Application of 1-naphthalenemethanol in the preparation of drugs for preventing and controlling rice blast fungus

Abstract

The invention discloses the application of 1-naphthalene methanol in preparing a medicine for preventing and treating rice blast fungi, and belongs to the field of plant filamentous fungal disease prevention and treatment. The application of 1-naphthalene methanol provided by the invention in inhibiting rice blast fungi and preventing and treating rice blast fungi can effectively inhibit the pathogenicity of rice blast fungi spores within a concentration range of 200 to 1000 μM, and in particular, 1000 μM of 1-naphthalene methanol can completely prevent and treat rice blast fungi, laying a foundation for the development of new green pesticides.

Description

Technical Field

[0001] The invention belongs to the field of plant filamentous fungal disease prevention and control, and relates to the application of 1-naphthalene methanol in the preparation of a medicine for preventing and controlling rice blast fungi. Background Art

[0002] At present, rice is one of the main food crops in the world with a huge planting area and a high annual yield. Plant diseases are one of the factors threatening the production of food crops, which can lead to a significant reduction in the yield of gramineous crops including rice. The types of rice diseases include rice blast, bacterial blight, sheath blight, etc. Among them, the rice blast fungus has the strongest destructive power, making rice blast the first of the three major diseases. The pathogenic fungus of rice blast, Magnaporthe oryzae (syn. Pyricularia oryzae), belongs to the genus Pyricularia of the Ascomycetes. It often spends the winter on rice straw through conidia and mycelium, and the conidia will invade the host again in the next year with the spread of air flow and rain, forming an infection cycle. 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%. Rice blast fungus also has the characteristics of wide regional distribution, strong environmental adaptability, and rapid mutation. Therefore, the comprehensive prevention and control of rice blast has always been a hot spot and difficulty in plant disease prevention and control.

[0003] Traditional prevention and control methods include the selection of disease-resistant and high-yield varieties, elimination of fungal sources, chemical control, strengthening field management, strengthening water and fertilizer management, and handling diseased rice and straw. Chemical control is a very important disease prevention measure. Because it still has significant effects when the plant is seriously ill, it is highly valued by agricultural scientists and favored by agricultural producers. However, in recent decades, with the frequent use of chemical pesticides, the soil properties of field plots have deteriorated sharply, and the ecological environment of farmland has also deteriorated. At the same time, due to the long-term selection of pesticide pressure, a large number of disease-resistant physiological subspecies have emerged in pathogenic fungi, which has increased the difficulty of rice blast prevention and control. In order to solve the above problems, it is urgent to accelerate the research and development of eco-friendly green and environmentally friendly pesticides by deeply exploring the potential pathogenic targets of rice blast, so as to achieve the pesticide control effect that can effectively resist rice blast infection, protect soil ecology, and ensure the yield and quality of rice.

[0004] 1-Naphthalenemethanol, also known as 1-naphthalenemethanol in Chinese, is a natural compound with antibacterial activity that can be extracted from the root of the Senegalese sugar apple (Annonasenegalensis). Its structural formula is as follows:

[0005]

[0006] However, the research and application of the compound 1-Naphthalenemethanol in preventing and controlling plant diseases has not been reported. Summary of the invention

[0007] The present invention aims to provide an application of 1-naphthalenemethanol in the preparation of a drug for preventing and controlling rice blast fungus, wherein the EC of 1-naphthalenemethanol, i.e., the compound 1-Naphthalenemethanol, on the mycelial growth of rice blast fungus is s0 It is 671.4289 μM, and it has the advantages of significant effect, long action time, safety and simple application method in the prevention and control of rice blast.

[0008] To achieve the above object, the technical solution of the present invention is as follows:

[0009] The invention provides application of 1-naphthalenemethanol in preparing a medicine for preventing and controlling rice blast fungus (Magnaporthe oryzae).

[0010] The invention also provides the use of 1-naphthalenemethanol in preparing a medicine for inhibiting the mycelial growth or spore germination of rice blast fungus.

[0011] Preferably, the concentration of the 1-naphthalenemethanol is 200-1000 μM.

[0012] The final concentration of 1-naphthalenemethanol in the present invention is 200-1000 μM.

[0013] In the present invention, 1-naphthalenemethanol can effectively inhibit the pathogenicity of rice blast fungus spores within a concentration range of 200 to 1000 μM.

[0014] More preferably, the concentration of 1-naphthalenemethanol is 600-1000 μM.

[0015] The 1-naphthalenemethanol at a concentration of 600-1000 μM has a significant inhibitory effect on in vitro barley blast.

[0016] Most preferably, the concentration of 1-naphthylmethanol is 1000 μM.

[0017] The 1-naphthalene carbinol at a concentration of 1000 μM can completely inhibit barley blast in vitro.

[0018] The invention also provides a medicine for preventing and controlling rice blast fungus, comprising 1-naphthalenemethanol.

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

[0020] Preferably, the concentration of 1-naphthalenemethanol in the drug for preventing and controlling rice blast fungus is 200-1000 μM.

[0021] More preferably, the concentration of 1-naphthalenemethanol is 600-1000 μM.

[0022] The 1-naphthalenemethanol at a concentration of 600-1000 μM has a significant inhibitory effect on in vitro barley blast.

[0023] Most preferably, the concentration of 1-naphthylmethanol is 1000 μM.

[0024] The 1-naphthalene carbinol at a concentration of 1000 μM can completely inhibit barley blast in vitro.

[0025] Beneficial effects of the present invention:

[0026] The application of 1-naphthalenemethanol provided by the invention in inhibiting rice blast fungi and preventing and controlling rice blast disease can effectively inhibit the pathogenicity of rice blast fungi spores within a concentration range of 200 to 1000 μM, and in particular, 1000 μM 1-naphthalenemethanol can completely prevent and control rice blast disease, thus laying a foundation for the development of new green pesticides. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The figure shows the inhibition of different concentrations of 1-naphthalenemethanol on the plate growth of rice blast fungus.

[0028] Figure 2 The figure is a comparison of the growth diameters of rice blast fungus colonies on plates under treatment with different concentrations of 1-naphthalenemethanol; in the figure, the inhibition rates of different concentrations of 1-naphthalenemethanol with significant differences are represented by different letters a, b, c, d, e or f; p<0.05; the data in the figure were obtained by one-way ANOVA.

[0029] Figure 3 The figure is a comparison of the mycelial inhibition rate of the rice blast fungus colony growth on the plate under the treatment of different concentrations of 1-naphthalenemethanol; in the figure, the inhibition rates of different concentrations of 1-naphthalenemethanol 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 ANOVA.

[0030] Figure 4 This is the disease occurrence of detached barley leaves when different concentrations of 1-naphthalenemethanol are added to the spore solution of rice blast fungus.

[0031] Figure 5 This is the disease occurrence of detached rice leaves when different concentrations of 1-naphthalenemethanol are added to the spore liquid of rice blast fungus.

[0032] Figure 6This is the disease occurrence of living rice leaves after spraying 1000 μM 1-naphthylmethanol at different stages of inoculation with spores of rice blast fungus.

[0033] Figure 7 This is a comparison of the relative lesion areas of living rice leaves after spraying 1000 μM 1-naphthalenemethanol 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: 10 g, peptone-140: 2 g, casamino 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, p-aminobenzoic acid: 0.1 mg, Na2MoO4·5 H2O: 1.5mg, CuSO4·5H2O: 1.6mg, CoCl2·6H2O: 1.7mg, MnCl2·4H2O: 5mg, FeSO4·7H2O: 5mg, H3BO3: 11mg, ZnSO4·7H2O: 22mg, Na4EDTA·2H2O: 50mg, agar powder: 15g, make up to 1L with deionized water, adjust the pH to 6.5 with NaOH, and sterilize at 121℃ and high temperature and high pressure for 15min.

[0036] 1-Naphthalenemethanol is a compound 1-Naphthalenemethanol, CAS No. 4780-79-4, purchased from MedChemexpress Biotechnology Company in the United States, catalog No. HY-W017241. The molecular structure of 1-naphthalenemethanol is as follows:

[0037]

[0038] Preparation and storage of 1-naphthalenemethanol stock solution: Dissolve 500 mg 1-naphthalenemethanol in 5267.594 μL DMSO to a final concentration of 600 mM. Filter through a 0.22 μm aqueous filter membrane for sterilization, store at -20°C for short-term storage, and store at -80°C for long-term storage.

[0039] Example 1: Toxicity determination of 1-naphthalene methanol against rice blast fungus and results

[0040] Test method: hyphae growth rate method. The rice blast fungus was cultured on a CM plate at 25℃ for 16h light / 8h dark for 7-9 days; 1-naphthalene methanol was added in gradient doses (0, 200, 400, 600, 800, 1000μM) to the solid CM medium cooled to 40-50℃ to prepare a drug-containing plate; in the sterile environment of the clean bench, a 5mm diameter round fungus cake with relatively consistent growth conditions was punched at the edge of the hyphae of the rice blast fungus cultured for 7-9 days with a hole puncher, and the fungus cake was picked up with a sterilized toothpick to the center of the drug-containing plate, so that the aerial hyphae surface was attached to the culture medium; each concentration treatment was repeated 3 times; CM culture medium containing the same concentration of DMSO was used as a control; the culture dish was inverted in an incubator at 25℃ for 8h darkness / 16h light to culture until the control group was almost full, and the growth of the mycelium of the rice blast fungus in the experimental group was observed. The mycelial diameter was measured by the cross method, and the mycelial growth rate method was used to calculate the mycelial growth inhibition rate of rice blast fungus under different gradient concentrations of 1-naphthalenemethanol: mycelial growth inhibition rate (%) = (average colony diameter of the control group - average colony diameter of the treatment group) / (average colony diameter of the control group - 0.5) × 100. The DPS statistical software was used to analyze and establish the toxicity regression equation to calculate the EC value of 1-naphthalenemethanol against rice blast fungus. 50 The correlation coefficient r was calculated and the toxicity level of 1-naphthalenemethanol to rice blast fungus was evaluated.

[0041] Table 1

[0042]

[0043] Result analysis: The toxicity of 1-naphthalenemethanol at different concentration gradients to rice blast fungus was tested. Figure 1 As shown in the figure, the five concentrations of 1-naphthalene methanol in the experiment all had a certain inhibitory effect on the mycelial growth of rice blast fungus. The higher the concentration, the smaller the average diameter of the colony ( Figure 2 ), the higher the mycelial growth inhibition rate ( Figure 3 ). Within this concentration gradient range, the inhibition rate of 1-naphthalenemethanol on rice blast fungus ranges from 10% to 80%, indicating that the accuracy and credibility of the experimental data are high; the EC of 1-naphthalenemethanol on the mycelial growth of rice blast fungus 50The value is 671.4289 μM. (Table 1). When the concentration of 1-naphthalene methanol is 1000 μM, it has the most significant antibacterial effect on rice blast fungus, and the relative inhibition rate is 76.76% (Table 1).

[0044] Example 2: Pathogenicity determination of 1-naphthalene methanol to rice blast fungus and results

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

[0046] (1) In vitro barley inoculation:

[0047] The rice blast fungus was cultured on CM medium at 25°C with 16 h of light / 8 h of darkness for 7-9 days, then double distilled water was added to the dish, and the surface of the colony was gently scraped with a sterilized coating stick to obtain spore liquid; gradient doses of 1-naphthalene methanol (0, 200, 400, 600, 800, 1000 μM) were mixed with the spore liquid to prepare a drug-containing spore liquid (the final spore concentration was 1×10 5 CFU / mL), the difference in 1-naphthalenecarbinol dosage in different treatment groups was made up with DMSO to make the final DMSO concentration consistent; one-week-old barley (variety: Hordeum vulgareZJ-8) with good growth and flat leaves were cut; 25μL of drug-containing spores were dropped on the barley leaves; 3 drops were placed on each leaf, and each treatment was repeated 3 times; the spores in the control group contained the same concentration of DMSO; the leaves were cultured at 25℃, moisturizing, and 16h / 8h light and dark alternation for about 4 days, and the disease condition of the leaves was observed.

[0048] Result analysis: The inhibitory effect of gradient concentrations of 1-naphthalenemethanol on blast disease in detached barley leaves was tested. Figure 4 As shown, 600-1000 μM 1-naphthalenemethanol treatment has a certain inhibitory effect on the infection of rice blast fungus on detached barley leaves; and within this range, the higher the tested concentration, the stronger the inhibitory effect; among them, the 1-naphthalenemethanol with a concentration of 800-1000 μM has a significant inhibitory effect on the rice blast fungus spores on the detached barley leaves; the 1-naphthalenemethanol with a concentration of 1000 μM can completely inhibit the occurrence of rice blast in detached barley leaves.

[0049] (2) In vitro rice inoculation: Cut the healthy second leaf of three-leaf rice (cultivar: Oryza sativa CO39, grown by China National Rice Research Institute) (about 15 days of culture). The specific operation method of the drug-containing spore drop method is the same as that of in vitro barley inoculation.

[0050] Result analysis: The inhibitory effect of gradient concentrations of 1-naphthalenemethanol on rice blast in vitro was tested. Figure 5As shown, among the five concentrations of 1-naphthalenemethanol used in the experiment, 200-1000 μM 1-naphthalenemethanol treatments all had a certain inhibitory effect on detached rice blast; and within this range, the higher the tested concentration, the stronger the inhibitory effect; among them, the 1-naphthalenemethanol with a concentration of 400-1000 μM had a significant inhibitory effect on detached rice leaves blast; the 1-naphthalenemethanol with a concentration of 600 μM could completely inhibit the occurrence of detached rice leaves blast. Figure 4 , Figure 5 As shown, at the same concentration of the compound, 1-naphthalenemethanol had a stronger control effect on rice blast in detached rice leaves than in detached barley leaves.

[0051] 2. Experimental method: Living rice spray method.

[0052] First, rice varieties of Oryza sativa CO39 were sown (30 seeds per pot) and cultured outdoors under sufficient light until they were two weeks old. Rice blast fungus was cultured on CM medium for 7-9 days to obtain spore solution. 1-naphthalene methanol, gelatin solution and spore solution were mixed to prepare spore solution containing drug and gelatin (the final spore concentration was 1×10 5 CFU / mL, gelatin final concentration of 0.4%), so that the final concentration of 1-naphthalene methanol is 1000μM, 1-naphthalene methanol dose difference is supplemented with DMSO to make the final concentration of DMSO consistent; 2mL of mixed gelatin spore solution containing drugs was evenly sprayed on rice leaves with a small sprayer, and 3 pots of rice were inoculated for each treatment; at the same time, in order to confirm that 1-naphthalene methanol has no obvious toxicity to rice, we also set up a control group sprayed with DMSO and gelatin solution containing the same concentration, and sprayed with 1-naphthalene methanol (1000μM) and gelatin solution containing the same concentration; the living rice after inoculation with spore solution was first placed in a dark incubator at 22℃ for 2d, and then placed in a light-dark alternating incubator at 25℃ and 16h / 8h for 3-4d; the disease situation of the living rice leaves 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.

[0053] The control effect of 1-naphthalenemethanol on live rice blast fungus may vary at different application periods. Therefore, based on the in vitro rice leaf inoculation implementation plan, in order to explore the optimal application period of 1-naphthalenemethanol, we conducted a live rice spray method. We selected the optimal concentration of 1-naphthalenemethanol to inhibit the occurrence of rice blast disease as 1000μM, and applied 1000μM 1-naphthalenemethanol at different time points after inoculation of spores of rice blast fungus. Three groups of experiments were set up: (1) -24h: 1-naphthalenemethanol was applied 24 hours before inoculation of spores of rice blast fungus; (2) 0h: 1-naphthalenemethanol was applied at the same time as inoculation of spores of rice blast fungus, that is, 1-naphthalenemethanol was mixed with spores of rice blast fungus; (3) +24h: 1-naphthalenemethanol was applied 24 hours after inoculation of spores of rice blast fungus. Three pots of rice were used for each treatment as replicates, and DMSO containing the same concentration was applied at the time point when 1-naphthalenemethanol was not applied as a control; the control group was applied with DMSO containing the same concentration at the corresponding time points.

[0054] Results analysis: The effect of applying 1-naphthalene methanol on rice blast in vivo at different time points was tested. At the same time, compared with the control group without compound application, the -24h, 0h and +24h treatment groups were able to significantly inhibit the growth of rice blast in vivo ( Figure 6 , Figure 7 ), which has practical significance for preventing rice blast. At the same time, it was observed that the rice growth trend in the control group with only the same concentration of DMSO or the same concentration of 1-naphthalene methanol was good, with no obvious adverse effects, indicating that the compound may be safe for rice ( Figure 6 ).

Claims

1. Use of 1-naphthalenemethanol in the preparation of a drug for controlling rice blast fungus (Magnaporthe oryzae). 2.1-The application of naphthalenemethanol in the preparation of drugs for inhibiting the mycelial growth or spore germination of rice blast fungus.

3. The use according to claim 1 or 2, characterized in that: The concentration of the 1-naphthalenemethanol is 200-1000 μM.

4. The use according to claim 3, characterized in that The concentration of the 1-naphthalenemethanol is 600-1000 μM.

5. A method for preventing and controlling rice blast fungus, characterized in that: Spray 1-naphthalene carbinol on the leaves of the plants.

6. The method for controlling rice blast fungus according to claim 5, characterized in that: The concentration of the 1-naphthalenemethanol is 200-1000 μM.

7. The method for controlling rice blast fungus according to claim 6, characterized in that: The concentration of the 1-naphthalenemethanol is 600-1000 μM.

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