiments, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the field, and the methods used are all conventional methods in the field.

[0039] The rice blast fungus used in this experiment was the wild-type strain Guy11 preserved in the laboratory (ATCC Strain Center, https: / / www.atcc.org / products / 201236), and the culture medium for culturing the rice blast fungus in the laboratory was complete medium (CM). CM culture medium formula: Glucose 10 g, Peptone-140 2 g, Casein amino acids 1 g, Yeast extract 1 g, NaNO3 6 g, KH2PO4 1.52 g, KCl 0.52 g, MgSO4·7H2O 0.52 g, Biotin 0.1 mg, Vitamin B 0.1 mg, Thiamine 0.1 mg, Riboflavin 0.1 mg, Niacin 0.1 mg, Para-aminobenzoic acid 0.1 mg, 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, Na4EDTA·2H2O 50 mg, Agar powder 15 g, adjust pH to 6.5 with NaOH, bring volume to 1 L with H2O, and autoclave at 121 °C for 15 min.

[0040] Compound HLF1-11, CAS No. 183623-03-2, was purchased from MedChemexpress Biotechnology Company, USA (Catalog No.: HY-106228).

[0041] Preparation and storage of the stock solution of compound HLF1-11: Dissolve 50 mg of compound HLF1-11 in 606 μL ddH2O to achieve a final concentration of 60 mM. Sterilize by filtration through a 0.22 μm aqueous membrane. Store for short periods at -20 ℃ and for long-term storage in aliquots at -80 ℃.

[0042] Example 1: Determination of the inhibitory effect of compound HLF1-11 on the development of appressorium of rice blast fungus. For rice blast fungus cultured on CM plates (25 °C, 16 h light / 8 h dark) for 7-9 days, spores were collected by gently scraping with sterile water using a sterile spreader. Then, 1 mL of the spore suspension was pipetted into a three-layer filter paper for filtration. The filtered spores were collected in a 1.5 mL centrifuge tube and centrifuged (7500 rpm) for 5 min. After centrifugation, 1 mL of ddH2O was injected. 。 The spores were then counted under a regular optical microscope, and the concentration of the spore solution was adjusted to 1×10⁻⁶. 5The extract was prepared in two tubes at a concentration of 1 mM (number of cells / mL). One tube served as the control group and was treated with H2O, while the other tube was treated with HLF1-11 to a concentration of 1 mM. To prevent bacterial contamination, cephalosporins and streptomycin were also added to each tube at a concentration of 50 mg / L. Clean, anhydrous glass slides and hydrophobic rigid membranes were used to induce appressorium growth. The control and experimental groups were tested for 4 h, 8 h, and 24 h, respectively, with each treatment repeated three times.

[0043] Results Analysis: The effect of compound HLF1-11 at a concentration of 1 mM on the development of appressorium of rice blast fungus was tested. Figure 1 As shown, the compound HLF1-11 in the experiment had a significant inhibitory effect on the development of appressorium of rice blast fungus at 4 h, 8 h and 24 h, resulting in the inability of rice blast fungus to form appressorium within 24 h, and even the formation of germ tubes was strongly inhibited.

[0044] Example 2: Pathogenicity determination and results of compound HLF1-11 against rice blast fungus. Experimental method: In vitro inoculation method.

[0045] In vitro barley inoculation: Rice blast fungus was cultured on CM medium (25 °C, 16 h light / 8 h dark) for 7-9 days. Double-distilled water was then added to the culture dish, and spores were collected by gently scraping the surface of the colonies with a sterile spreader. Different doses (0, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 μM) of compound HLF1-11 were mixed with the spores to prepare a drug-containing spore solution (final spore concentration 1×10⁻⁶). 5 CFU / mL), the dose difference of compound HLF1-11 in different treatment groups was made up with H2O. One-week-old barley (variety: Hordeum vulgare For well-grown, flat leaves of barley (ZJ-8), drop 25 μL of spore-containing solution onto each barley leaf (3 drops per leaf), with each treatment repeated 3 times. The control group contained the same volume of H2O and was cultured at 25℃ under humid conditions for approximately 4 days under alternating light and dark conditions (16 h / 8 h). The disease incidence on the leaves was then observed.

[0046] Results Analysis: The inhibitory effects of gradient concentrations of compound HLF1-11 on rice blast disease in detached barley leaves were tested. Figure 2 As shown, treatment with compound HLF1-11 at 300–1000 μM had a certain inhibitory effect on the infection of detached barley leaves by rice blast fungus; however, within this range, the tested concentration and the inhibitory effect did not seem to be positively correlated; among them, compound HLF1-11 at 800–1000 μM had a more obvious inhibitory effect on rice blast infection of detached barley leaves.

[0047] Example 3: Determination and results of the pathogenicity of compound HLF1-11 against rice blast fungus in net housing. Experimental method: live rice spraying method.

[0048] The net-house experiment selected rice (variety: ) that had grown for 25 days. Oryza sativa Spray the leaves of CO39 with a spore solution containing 1 mM HLF1-11 (5 × 10⁻⁶). 4 Spores / mL, 0.2% (w / v) gelatin), symptom scores were examined and images were taken after 12 days. Kasugamycin, tricyclazole, and carbendazim were selected as positive control groups, and water was used as a blank control group. Each treatment was set up in 3 replicates, and 30 leaves were taken from each replicate, for a total of 90 leaves. The disease progression of leaf blast in net houses was investigated according to the standard evaluation system of rice (International Rice Research Institute, 2002).

[0049] Results analysis: like Figure 3 As shown, in the net greenhouse experiment, rice seedlings treated with plain water exhibited typical leaf blast symptoms (CK in the figure), while seedlings treated with HLF1-11 or three other fungicides showed a significant reduction in leaf blast. Figure 4 As shown, in the net greenhouse experiment, the proportion of grade 3 diseases in the experimental group decreased significantly compared to the control group, while the proportions of grade 0 and 1 diseases increased significantly compared to the control group; Figure 5 As shown, the average disease index of the control group was 22.54. After drug treatment, the disease indexes were 7.75, 4.09, 2.45 and 3.91, respectively, which were 65.60%, 81.83%, 89.14% and 82.63% lower than those of the control group.

[0050] Example 4: Determination and results of the pathogenicity of compound HLF1-11 against rice blast fungus in the field (specifically, leaf blast and neck blast). Experimental method: Field rice spraying The field blast experiment was conducted in a rice blast fungus nursery (Lin'an District, Hangzhou, Zhejiang Province). Rice (variety: ) was sown on June 4, 2024. Oryza sativa CO39 plants that had grown for 28 days were selected and sprayed with a spore solution containing 1 μM HLF1-11. Leaf blast symptoms were examined and images were taken on day 8 after spraying. Kasugamycin, tricyclazole, and carbendazim were selected as positive control groups, and water was selected as a blank control group. Each treatment was repeated in triplicate, and 30 leaves were taken from each replicate, for a total of 90 leaves. The disease progression of leaf blast in the field was investigated according to the standard evaluation system of rice (International Rice Research Institute, 2002).

[0051] The field neck blast experiment was conducted in a rice blast fungus nursery (Lin'an District, Hangzhou, Zhejiang Province). Yongyou rice varieties were sown on June 4, 2024. Yongyou varieties that had grown for 90 days were selected and sprayed with 1 μM HLF1-11 (dissolved in water). Neck blast symptoms were examined and imaged on day 14 post-spraying. Kasugamycin, tricyclazole, and carbendazim were selected as positive control groups, and water was used as a blank control group. Each treatment was replicated in triplicate.

[0052] Seedlings for each treatment were planted in three randomized plots (approximately 30 m² per treatment). 2 It can accommodate approximately 1000 seedlings, with a spraying rate of 70 mL / m². 2 The disease progression of neck blast was investigated according to the standard evaluation system of rice (International Rice Research Institute, 2002).

[0053] Results analysis: such as Figure 6 and Figure 9 As shown, in field trials, rice seedlings treated with water exhibited typical leaf blast / neck blast symptoms, while seedlings treated with HLF1-11 or three other fungicides showed a significant reduction in leaf blast / neck blast. Figure 7 As shown, in the field experiment, the proportion of leaf blast disease grade 3 in the experimental group was significantly lower than that in the control group, while the proportion of disease grade 0 was significantly higher than that in the control group; Figure 10 As shown, in the field experiment, the proportions of disease grades 5, 7, and 9 in the neck blast experimental group were significantly lower than those in the control group, while the proportions of disease grades 0 and 1 were significantly higher. Figure 8 As shown, the average disease index of the leaf blight control group was 12.05. After drug treatment, the disease indices were 3.97, 3.32, 0.97, and 1.19, respectively, representing decreases of 67.07%, 72.46%, 91.94%, and 90.15% compared to the control group. Figure 11 As shown, the average disease index of the control group of rice blast was 34.31. After drug treatment, the disease indices were 8.73, 4.55, 3.39 and 2.78, respectively, which were 74.55%, 86.73%, 90.12% and 91.91% lower than those of the control group.

Claims

1. Compound HLF1-11 was used in the preparation of a fungicide for controlling rice blast fungus. Magnaporthe oryzae Its application in drugs.

2. The application according to claim 1, characterized in that, The concentration of the compound HLF1-11 is 300~1000 μM.

3. The application according to claim 2, characterized in that, The concentration of the compound HLF1-11 is 800~1000 μM.

4. The application according to claim 1, characterized in that, The rice blast fungus infects grass plants. When applying the drug, it is sprayed onto the leaves of the grass plants.

5. Application of compound HLF1-11 in the preparation of drugs that inhibit the germination of rice blast fungus spores.

6. The application according to claim 5, characterized in that, The concentration of the compound HLF1-11 is 300~1000 μM.

7. The application according to claim 6, characterized in that, The concentration of the compound HLF1-11 is 800~1000 μM.

8. A pesticide for controlling rice blast fungus, characterized in that, The active substance is compound HLF1-11.

9. A method for controlling rice blast fungus, characterized in that, The pesticide for controlling rice blast fungus as described in claim 8 is sprayed onto the leaves of grass plants.

10. The method for controlling rice blast fungus according to claim 9, characterized in that, The concentration of compound HLF1-11 in the pesticide used to control rice blast fungus is 300~1000 μM.

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