Application of trigonelline in improving resistance of rice to rice blast

Abstract

The invention discloses application of trigonelline in improving resistance of rice to rice blast, and relates to the technical field of biology. By identifying metabolites influencing the resistance of rice to the rice blast and detecting the influence of the metabolites on the disease resistance of the rice in the field in a simultaneous, prevention and treatment mode, the trigonelline is found to be capable of remarkably enhancing the resistance of the rice to the rice blast. The qRT-PCR detection finds that the expression quantity of the PR gene in the rice is obviously increased after the trigonelline is externally applied, which indicates that the trigonelline is a potential metabolite for enhancing the immunity of the rice and can be used for preventing and treating the rice blast.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to the application of trigonelline in improving the resistance of rice to rice blast. Background Art

[0002] As an important food crop, rice feeds more than half of the world's population. Rice blast, caused by the fungus Magnaporthe oryzae, is one of the most serious diseases of rice. Rice blast occurs in rice production areas all over the world, seriously affecting the yield and quality of rice. Therefore, how to prevent and control rice blast has always been the research focus and hotspot in related fields at home and abroad. At present, breeding resistant varieties by using resistance (R) genes is the most economical and effective method to reduce the damage of rice blast, but the breeding cycle of resistant varieties is long and the cost is high. In addition, chemical pesticides are used to control rice blast. Currently, the commonly used pesticides for controlling rice blast mainly include tricyclazole, amisulbrom, and wenteeling. Chemical pesticides are effective quickly, but they cause large environmental pollution and are likely to make pathogenic bacteria develop drug resistance. Therefore, it is urgent to find new methods to prevent and control rice blast.

[0003] At present, although mining R genes has great application potential in breeding, the rice blast resistance mediated by R genes usually has race specificity and is easily lost due to the change of dominant races, resulting in large-scale diseases. In addition, the breeding period of resistant varieties is long, and the consumption of funds and manpower is large. Using chemical pesticides to control rice blast, although it is effective quickly, it causes relatively large environmental pollution, kills natural enemies and affects the ecological environment, and long-term application will lead to the development of drug resistance in pathogenic bacteria.

[0004] Therefore, it is particularly important to find new methods to prevent and control the damage of rice blast. When plant metabolites improve the defense ability of plants, they usually have no selectivity for pathogenic bacteria. Compared with chemical control agents for rice blast, biopesticides made from plant-derived metabolites are not likely to make rice blast bacteria develop drug resistance and have no safety hazards of pesticide residues. Therefore, mining metabolites related to rice disease resistance has very important value for preventing and controlling rice blast. Summary of the Invention

[0005] The purpose of the present invention is to provide the application of trigonelline in improving the resistance of rice to rice blast to solve the problems existing in the above-mentioned prior art. The present invention has found through research that trigonelline can significantly enhance the resistance of rice to rice blast and can be applied to the prevention and control of rice blast.

[0006] To achieve the above purpose, the present invention provides the following solution:

[0007] The present invention provides the application of trigonelline in improving the resistance of rice to rice blast.

[0008] Furthermore, trigonelline enhances the resistance of rice to rice blast by increasing the expression levels of resistance genes OsPBZ1, OsPR10, and OsPR1a.

[0009] The present invention also provides the application of trigonelline in the preparation of pesticides for controlling rice blast.

[0010] The present invention also provides a pesticide for controlling rice blast, the active ingredient of which includes trigonelline.

[0011] Furthermore, the pesticide further includes agriculturally acceptable excipients.

[0012] Furthermore, the excipients include emulsifiers, dispersants, and / or solubilizers.

[0013] The present invention also provides the application of the above pesticide in the control of rice blast.

[0014] The present invention also provides a method for controlling rice blast, which includes the step of applying trigonelline to rice plants to control rice blast.

[0015] Furthermore, the application method is foliar spraying.

[0016] Furthermore, the trigonelline is foliar sprayed at a concentration of 100 - 300 μM.

[0017] The present invention discloses the following technical effects:

[0018] By identifying metabolites that affect the resistance of rice to rice blast and using simultaneous, preventive, and therapeutic methods to detect the effects of metabolites on the disease resistance of rice in the field, the present invention finds that trigonelline can significantly enhance the resistance of rice to rice blast. Through qRT-PCR detection, it is found that the expression levels of PR genes in rice increase significantly after exogenous application of trigonelline, indicating that trigonelline is a potential metabolite for enhancing rice immunity and can be used for the control of rice blast. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the following described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0020] Figure 1 Representative pictures of diseased leaves under different treatment methods; among them, A is the simultaneous treatment method; B is the preventive treatment method; C is the therapeutic treatment method;

[0021] Figure 2Statistical chart of the detection results of the relative expression level of rice OsPR1a gene;

[0022] Figure 3 Statistical chart of the detection results of the relative expression level of rice OsPBZ1 gene;

[0023] Figure 4 Statistical chart of the detection results of the relative expression level of rice OsPR10 gene. Detailed implementation manners

[0024] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be regarded as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation manners of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0027] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are only exemplary.

[0028] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0029] Term description:

[0030] Trigonelline is an alkaloid extracted from the dried seeds of fenugreek, and its chemical structural formula is C 7 H 7 NO2 , MW = 137.14, and its structural formula is:

[0031]

[0032] In the medical field, it has been found that trigonelline is an alkaloid with potential anti-diabetic activity. It can reduce diabetic auditory neuropathy and platelet aggregation by promoting the regeneration of β-cells, regulating insulin secretion, altering the activity of enzymes related to glucose metabolism, affecting the level of reactive oxygen species, promoting axonal extension, and adjusting neuronal excitability. It is also an inhibitor of E2-related factor 2 (Nuclear factor E2-related factor 2, Nrf2), which can block NRF2-dependent proteasome activity, thereby enhancing pancreatic cancer cell apoptosis. In addition, trigonelline also has activities such as anti-herpes simplex virus; it can induce ferroptosis; protect cardiomyocytes from hydrogen peroxide-induced apoptosis; and can also serve as a precursor of NAD + to enhance muscle function during the aging process.

[0033] Example 1

[0034] 1. Experimental materials

[0035] Trigonelline was purchased from Shanghai Yuanye Bio-Technology Co., Ltd., with the product number Cat#B20521. It was dissolved in sterilized water.

[0036] Rice material: Lijiangxintuanheigu (LTH), which is highly susceptible to most physiological races of Magnaporthe oryzae.

[0037] Pathogen: Magnaporthe oryzae physiological race Guy11. Spores were added to sterile water to prepare a spore suspension for standby.

[0038] 2. Experimental methods

[0039] 2.1 Effects of trigonelline on the incidence of rice blast under different treatment methods (simultaneous, preventive, and therapeutic)

[0040] (1) Simultaneous treatment

[0041] Rice plants at the four-leaf stage with consistent growth were divided into 4 groups, and each group was treated as shown in Table 1. After 7 days, based on the number of lesions on the leaves after spraying the pathogen, the incidence of the water treatment group was set as 100%, and the incidence of each treatment group was statistically analyzed.

[0042] Incidence = (number of lesions in the experimental group / number of lesions in the water treatment group) × 100%.

[0043] Table 1 Experimental treatments for simultaneous treatment

[0044]

[0045] (2) Preventive treatment

[0046] Select rice plants with consistent growth at the four-leaf stage, divide them into 4 groups, and each group is treated as shown in Table 2. After 7 days, based on the number of lesions on the leaves after spraying the pathogen, the incidence rate of the water treatment group is set at 100%, and the disease incidence of each treatment group is counted.

[0047] Table 2 Experimental treatments of preventive treatment methods

[0048]

[0049] (3) Therapeutic treatment

[0050] Select rice plants with consistent growth at the four-leaf stage, divide them into 4 groups, and each group is treated as shown in Table 3. After 7 days, based on the number of lesions on the leaves after spraying the pathogen, the incidence rate of the water treatment group is set at 100%, and the disease incidence of each treatment group is counted.

[0051] Table 3 Experimental treatments of therapeutic treatment methods

[0052]

[0053]

[0054] 2. qRT-PCR analysis of PR genes in rice after exogenous application of trigonelline.

[0055] 2.1 Fluorescent quantitative PCR analysis of PR genes

[0056] Select rice plants with consistent growth at the four-leaf stage, divide them into two groups: one group is sprayed with 300 μM trigonelline on the leaf surface, and the spraying amount is 0.0125 mL / cm 2 ; the other group is sprayed with water as a control on the leaf surface, and the spraying amount is 0.0125 mL / cm 2 . After the two groups are treated with leaf surface spraying, take the leaves at 2 hours, 6 hours, and 9 hours respectively, extract RNA, reverse transcribe to obtain cDNA, and use a fluorescent quantitative PCR instrument to detect the relative expression levels of OsPBZ1, OsPR10, and OsPR1a. Using the rice Ubiquitin gene as an internal reference, calculate the relative expression levels of each gene. The specific PCR primer sequences, reaction systems, and reaction procedures are as follows:

[0057] 2.1.1 PCR primer sequences

[0058] The PCR primer sequences are shown in Table 4.

[0059] Table 4 PCR Primer Sequences

[0060]

[0061] 2.1.2 Reaction System

[0062] SYBR Green dye was used to perform real-time fluorescence quantitative reverse transcription polymerase chain reaction (qRT-PCR). The PCR reaction system is shown in Table 5.

[0063] Table 5 PCR Reaction System

[0064]

[0065] 2.1.3 Reaction Program

[0066] After the system was configured, it was necessary to shake it to ensure thorough mixing, followed by centrifugation. Then, subsequent operations were carried out using the CFX96 Real-Time System (Bio-Rad). The reaction program was as follows: 95°C / 30 s, 95°C / 10 s, 58°C / 20 s, for a total of 39 cycles, and fluorescence was collected once per cycle. Starting from 65°C, the temperature was increased by 0.5°C per second until 95°C, and fluorescence was collected each time the temperature was increased to obtain the melting curve. Finally, the CFX Manager TM software was used for calculation and processing by the 2 -ΔΔCt method.

[0067] 3. Experimental Results

[0068] (1) Incidence Statistics Results

[0069] Under different treatment methods, the incidence statistics results of each group are shown in Table 6. Among them, representative pictures of diseased leaves under the simultaneous treatment, preventive treatment, and therapeutic treatment methods are shown in Figure 1 . The results showed that foliar spraying of trigonelline could improve the resistance of rice to rice blast, and with the increase of trigonelline concentration, the incidence of rice blast decreased significantly.

[0070] Table 6 Incidence Statistics Results under Different Treatment Methods

[0071] Treatment method Water 100 μM 200 μM 300 μM Concurrent treatment 100％±3.90％ 94.50％±5.9％ 86.83％±3.4％ 81.10％±1.0％ Preventive treatment 100％±4.8％ 95.17％±0.5％ 83.71％±1.6％ 77.37％±3.6％ Therapeutic treatment 100％±3.6％ 100％±4.8％ 93.35％±4.4％ 87.09％±4.4％

[0072] (2) Fluorescence Quantitative PCR Analysis Results of PR Genes

[0073] After spraying on the leaves of two groups for 2, 6, and 9 h, the results of detecting the relative expression levels of the resistance genes OsPR1a, OsPBZ1, and OsPR10 are shown in Figures 2 - 4 . According to Figures 2 - 4It can be seen that after spraying trigonelline, the expression levels of rice OsPR1a, OsPBZ1, and OsPR10 genes increased significantly, indicating that trigonelline can enhance the resistance of rice to rice blast and can be applied to the prevention and control of rice blast.

[0074] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of trigonelline in improving rice resistance to rice blast.

2. The application according to claim 1, characterized in that: The trigonelline improves the resistance of rice to rice blast by increasing the expression levels of the resistance genes OsPBZ1, OsPR10 and OsPR1a.

3. Application of trigonelline in the preparation of pesticides for preventing and controlling rice blast.

4. A pesticide for preventing and controlling rice blast, characterized in that: Active ingredients include trigonelline.

5. The pesticide according to claim 4, characterized in that The pesticide also includes pesticide-acceptable adjuvants.

6. The pesticide according to claim 5, characterized in that The auxiliary materials include emulsifiers, dispersants and / or cosolvents.

7. Use of the pesticide according to any one of claims 4 to 6 in preventing and controlling rice blast.

8. A method for preventing and controlling rice blast, characterized in that: The invention comprises the steps of applying trigonelline to rice plants to prevent and control rice blast.

9. The method according to claim 8, characterized in that The application method is foliar spraying.

10. The method according to claim 9, characterized in that The trigonelline is sprayed on the leaves at a concentration of 100-300 μM.

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