Preparation and application of 5-aminolevulinic acid solution in preventing and controlling tobacco mosaic virus disease

Abstract

The invention discloses a preparation and application of a 5-aminolevulinic acid solution in preventing and treating tobacco mosaic virus disease, including: preparing a stock solution with the 5-aminolevulinic acid as a raw drug, diluting with sterile water to obtain a 5-aminolevulinic acid agent solution, and spraying the 5-aminolevulinic acid agent solution on tobacco seedlings. The present invention uses 5-aminolevulinic acid to prevent and treat tobacco mosaic virus disease. Since 5-aminolevulinic acid itself does not act on tobacco mosaic virus, it does not cause drug resistance in viruses, etc., and is different from traditional chemical pesticides and biological pesticides. The problem of pesticide residues and excessive pesticides does not occur, which meets the greatest needs of my country's agricultural sustainable development and environmental protection.

Description

Technical Field

[0001] The invention relates to the technical field of plant protection, and in particular to the preparation and application of a 5-aminolevulinic acid solution in preventing and controlling tobacco mosaic virus disease. Background Art

[0002] Traditional tobacco mosaic virus (TMV) is the pathogen of tobacco mosaic disease and belongs to the Tobamovirus group. TMV has a wide host range and can infect more than 400 plant species from 38 families. When TMV infects plants, its genetic material will rapidly replicate after invading tobacco plant cells. Since tobacco does not have a complete defense system to resist viruses, the infected tobacco plants remain in a susceptible state. The chlorophyll of the susceptible plants is destroyed, which weakens the photosynthesis of the plants and prevents them from growing healthily, resulting in deformities, dwarfing and even death. The tobacco leaves are basically no longer of use value, causing hundreds of millions of dollars in losses every year worldwide.

[0003] At present, the main method for preventing and controlling tobacco mosaic virus disease is chemical control, but there are few effective anti-plant virus preparations in production, and the control effect is poor, with the field control effect mostly below 50%. The excessive use of chemical pesticides has led to a series of problems such as increased resistance, pesticide residues and environmental pollution, which have attracted widespread attention, and biological control has been increasingly valued.

[0004] 5-Aminolevulinic acid is a synthetic precursor of all porphyrin compounds such as chlorophyll, hemoglobin, cytochrome and vitamin B12. Because it is highly efficient and pollution-free, it is widely used in agricultural production as a plant growth regulator, stress resistance agent, pigment synthesizer, pesticide synergist, blue light inducer, herbicide and insecticide.

[0005] Therefore, 5-aminolevulinic acid, as a safe and effective substance for resisting plant viral diseases, conforms to the needs of my country's agricultural sustainable development and environmental protection. Summary of the invention

[0006] In order to solve the above technical problems, the purpose of the present invention is to provide a preparation and application of a 5-aminolevulinic acid solution in the prevention and treatment of tobacco mosaic virus disease, so as to solve the traditional chemical pesticide control and biological pesticide control.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] The preparation of 5-aminolevulinic acid solution for preventing and treating tobacco mosaic virus disease comprises the steps of: using the 5-aminolevulinic acid as a raw drug to prepare a raw solution, namely weighing the 5-aminolevulinic acid as a raw drug, then adding sterile water to dissolve the raw drug to obtain a 1-5 g / L mother solution, and then diluting the mother solution with sterile water to obtain a 5-aminolevulinic acid agent solution.

[0009] Furthermore, the mother solution is diluted with sterile water to a concentration of 30 to 70 mg / L of 5-aminolevulinic acid.

[0010] Furthermore, in the 5-aminolevulinic acid pharmaceutical solution, the concentration of 5-aminolevulinic acid is 50 mg / L.

[0011] Furthermore, the use of the 5-aminolevulinic acid solution in preventing and treating tobacco mosaic virus disease comprises spraying the obtained 5-aminolevulinic acid agent solution on tobacco seedling plants that are 4 to 8 weeks old.

[0012] Furthermore, heartleaf tobacco plants with consistent growth were selected, and a 5-aminolevulinic acid solution with a concentration of 30-70 mg / L was sprayed on the leaves, and sterile water was used as a control; the spraying amount of the 5-aminolevulinic acid solution was sprayed on the tobacco seedlings until the solution on the leaf surface began to drip; 24 hours later, the third and fourth leaves were inoculated with TMV virus particles; emery was sprinkled on the leaves, 40 μL TMV or 80 μL TMV at a concentration of 0.8 mg / ml per leaf, and the bottles were washed after TMV inoculation; cultured under light at 25°C, with 10 to 20 plants as one treatment, and repeated three times; qRT-PCR was used to detect TMV conditions on the leaves at 48h, 72h, and 96h after TMV inoculation.

[0013] Furthermore, the 5-aminolevulinic acid solution was sprayed on the leaves of tobacco seedlings to induce the expression of tobacco disease resistance-related genes PR1, NPR1 and PR5.

[0014] Furthermore, 5-aminolevulinic acid can activate the immune system in the host plant and induce systemic resistance in the plant host.

[0015] Compared with the prior art, one or more embodiments of the present invention may have the following advantages:

[0016] The application of 5-aminolevulinic acid in preventing and treating tobacco mosaic virus disease of the present invention is that by spraying the 5-aminolevulinic acid agent solution on the leaves of tobacco seedlings, 5-aminolevulinic acid can promote the expression of tobacco disease resistance related genes PR1, NPR1 and PR5, stimulate the tobacco system to acquire resistance, thereby enhancing the immune ability of tobacco, resisting the infection of tobacco mosaic virus, and preventing the occurrence of tobacco mosaic virus disease. However, 5-aminolevulinic acid itself will not act on tobacco mosaic virus, will not make viruses and the like produce drug resistance, and different from traditional chemical pesticides and biological pesticides, will not cause pesticide residues and excessive pesticides, and meets the greatest needs of my country's agricultural sustainable development and environmental protection.

[0017] In addition to the effects on TMV described above, the 5-aminolevulinic acid solution is also effective against other viral diseases such as tobacco cucumber mosaic virus (CMV) and tomato chlorosis virus (ToCV). BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a schematic diagram of the necrosis of the heartleaf tobacco leaves after being treated with 50 mg / L 5-aminolevulinic acid solution in Example 1 and the control group;

[0019] Figure 2 Schematic diagram of the results of TMV-GFP at the top of Nicotiana benthamiana leaves in Example 2 and the control group;

[0020] Figure 3 Schematic diagram of the results of detecting TMV in tobacco system leaves using qRT-PCR in Example 3 and the control group;

[0021] Figure 4 It is a schematic diagram of the expression results of the disease resistance-related gene PR1 of the plants in Example 3 and the control group after TMV infection detected by qRT-PCR;

[0022] Figure 5 It is a schematic diagram of the expression results of the disease resistance-related gene NPR1 of the plants in Example 3 and the control group after TMV infection detected by qRT-PCR;

[0023] Figure 6 It is a schematic diagram of the expression results of the disease resistance-related gene PR5 of Example 3 and the control group after TMV infection detected by qRT-PCR. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with embodiments and drawings.

[0025] This embodiment provides the preparation and application of a 5-aminolevulinic acid solution in preventing and treating tobacco mosaic virus disease.

[0026] 5-aminolevulinic acid, abbreviated as 5-ALA, also known as δ-aminolevulinic acid, δ-aminovaleric acid, is a common precursor of tetrahydropyrrole compounds such as porphyrin, (ferrous) heme and vitamin B12. It is a non-protein amino acid widely present in the cells of bacteria, fungi, animals and plants. 5-ALA is a complement factor that constitutes the respiratory system complex, photosynthesis complex, peroxidase, etc. Its molecular structure is;

[0027]

[0028] The present invention finds that 5-aminolevulinic acid, as a non-protein amino acid, can promote the expression of tobacco disease resistance-related genes PR1, NPR1 and PR5, stimulate the tobacco production system to acquire resistance, improve its immune ability, and thus resist the infection of tobacco mosaic virus. Therefore, spraying tobacco plants with 5-aminolevulinic acid solution on the leaves in advance can prevent the occurrence of tobacco mosaic virus disease. However, 5-aminolevulinic acid itself does not work on tobacco mosaic virus, and does not cause viruses and the like to develop drug resistance. It is different from traditional chemical pesticides and biological pesticides for prevention and control, and meets the greatest needs of my country's agricultural sustainable development and environmental protection.

[0029] In this embodiment, the application method of 5-aminolevulinic acid for preventing and controlling tobacco mosaic virus disease is as follows: 5-aminolevulinic acid is used as the original drug, water is added to prepare a mother solution of 1 to 5 g / L, and then diluted with water to obtain a 5-aminolevulinic acid agent solution, and the 5-aminolevulinic acid agent solution is sprayed on the leaves of tobacco seedlings.

[0030] The 5-aminolevulinic acid solution is sprayed on the leaves of tobacco seedlings to induce the expression of tobacco disease resistance-related genes PR1, NPR1 and PR5. Therefore, 5-aminolevulinic acid needs to be made into a solution first, and then the tobacco plants are treated by foliar spraying.

[0031] Preferably, in the 5-aminolevulinic acid solution, the concentration of 5-aminolevulinic acid is 30-70 mg / L. Through experimental verification, it is found that when the concentration of 5-aminolevulinic acid is 30, 50, and 70 mg / L, tobacco can produce resistance to tobacco mosaic virus disease, achieving the effect of preventing and controlling tobacco mosaic virus disease. Among them, spraying tobacco with a 50 mg / L solution on the leaves has the best prevention and control effect on tobacco mosaic virus disease and is the most cost-effective.

[0032] In this embodiment, the tobacco seedlings are 4 to 8 weeks old. The seedlings at this stage are in the early stage of tobacco growth and are more susceptible to tobacco mosaic virus infection. At this stage, the 5-aminolevulinic acid solution is sprayed on the leaves to stimulate the expression of disease-resistant genes related to the tobacco system, which can prevent tobacco mosaic virus disease.

[0033] In this embodiment, the foliar spraying amount of the 5-aminolevulinic acid solution is to spray the tobacco seedlings with the foliar spray until the foliar solution begins to drip. The mechanism of 5-aminolevulinic acid in preventing and treating tobacco mosaic virus disease is to promote the expression of tobacco disease-resistant related genes PR1, NPR1 and PR5. If too little is applied, it will not promote the expression of disease-resistant genes. If too much is applied, it will not only be costly, but also may cause damage to crops. The above-mentioned spraying amount can achieve the effect of preventing and treating tobacco mosaic virus disease without significantly increasing the cost of prevention and treatment and causing damage to crops.

[0034] The following is a further description of the effect of 5-aminolevulinic acid on the TMV resistance of different tobacco varieties. The following examples only select two tobacco varieties, Nicotiana benthamiana and Nicotiana sinensis, but it can be understood that the present method is also applicable to the prevention and treatment of tobacco mosaic virus disease in other tobacco varieties.

[0035] Preparation of 5-aminolevulinic acid solution: To prepare 5-ALA solution more accurately, first weigh 5-aminolevulinic acid as the original drug, then add water to dissolve it to prepare a 1 g / L mother solution.

[0036] The mother solution was diluted with sterile water to concentrations of 30, 50, and 70 mg / L, respectively, to prepare 5-aminolevulinic acid pharmaceutical solutions.

[0037] Example 1

[0038] Experiment on TMV resistance of Nicotiana tabacum induced by 5-aminolevulinic acid.

[0039] 4 to 8 weeks after transplanting the heartleaf tobacco, select the heartleaf tobacco plants with the same growth, spray the prepared 5-aminolevulinic acid solution of different concentrations on the leaves, use sterile water as a control, and other conditions are the same. Spray the tobacco seedlings with 5-aminolevulinic acid solution on the leaves until the solution on the leaves begins to drip; 24 hours later, the third and fourth leaves are inoculated with TMV virus particles. Sprinkle emery on the leaves, 80μL per leaf, TMV concentration is (0.8mg / ml), wash the bottle after TMV inoculation. Cultivate under light at 25℃, treat 10 plants per treatment, and repeat three times. Count the number of dead spots after 72 hours.

[0040] Table 1 shows the average number of dead spots of heartleaf tobacco treated with 5-aminolevulinic acid solutions of different concentrations and then inoculated with TMV. Figure 1 This is a schematic diagram of the necrotic conditions on the leaves of the heartleaf tobacco after treatment with 50 mg / L 5-aminolevulinic acid solution and the control group.

[0041] Table 1 Statistics of average number of dead spots after TMV grafting

[0042]

[0043] See Table 1 and Figure 1 The data of the 5-aminolevulinic acid-induced TMV resistance test on heartleaf tobacco showed that 72 hours after the plants were infected with the virus, the number of dead spots on the leaf surface of the heartleaf tobacco in the 5-aminolevulinic acid treatment group was significantly lower than that in the control group, indicating that 5-aminolevulinic acid at a concentration of 30-70 mg / L is effective in preventing and controlling tobacco mosaic virus disease, and 50 mg / L of 5-aminolevulinic acid has a relatively higher effect on preventing and controlling tobacco mosaic virus disease.

[0044] Example 2

[0045] Accumulation test of 5-aminolevulinic acid in TMV of Nicotiana benthamiana.

[0046] 4-5 weeks after transplanting N. benthamiana, select N. benthamiana plants with uniform growth and spray 50 mg / L 5-aminolevulinic acid solution on the leaves. Sterile water is used as the control, and other conditions are the same. Spray the tobacco seedlings with 5-aminolevulinic acid solution on the leaves until the solution on the leaves begins to drip; 24 hours later, rub the third and fourth leaves with infectious cloned TMV virus particles marked with GFP. Sprinkle emery on the leaves, 40 μL TMV per leaf, TMV concentration is (0.8 mg / ml), wash the bottle after TMV inoculation. Cultivate under light at 25℃, treat 10 plants per time, and repeat three times. Observe the GFP situation at the top of the leaves after 7 days.

[0047] Figure 2 Schematic diagram of the results of TMV-GFP at the leaf tips of Nicotiana benthamiana leaves in implementation 2 and the control group.

[0048] See also Figure 2 , 7 days later, the GFP results under ultraviolet light showed that compared with the control group, the TMV accumulation of the apical system leaves treated with 50 mg / L 5-aminolevulinic acid was lower.

[0049] Example 3

[0050] Experiment on the TMV resistance of Nicotiana benthamiana induced by 5-aminolevulinic acid

[0051] 4-5 weeks after transplanting N. benthamiana, select N. benthamiana plants with uniform growth and spray 70 mg / L 5-aminolevulinic acid solution on the leaves. Sterile water is used as the control, and other conditions are the same. Spray the tobacco seedlings with 5-aminolevulinic acid solution on the leaves until the solution on the leaves begins to drip; 24 hours later, inoculate TMV on the third and fourth leaves. Sprinkle emery on the leaves, 40 μL TMV per leaf, TMV concentration is (0.8 mg / ml), and wash the bottle after TMV inoculation. Cultivate under light at 25℃, treat 20 plants per plant, and repeat three times. qRT-PCR detects TMV on leaves at 48h, 72h, and 96h after TMV inoculation; detect the expression of disease-resistant related genes PR1, NPR1, and PR5 at 0h, 12h, 24h, and 48h. Figure 3 This is a schematic diagram of the results of using qRT-PCR to detect TMV in leaves of Example 3 and the control group. Figure 4 is the expression level of the disease resistance-related gene PR1 of the plants in Example 3 and the control group after TMV infection detected by qRT-PCR, Figure 5 is the expression level of the disease resistance-related gene NPR1 in Example 3 and the control group after TMV infection detected by qRT-PCR, Figure 6It is the expression level of the disease resistance-related gene PR5 of the plants in Example 3 and the control group after TMV infection detected by qRT-PCR.

[0052] Depend on Figure 4 It can be seen that the qRT-PCR detection of TMV content in leaves at 48h, 72h, and 96h showed that the relative expression of TMV in plants treated with 5-aminolevulinic acid was significantly lower than that in the control group at 48h and 72h. It can be concluded that 5-aminolevulinic acid inhibited the proliferation of tobacco mosaic virus in the early stage of TMV infection, and at 96h, it was not much different from the control group, indicating that the onset time of 5-aminolevulinic acid was about 72h; combined with Figure 4 , Figure 5 and Figure 6 It can be seen from qRT-PCR detection that the expression levels of disease-resistant related genes PR1, NPR1 and PR5 of plants treated with 5-aminolevulinic acid were significantly higher than those of the control group 24 hours after TMV infection. It can be concluded that 5-aminolevulinic acid induces TMV resistance in Nicotiana benthamiana by inducing the expression of disease-resistant related genes of tobacco itself, thereby inhibiting the proliferation of tobacco mosaic virus in the early stage of TMV infection, thereby delaying the onset of the disease and reducing the incidence rate; the onset time of 5-aminolevulinic acid is about 72 hours, so in field operations, it can be applied every 72 hours (every three days) during the tobacco seedling stage until the entire seedling stage is passed.

[0053] The above is only a preferred embodiment of the present invention. The laboratory also found that 5-ALA is effective against other viral diseases such as tobacco cucumber mosaic virus (CMV) and tomato chlorosis virus (ToCV). For those skilled in the art, the present invention can have various changes and variations. The described content is only an implementation method adopted to facilitate the understanding of the present invention, and is not intended to limit the present invention. Any technician in the technical field to which the present invention belongs can make any modifications and changes in the form and details of the implementation without departing from the spirit and scope disclosed by the present invention, but the scope of patent protection of the present invention shall still be based on the scope defined by the attached claims.

Claims

The use of 1.5-aminolevulinic acid solution in preventing and controlling tobacco mosaic virus is characterized in that: Spray 50 mg / L 5-aminolevulinic acid solution on the leaves of heartleaf tobacco; The 5-aminolevulinic acid can activate the immune system in the host plant and induce the plant host to produce systemic resistance; The preparation of the 5-aminolevulinic acid solution comprises: taking 5-aminolevulinic acid as a raw material, adding sterile water to dissolve it to obtain a 1-5 g / L mother solution, and diluting the mother solution with sterile water to obtain a 50 mg / L 5-aminolevulinic acid solution.

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