Application of compound SBP-7455 in the preparation of drugs for controlling rice blast fungus

Abstract

The invention discloses the application of compound SBP-7455 in the preparation of a medicine for preventing and controlling rice blast fungi, and belongs to the field of plant disease prevention and control. The compound SBP-7455 provided by the invention is used in inhibiting rice blast fungi and preventing and controlling rice blast, and can effectively inhibit the pathogenicity of rice blast fungi spores within a concentration range of 0.5-4 μM, and in particular, the compound SBP-7455 at a concentration of 4 μM can completely prevent and control rice blast. This lays a foundation for the development of new green pesticides.

Description

Technical Field

[0001] The invention belongs to the field of plant disease prevention and control, and specifically relates to application of a compound SBP-7455 in preparing a medicine for preventing and controlling rice blast fungus. Background Art

[0002] Plant diseases are one of the factors that threaten the production of food crops, among which rice blast can lead to a significant reduction in the yield of gramineous crops including rice. Among fungal diseases, rice blast caused by the rice blast fungus Magnaporthe oryzae (syn. Pyricularia oryzae) has great destructive power on rice production. The host range of rice blast fungus is relatively wide, and different strains can infect at least 50 species of gramineous plants, including major food crops such as rice, wheat, barley and oats. When the disease is serious, the crop yield loss can reach 100%. The rice blast fungus also has the characteristics of wide regional distribution, strong environmental adaptability and fast mutation rate. Therefore, the comprehensive prevention and control of rice blast has always been a hot spot and difficulty in plant disease prevention and control.

[0003] The existing methods of preventing and controlling rice blast mainly include breeding disease-resistant varieties, eliminating fungal sources, strengthening cultivation management, and chemical control. Among them, chemical control is the most effective prevention and control measure recognized by most technicians and farmers. However, the abuse of a large number of chemical pesticides has gradually increased the resistance of rice blast fungi and caused a series of environmental problems. Therefore, finding pathogenic molecular targets and developing new green pesticides are important strategies for preventing and controlling rice blast, which are of great significance to ensuring food production.

[0004] Compound SBP-7455 is a compound with the potential to treat triple-negative breast cancer. It is artificially modified from SBI-0206965, an inhibitor of the autophagy-related kinase ULK1. Its structural formula is as follows:

[0005]

[0006] Studies have shown that compared with SBI-0206965, compound SBP-7455 has a higher binding affinity for ULK1 / 2, and can effectively inhibit ULK1 / 2 activity both in vivo and in vitro, thereby reducing the phosphorylation levels of downstream Beclin1 and Vps34, inhibiting starvation-induced autophagy flux in triple-negative breast cancer cells that rely on autophagy for survival (Huiyu Ren, et al. Design, Synthesis, and Characterization of an Orally Active Dual-Specific ULK1 / 2 Autophagy Inhibitor that Synergizes with the PARP Inhibitor Olaparib for the Treatment of Triple-Negative Breast Cancer. J Medd Chem. 2020 Dec 10; 63(23): 14609-14625.), reducing the survival rate of triple-negative breast cancer cells, and has oral bioavailability in mice, which is clinically practical. However, the research and application of compound SBP-7455 in the prevention and control of plant diseases have not been reported. Summary of the invention

[0007] Since the pathogenicity of rice blast fungus is regulated by autophagy core proteins, and the MoAtg1 protein homologous to ULK1 is related to the pathogenicity of rice blast fungus, the applicant speculates that compound SBP-7455 may have the potential to inhibit rice blast fungus and prevent and control rice blast disease.

[0008] The present invention aims to provide a compound SBP-7455 for use in inhibiting rice blast fungus and preventing and treating rice blast disease. The compound SBP-7455 has an IC of 1.1475 for the mycelial growth of rice blast fungus. 50 It is 0.1780μM, and has the advantages of significant effect, long action time, safety and simple application method in the prevention and control of rice blast.

[0009] To achieve the above object, the technical solution of the present invention is as follows:

[0010] The present invention provides application of compound SBP-7455 in preparing medicine for preventing and controlling rice blast fungus (Magnaporthe oryzae).

[0011] The present invention also provides the use of the compound SBP-7455 in preparing a medicine for inhibiting the mycelial growth or spore germination of rice blast fungus.

[0012] Preferably, the concentration of the compound SBP-7455 is 0.5-4 μM.

[0013] The compound SBP-7455 of the present invention can effectively inhibit the pathogenicity of rice blast fungus spores within a concentration range of 0.5 to 4 μM.

[0014] More preferably, the concentration of the compound SBP-7455 is 2-4 μM.

[0015] The compound SBP-7455 at a concentration of 2 to 4 μM has a significant inhibitory effect on in vitro barley blast.

[0016] Most preferably, the concentration of the compound SBP-7455 is 4 μM.

[0017] The compound SBP-7455 at a concentration of 4 μM was able to completely inhibit barley blast in vitro.

[0018] The present invention also provides a drug for preventing and controlling rice blast fungus, comprising compound SBP-7455.

[0019] The present invention also provides a method for preventing and controlling rice blast fungi, and the medicine for preventing and controlling rice blast fungi is sprayed on leaves of plants.

[0020] Preferably, the concentration of compound SBP-7455 in the drug for controlling rice blast fungus is 0.5-4 μM. 0.5-4 μM compound SBP-7455 treatment has a certain inhibitory effect on in vitro barley rice blast fungus.

[0021] More preferably, the concentration of the compound SBP-7455 is 2-4 μM.

[0022] The compound SBP-7455 at a concentration of 2 to 4 μM has a significant inhibitory effect on in vitro barley blast.

[0023] Most preferably, the concentration of the compound SBP-7455 is 4 μM.

[0024] The compound SBP-7455 at a concentration of 4 μM was able to completely inhibit barley blast in vitro.

[0025] Beneficial effects of the present invention:

[0026] The compound SBP-7455 provided by the present invention is used in inhibiting rice blast fungi and preventing and controlling rice blast disease. The compound SBP-7455 can effectively inhibit the pathogenicity of rice blast fungi spores in the concentration range of 0.5-4 μM, and in particular, the compound SBP-7455 at a concentration of 4 μM can completely prevent and control rice blast disease, laying a foundation for the development of new green pesticides. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1The inhibition of different concentrations of compound SBP-7455 on the plate growth of rice blast fungus.

[0028] Figure 2 The figure is a comparison of the diameter of the rice blast fungus plate under the treatment of different concentrations of compound SBP-7455; in the figure, the inhibition rates of different concentrations of compound SBP-7455 are significantly different, which are represented by different letters a, b, c, d or e; p < 0.05; the data in the figure are obtained by one-way ANOVA.

[0029] Figure 3 The figure is a comparison of the inhibition rate of rice blast fungus plate growth under the treatment of different concentrations of compound SBP-7455; in the figure, the inhibition rates of different concentrations of compound SBP-7455 are significantly different, which are represented by different letters a, b, c, d or e; p<0.05; the data in the figure are obtained by one-way analysis of variance.

[0030] Figure 4 This is the disease occurrence of detached barley leaves when different concentrations of the compound SBP-7455 were added to the spore liquid of the rice blast fungus.

[0031] Figure 5 The disease condition of detached rice leaves when different concentrations of compound SBP-7455 were added to the spore solution of rice blast fungus;

[0032] Figure 6 The graph shows the disease occurrence of live rice leaves after spraying compound SBP-7455 at different stages of inoculation with spores of rice blast fungus.

[0033] Figure 7 This is a comparison of the proportion of lesion area on rice leaves after spraying compound SBP-7455 at different stages of inoculation with rice blast fungus spores. ** indicates p<0.0001. DETAILED DESCRIPTION

[0034] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following embodiments, unless otherwise specified, are all conventional methods in the art.

[0035] The rice blast fungus used in this experiment was the wild-type strain Guy11 preserved in the laboratory (ATCC, https: / / www.atcc.org / products / 201236). The culture medium for rice blast fungus in the laboratory was complete medium (CM for short). CM medium formula: glucose 10g, peptone-1402g, casamino acids 1g, yeast extract 1g, NaNO36g, KH2PO41.52g, KC1 0.52g, MgSO4·7H2O 0.52g, biotin 0.1mg, vitamin B 0.1mg, thiamine 0.1mg, riboflavin 0.1mg, niacin 0.1mg, p-aminobenzoic acid 0.1mg, Na2MoO4·5H2O1.5mg, CuSO4·5H2O1.6mg, CoCl2·6H2O1.7mg, MnCl2·4H2O5mg, FeSO4·7H2O5mg, H3BO311mg, ZnSO4·7H2O22mg, Na4EDTA·2H2O50mg, agar powder 15g, adjust the pH to 6.5 with NaOH, make up to 1L with H2O, and sterilize at 115℃ and high temperature and high pressure for 30min.

[0036] Compound SBP-7455, CAS No. 1884222-74-5, was purchased from MCE (Cat. No. HY-137742), and its structural formula is as follows:

[0037]

[0038] Preparation method of compound SBP-7455 stock solution: 5 mg of compound SBP-7455 was diluted with 1.4111 mL of DMSO at a concentration of 10 mM.

[0039] Example 1 Toxicity determination of compound SBP-7455 against rice blast fungus and results

[0040] Test method: mycelium growth rate method. The rice blast fungus was cultured on a CM plate at 25°C for 7-9 days; different doses (0, 0.1, 0.2, 0.3 and 0.4 μM) of the compound SBP-7455 were added to the solid CM culture medium cooled to 40-50°C to prepare a drug-containing plate; under a sterile operating environment, a round fungus cake (5 mm in diameter) with relatively consistent growth conditions was punched at the edge of the mycelium of the rice blast fungus cultured for 7-9 days with a hole puncher, and then a sterilized toothpick was used to pick it to the center of the drug-containing plate, with the aerial mycelium surface facing the culture medium; each treatment was repeated 3 times; the control group contained the same concentration of DMSO; the culture dish was inverted and cultured in a dark incubator at 25°C, and when the control group was nearly full, the growth of the mycelium was observed and measured, and the mycelium diameter was measured by the cross method, and the growth inhibition rate was calculated: growth inhibition rate (%) = (average diameter of the control group-diameter of the treatment group) / (average diameter of the control group-diameter of the fungus cake) × 100, and the toxicity regression equation, correlation coefficient R and IC were obtained by analysis using the DPS statistical analysis system. 50 The toxicity of compound SBP-7455 to rice blast fungus was evaluated.

[0041] Results: The toxicity of compound SBP-7455 to rice blast fungus was tested at different concentrations. Figure 1 As shown in the figure, the four concentrations of compound SBP-7455 in the experiment all had a certain inhibitory effect on the mycelial growth of rice blast fungus, and within this range, the higher the test concentration, the smaller the colony diameter ( Figure 2 ), the higher the mycelial growth inhibition rate ( Figure 3 ); IC of compound SBP-7455 on mycelial growth of rice blast fungus 50 When the concentration of compound SBP-7455 was 0.4 μM, it had excellent antibacterial effect on rice blast fungus, and the relative inhibition rate was 100% (Table 1).

[0042] Table 1 Indoor toxicity of compound SBP-7455 at different concentrations against rice blast fungus

[0043]

[0044] Example 2 Pathogenicity test and results of compound SBP-7455 against rice blast fungus

[0045] 1. Experimental method: in vitro inoculation method.

[0046] (1) In vitro barley inoculation:

[0047] Healthy leaves of one-week-old barley (cultivar: Hordeum vulgar, grown at the Institute of Biotechnology, Zhejiang University) were cut; the rice blast fungus was cultured on CM medium for 7-9 days to obtain spore liquid; different doses (0, 0.1, 0.5, 1, 2 and 4 μM) of compound SBP-7455 were mixed with the spore liquid to prepare drug-containing spore liquid (final spore concentration was 1×10 5 CFU / mL), the dose difference of compound SBP-7455 in different treatment groups was made up with DMSO to make the final DMSO concentration consistent; 25 μL of drug-containing spores were dropped on barley leaves; 3 drops were placed on each leaf, and each treatment was repeated 3 times; the control group contained the same concentration of DMSO; the leaves were cultured at 25℃, moisturizing, and 16h / 8h light / dark alternation for about 4 days, and the disease condition of the leaves was observed.

[0048] Result analysis: The inhibitory effect of compound SBP-7455 at different concentrations on barley blast in vitro was tested. Figure 4 As shown, 0.5-4 μM compound SBP-7455 treatment has a certain inhibitory effect on in vitro barley blast; and within this range, the higher the tested concentration, the stronger the inhibitory effect; among them, the compound SBP-7455 with a concentration of 2-4 μM has a significant inhibitory effect on in vitro barley blast; the compound SBP-7455 with a concentration of 4 μM can completely inhibit in vitro barley blast.

[0049] (2) In vitro rice inoculation:

[0050] The healthy second leaf of three-leaf rice (cultivated by China National Rice Research Institute, variety: Oryza sativa CO39) was selected, and the specific implementation method was the same as that of in vitro barley inoculation.

[0051] Result analysis: The inhibitory effect of compound SBP-7455 at different concentrations on rice blast in vitro was tested. Figure 5 As shown, the five concentrations of compound SBP-7455 in the experiment all had a certain inhibitory effect on rice blast in vitro; among them, the compound SBP-7455 at a concentration of 0.5 μM had a significant inhibitory effect on rice blast in vitro. At the same concentration, the compound SBP-7455 had a stronger inhibitory effect on rice in vitro than on barley in vitro ( Figure 4 and Figure 5 ).

[0052] 2. Test method: Living rice spray method.

[0053] Oryza sativa CO39 rice was sown at 30 seeds per pot and cultured outdoors until two weeks old. Rice blast fungus was cultured on CM medium for 7-9 days to obtain spore solution. Different doses of compound SBP-7455, gelatin solution and spore solution were mixed to prepare spore solution containing drug and gelatin (final spore concentration was 1×10 5 CFU / mL, gelatin final concentration of 0.2%), the difference in the dosage of compound SBP-7455 in different treatment groups was made up with DMSO to make the final concentration of DMSO consistent; 2mL of the mixed spore solution was evenly sprayed on the rice leaves with a small sprayer, and 3 pots of rice were inoculated for each treatment. In order to explore the control effect of compound SBP-7455 on live rice blast fungus at the application time, the optimal concentration of compound SBP-7455 for inhibiting the occurrence of rice blast was selected as 4μM, and 4μM compound SBP-7455 was applied at different time points of inoculation of spore solution of rice blast fungus, and three implementation plans were set up: (1) -24h: compound SBP-7455 was applied 24 hours before inoculation of spore solution of rice blast fungus; (2) 0h: compound SBP-7455 was applied at the same time as inoculation of spore solution of rice blast fungus; (3) +24h: compound SBP-7455 was applied 24 hours after inoculation of spore solution of rice blast fungus. Three pots of rice were treated in each treatment, and DMSO containing the same concentration was applied at the time point when compound SBP-7455 was not applied; the control group was also applied with DMSO containing the same concentration at the corresponding time point. In order to detect whether compound SBP-7455 has an effect on rice growth, a control group was set up in which compound SBP-7455 (4 μM) and gelatin solution were sprayed only. The inoculated rice was first cultured in a dark incubator at 22°C for 2 days, and then cultured in a 25°C, 16h / 8h light-dark alternating incubator for 3-4 days; the leaf disease was observed and photographed, and the lesion area was calculated using Image J software, and the lesion area ratio was calculated: lesion area ratio (%) = lesion area / leaf area × 100.

[0054] Result analysis: The control effect of compound SBP-7455 on rice blast disease in living rice was tested at different time points. Figure 6 As shown, compared with the control group without compound SBP-7455, the -24h, 0h and +24h treatment groups were able to significantly inhibit the growth of rice blast ( Figure 7 ); In addition, compound SBP-7455 had no obvious adverse effects on rice growth, indicating that it may be safe for rice and has practical significance for preventing rice blast disease.

Claims

1. Use of compound SBP-7455 in the preparation of a drug for controlling rice blast fungus (Magnaporthe oryzae), wherein the structural formula of the compound SBP-7455 is as follows:

2. Use of compound SBP-7455 in the preparation of a drug for inhibiting mycelial growth or spore germination of rice blast fungus, wherein the structural formula of the compound SBP-7455 is as follows:

3. The use according to claim 1 or 2, characterized in that: The concentration of the compound SBP-7455 is 0.5-4 μM.

4. The use according to claim 3, characterized in that The concentration of the compound SBP-7455 is 2-4 μM.

5. A method for preventing and controlling rice blast fungus, characterized in that: The compound SBP-7455 is sprayed on the leaves of the plants. The structural formula of the compound SBP-7455 is as follows:

6. The method for controlling rice blast fungus as claimed in claim 5, characterized in that: The concentration of the compound SBP-7455 is 0.5-4 μM.

7. The method for controlling rice blast fungus as claimed in claim 6, characterized in that: The concentration of the compound SBP-7455 is 2-4 μM.

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