Application of compound delatinib in preparation of medicine for preventing and treating magnaporthe oryzae

Abstract

The invention provides application of the compound delatinib in preparation of drugs for preventing and controlling magnaporthe oryzae and drugs for inhibiting mycelial growth or spore germination of the magnaporthe oryzae and further provides a method for preventing and controlling the magnaporthe oryzae, and the compound delatinib can inhibit mycelial growth or spore germination of the magnaporthe oryzae. The delatinib compound can inhibit pathogenicity of magnaporthe oryzae, can prevent and treat the magnaporthe oryzae, is long in inhibition effect duration, is environment-friendly, has no obvious adverse effect on rice growth, and lays a foundation for development of novel green pesticides.

Description

Technical Field

[0001] The invention belongs to the field of plant disease prevention and control, and specifically relates to the use of the compound delatinib in the preparation of a medicine for preventing and controlling rice blast fungus. Background Art

[0002] Plant diseases are one of the factors that threaten the production of grain crops. Among them, rice blast can cause a significant reduction in the yield of gramineous crops including rice. Among fungal diseases, rice blast caused by the rice blast fungus Magnaporthe oryzae has a great destructive power on rice production. The host range of rice blast fungus is relatively wide. Different strains can infect at least 50 species of gramineous plants, including major grain 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 means of preventing and controlling rice blast mainly include breeding disease-resistant varieties, eliminating bacteria sources, strengthening cultivation management, chemical control, etc., among which chemical control is the most effective control measure recognized by most technicians and farmers, but 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. For example, the patent application with publication number CN87100851A discloses a method for preventing and controlling crop diseases: TCCA (trichloroisocyanuric acid) is used as an agricultural bactericidal disinfectant to disinfect rice seeds, cotton seeds and various crop seeds, and field application of pesticides to prevent and control various crop diseases such as rice bacterial leaf streak, white leaf blight, rice blast, seedling disease, cotton rot, sheath blight, wheat rust, root rot, cucumber downy mildew, anthracnose, and gourd powdery mildew. Therefore, finding pathogenicity-related molecular targets and developing new green pesticides are important strategies for preventing and controlling rice blast diseases, which are of great significance to ensuring food production.

[0004] Derazantinib (also known as Derazantinib) is a selective fibroblast growth factor receptor (FGFR) inhibitor, mainly used to inhibit the proliferation and migration of tumor cells, especially in the treatment of certain types of cancer such as intrahepatic cholangiocarcinoma and other tumors. Its structural formula is as follows:

[0005]

[0006] Delatinib inhibits FGFR2 autophosphorylation and other proteins downstream of the FGFR pathway (FRS2α, AKT, ERK), and has the activity of inhibiting amplification, fusion and mutation in cell lines driven by FGFR dysregulation. In addition, delatinib induces a positive correlation between G1 cell cycle arrest and subsequent apoptosis induction, inhibits FGF-mediated extracellular matrix loss and induces premature senescence of chondrocytes. Delatinib effectively inhibits tumor growth in xenograft tumor models of FGFR transformation, SNU-16 and NCI-H716, gene amplification and fusion.

[0007] The patent application with publication number CN119451699A discloses a drug for treating or preventing ALK fusion gene-positive cancer with positive FGFR1 expression and positive FGF2 expression, which is a combination of an ALK inhibitor and an FGFR inhibitor, wherein the FGFR inhibitor is a compound or salt thereof selected from infigratinib, delatinib, pemitinib, erdatinib, ponatinib, Debio1347, fobactinib, tasugatinib, AZD4547 and PD173074. At present, the research and application of delatinib are mainly focused on human diseases such as oncology and fibrotic lesions, and do not involve the prevention and treatment of plant diseases. Summary of the invention

[0008] In order to solve the above-mentioned technical problems existing in the prior art, the present invention provides the use of the compound delatinib in the preparation of a drug for preventing and controlling rice blast fungus. The compound delatinib has the advantages of significant effect, long action time and safety in preventing and controlling rice blast fungus.

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

[0010] The present invention provides application of the compound delatinib in preparing a medicine for preventing and controlling rice blast fungus.

[0011] Preferably, the concentration of the compound delatinib is 5 to 100 μM, which can effectively prevent and control rice blast fungus.

[0012] Further preferably, the concentration of the compound delatinib is 10 to 100 μM.

[0013] Most preferably, the concentration of the compound delatinib is 100 μM, which has the best effect in preventing and controlling rice blast fungus.

[0014] Preferably, the rice blast fungus infects a gramineous plant, and when used, the drug is sprayed on the leaves of the gramineous plant.

[0015] The present invention also provides the use of the compound delatinib in the preparation of a drug for inhibiting the mycelial growth or spore germination of rice blast fungus.

[0016] Preferably, the concentration of the compound delatinib is 5 to 100 μM, which can effectively inhibit the mycelial growth or spore germination of rice blast fungus.

[0017] Further preferably, the concentration of the compound delatinib is 10 to 100 μM.

[0018] Most preferably, the concentration of the compound delatinib is 100 μM, which has the best effect of inhibiting the mycelial growth or spore germination of rice blast fungus.

[0019] The present invention also provides a method for preventing and controlling rice blast fungi, which comprises spraying a drug for preventing and controlling rice blast fungi containing the compound delatinib on leaves of gramineous plants, and the operation is simple and convenient.

[0020] Preferably, the concentration of the compound delatinib in the drug for controlling rice blast fungus is 5 to 100 μM.

[0021] Further preferably, the concentration of the compound delatinib is 10 to 100 μM.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides the use of the compound delatinib in the preparation of a drug for preventing and controlling rice blast fungi and a drug for inhibiting mycelial growth or spore germination of rice blast fungi, and also provides a method for preventing and controlling rice blast fungi. The compound delatinib can inhibit the mycelial growth or spore germination of rice blast fungi, can inhibit the pathogenicity of rice blast fungi, can prevent and control rice blast, and the inhibitory effect lasts for a long time. The compound delatinib is environmentally friendly and has no obvious adverse effect on rice growth, thus laying a foundation for the development of new green pesticides. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The inhibition of different concentrations of the compound delatinib on the plate growth of rice blast fungus.

[0025] Figure 2 This is a comparison of the diameters of rice blast fungi grown on plates treated with different concentrations of the compound delatinib. In the figure, those with significant differences in inhibition rate with different concentrations of the compound delatinib are represented by different letters a, b, c, d or e; p<0.05; the data in the figure were obtained by one-way ANOVA.

[0026] Figure 3This is a comparison chart of the inhibition rate of rice blast fungus plate growth under treatment with different concentrations of the compound delatinib; in the figure, the inhibition rates of different concentrations of the compound delatinib with significant differences are represented by different letters a, b, c, d or e; p<0.05; the data in the figure were obtained by one-way analysis of variance.

[0027] Figure 4 The disease occurrence of detached barley leaves when different concentrations of the compound delatinib were added to the spore liquid of rice blast fungus.

[0028] Figure 5 This is the disease occurrence of detached rice leaves when different concentrations of the compound delatinib are added to the spore liquid of rice blast fungus.

[0029] Figure 6 The figure shows the disease occurrence of living rice leaves after spraying the compound delatinib at different times after inoculation with spores of rice blast fungus (conidia).

[0030] Figure 7 This is a comparison of the proportion of lesion area on rice leaves after spraying the compound delatinib at different times after inoculation with rice blast fungus spores. **** indicates p<0.0001. DETAILED DESCRIPTION

[0031] In the following examples, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods used in the following examples, unless otherwise specified, are all conventional methods in the art.

[0032] 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: 10 g glucose, 2 g peptone-140, 1 g casamino acids, 1 g yeast extract, 1 g NaNO 3 6g, KH 2 PO 4 1.52 g, KCl 0.52 g, MgSO 4 7H 2 O 0.52g, biotin 0.1mg, vitamin B 0.1mg, thiamine 0.1mg, riboflavin 0.1mg, niacin 0.1mg, p-aminobenzoic acid 0.1mg, Na 2 MoO 4 ·5H 2 O 1.5 mg, CuSO 4 ·5H 2 O1.6mg, CoCl 2 6H2 O 1.7 mg, MnCl 2 ·4H 2 O 5mg, FeSO 4 7H 2 O 5mg, H 3 BO 3 11mg, ZnSO 4 7H 2 O22mg, Na 4 EDTA·2H 2 O 50 mg, agar powder 15 g, adjust pH to 6.5 with NaOH, 2 O to 1 L and sterilize at 115℃ for 30 min.

[0033] The compound delatinib, CAS No. 1234356-69-4, was purchased from MCE (Cat. No.: HY-19981).

[0034] Preparation method of compound delatinib stock solution: dissolve 5 mg of compound delatinib in 1.0671 mL of DMSO, and the solution concentration is 10 mM.

[0035] Example 1 Determination of the toxicity of the compound delatinib against rice blast fungus and the results

[0036] 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, 5, 10, 25, 50 and 100 μM) of the compound delatinib 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 cake (4.5 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 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. After the control group was almost full, the growth of the mycelium was observed and measured, and the mycelium diameter was measured by the cross method to calculate the growth inhibition rate: growth inhibition rate (%) = (average diameter of the control group - diameter of the treatment group) / (average diameter of the control group - diameter of the cake) × 100, and the virulence regression equation, correlation coefficient R and IC were obtained by analysis using the DPS statistical analysis system. 50 Values ​​were used to evaluate the toxicity of compound delatinib against rice blast fungus.

[0037] Results analysis: The toxicity of different concentrations of the compound delatinib to rice blast fungus was tested. Figure 1As shown in the figure, the four concentrations of the compound delatinib 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 ); The IC50 of the compound delatinib on the mycelial growth of rice blast fungus is 56.23 μM. (Table 1). When the concentration of the compound delatinib is 100 μM, it has an excellent antibacterial effect on rice blast fungus, with a relative inhibition rate of 70.5% (Table 1).

[0038] Table 1 Indoor toxicity of different concentrations of compound delatinib to rice blast fungus

[0039]

[0040] Example 2 Pathogenicity determination and results of compound delatinib against rice blast fungus

[0041] 1. Experimental method: in vitro inoculation method.

[0042] (1) In vitro barley inoculation:

[0043] 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 (1, 5, 10, 25, 50 and 100 μM) of the compound delatinib were mixed with the spore liquid to prepare the drug-containing spore liquid (the final spore concentration was 1×10 5 CFU / mL); drop 25 μL of drug-containing spores on barley leaves; 3 drops on each leaf, and each treatment was repeated 3 times; the control group contained the same concentration of DMSO; culture at 25℃, moisturizing, and 16h / 8h light / dark alternation for about 4 days, and then observe the disease condition of the leaves.

[0044] Result analysis: The inhibitory effects of different concentrations of the compound delatinib on barley blast in vitro were tested. Figure 4 As shown, treatment with 5 to 100 μM concentration of delatinib has a certain inhibitory effect on in vitro barley blast; and within this range, the higher the test concentration, the stronger the inhibitory effect; among them, the compound delatinib with a concentration of 10 to 100 μM has a significant inhibitory effect on in vitro barley blast; the compound delatinib with a concentration of 100 μM can basically completely inhibit in vitro barley blast.

[0045] (2) In vitro rice inoculation:

[0046] 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.

[0047] Result analysis: The inhibitory effects of different concentrations of the compound delatinib on rice blast in vitro were tested. Figure 5 As shown in the figure, the five concentrations of the compound delatinib in the experiment all had a certain inhibitory effect on rice blast in vitro; among them, the compound delatinib at a concentration of 5 μM had a significant inhibitory effect on rice blast in vitro. At the same concentration, compared with barley in vitro, the compound delatinib had a stronger inhibitory effect on rice in vitro ( Figure 4 and Figure 5 ).

[0048] 2. Test method: Living rice spray method.

[0049] 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. Compound delatinib with a concentration of 100 μM, gelatin solution and spore solution were mixed to prepare a spore solution containing drug and gelatin (the final spore concentration was 1×10 5 CFU / mL, final gelatin concentration was 0.2%).

[0050] In order to explore the effect of the application time of compound delatinib on the control of live rice blast fungus, the optimal concentration of compound delatinib for inhibiting the occurrence of rice blast was selected as 100μM, and 100μM compound delatinib was applied at different time points of inoculation of rice blast fungus spores, and three implementation plans were set up: (1) -24h: compound delatinib was applied 24 hours before inoculation of rice blast fungus spores; (2) 0h: compound delatinib was applied at the same time as inoculation of rice blast fungus spores; (3) +24h: compound delatinib was applied 24 hours after inoculation of rice blast fungus spores. Three pots of rice were treated in each group. The application method of compound GN44028 was to spray 2mL of mixed spores evenly on the rice leaves with a small sprayer, and DMSO with the same concentration was applied at the time point when compound delatinib was not applied; the control group was applied with DMSO with the same concentration at the corresponding time point. In order to detect whether the compound delatinib has an effect on rice growth, a control group was set up in which only the compound delatinib (100 μM) and gelatin solution were sprayed. 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.

[0051] Result analysis: The effect of applying compound delatinib at different time points on the control of rice blast in living rice was tested. Figure 6 and Figure 7As shown, compared with the control group without application of compound delatinib, the -24h, 0h and +24h treatment groups were able to significantly inhibit rice blast disease in vivo, indicating that compound delatinib can continue to exert its effect for a long time; in addition, compound delatinib 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 the compound delatinib in the preparation of a medicament for controlling rice blast fungus (Magnaporthe oryzae).

2. The use according to claim 1, characterized in that: The concentration of the compound delatinib is 5 to 100 μM.

3. The use according to claim 2, characterized in that: The concentration of the compound delatinib is 10-100 μM.

4. The use according to claim 1, characterized in that: The rice blast fungus infects gramineous plants. When used, the drug is sprayed on the leaves of the gramineous plants.

5. Use of the compound delatinib in the preparation of a drug for inhibiting the mycelial growth or spore germination of rice blast fungus.

6. The use according to claim 5, characterized in that: The concentration of the compound delatinib is 5 to 100 μM.

7. The use according to claim 6, characterized in that: The concentration of the compound delatinib is 10-100 μM.

8. A method for preventing and controlling rice blast fungus, characterized in that: A drug for controlling rice blast fungus containing the compound delatinib is sprayed on the leaves of grass plants.

9. The method for controlling rice blast fungus according to claim 8, characterized in that: The concentration of the compound delatinib in the drug for preventing and controlling rice blast fungus is 5 to 100 μM.

10. The method for controlling rice blast fungus according to claim 8, characterized in that: The concentration of the compound delatinib is 10-100 μM.

Images