CN110876380B - Application of p-coumaroyl homoserine lactone in prevention and treatment of tobacco mosaic virus - Google Patents
Application of p-coumaroyl homoserine lactone in prevention and treatment of tobacco mosaic virus Download PDFInfo
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- CN110876380B CN110876380B CN201911041197.2A CN201911041197A CN110876380B CN 110876380 B CN110876380 B CN 110876380B CN 201911041197 A CN201911041197 A CN 201911041197A CN 110876380 B CN110876380 B CN 110876380B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/06—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom five-membered rings
- A01N43/08—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom five-membered rings with oxygen as the ring hetero atom
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
Abstract
The p-coumaroyl homoserine lactone can promote the expression of tobacco disease-resistant related genes PR1, PR10 and WRKY8 and excite a tobacco system to obtain resistance, so that the immunity of tobacco is enhanced, the infection of tobacco mosaic virus is resisted, and the occurrence of the tobacco mosaic virus is prevented. The p-coumaroyl homoserine lactone is adopted to prevent and treat tobacco mosaic virus diseases, and the p-coumaroyl homoserine lactone does not act on tobacco mosaic virus, so that the tobacco mosaic virus does not generate drug resistance, is different from the traditional chemical pesticide and biological pesticide prevention and treatment, does not generate the problems of pesticide residue and pesticide standard exceeding, and meets the maximum requirements of agricultural sustainable development and environmental protection in China.
Description
Technical Field
The invention relates to the technical field of plant protection, in particular to application of p-coumaroyl homoserine lactone in prevention and treatment of tobacco mosaic virus diseases.
Background
China is a big tobacco planting country, and in recent years, with the expansion of tobacco planting area, tobacco mosaic virus diseases are expanded year by year. Tobacco Mosaic Virus (TMV) is a single-stranded RNA virus belonging to the Tobamovirus group, which infects over 36 plants of the Solanaceae family, such as more than 400 plants, and makes the infected leaves look mottled and stained. After the plants such as tobacco and the like are infected with diseases, the tissues of the lateral veins and branch veins of the young leaves are semitransparent, namely the bright veins and the mesophyll tissues at the two sides of the veins are gradually light green. The virus proliferates in leaf tissue to increase or increase partial mesophyll cells, and leaves are uneven in thickness, alternate yellow and green in color and in the shape of a mosaic. The mottle degree of the posterior leaves is increased, large area of dark brown necrotic spots appear, and the middle and lower old leaves are particularly serious and cause severe leaf shrinkage, deformity and distortion. The tobacco mosaic virus disease is widely distributed in various tobacco regions in China and is the main virus disease of tobacco.
In order to effectively control the spread of plant virus diseases and fundamentally prevent the occurrence of the virus diseases and reduce the loss, researchers adopt various measures, and at present, chemical pesticides and a transgenic tobacco method are mainly utilized for preventing and treating the tobacco mosaic virus diseases. The use of chemical pesticides in large quantities makes the resistance of viruses increasingly evident, and one has to increase the dosage, which is a vicious circle and, as a result, causes high amounts of pesticide residues. The residual pesticide can not only harm the health of tobacco users, but also directly influence the export and foreign exchange of tobacco in China. Also, although transgenic technology can effectively control the occurrence and development of viral diseases, the biological safety of transgenic technology has attracted high attention from experts and governments at home and abroad.
Therefore, people must find a safer and more effective method for resisting plant virus diseases, so as to meet the requirements of agricultural sustainable development and environmental protection in China.
Disclosure of Invention
The invention provides application of p-coumaroyl homoserine lactone in prevention and treatment of tobacco mosaic virus, and aims to solve the technical problems that traditional chemical pesticide prevention and biological pesticide prevention easily generate drug resistance, and pesticide residue and pesticide standard exceed are caused.
According to one aspect of the invention, the application of p-coumaroyl homoserine lactone in prevention and treatment of tobacco mosaic virus diseases is provided.
Further, the application method of p-coumaroyl homoserine lactone for preventing and treating tobacco mosaic virus diseases comprises the following steps: dissolving the p-coumaroyl homoserine lactone serving as a raw material in a solvent to prepare a mother solution, diluting the mother solution with sterile water to obtain a p-coumaroyl homoserine lactone medicament solution, and spraying the p-coumaroyl homoserine lactone medicament solution to tobacco seedlings on leaf surfaces.
Further, the solvent is one of acetone or methanol.
Further, the molar concentration of p-coumaroyl homoserine lactone in the mother liquor obtained after dissolving p-coumaroyl homoserine lactone is as follows: 40 to 60 mM.
Further, in the p-coumaroyl homoserine lactone medicament solution, the concentration of the p-coumaroyl homoserine lactone is 1-10 mu M.
Further, in the p-coumaroyl homoserine lactone medicament solution, the concentration of the p-coumaroyl homoserine lactone is 4-8 mu M.
Further, in the p-coumaroyl homoserine lactone drug solution, the concentration of the p-coumaroyl homoserine lactone is 5 μ M.
Further, the tobacco seedling leaves are tobacco seedlings growing for 4-8 weeks.
Further, the foliar spraying amount of the p-coumaroyl homoserine lactone insecticide solution is to spray the tobacco seedlings on the foliar until the foliar insecticide solution begins to drip.
Further, the p-coumaroylhomoserine lactone is a quorum sensing hormone produced by rhodopseudomonas palustris.
The invention has the following beneficial effects:
the p-coumaroyl homoserine lactone is applied to prevention and treatment of tobacco mosaic virus diseases, the p-coumaroyl homoserine lactone medicament solution is sprayed on tobacco seedlings on leaf surfaces, the p-coumaroyl homoserine lactone can promote expression of tobacco disease resistance related genes PR1, PR10 and WRKY8, a tobacco system is stimulated to obtain resistance, so that the immunity of tobacco is enhanced, infection of tobacco mosaic virus is resisted, and the tobacco mosaic virus diseases are prevented. The p-coumaroyl homoserine lactone does not act on tobacco mosaic virus, does not cause drug resistance of virus and the like, is different from the traditional chemical pesticide and biological pesticide control, does not cause the problems of pesticide residue and pesticide standard exceeding, and meets the maximum requirements of agricultural sustainable development and environmental protection in China.
In addition to the objects, features and advantages described above, other objects, features and advantages of the present invention are also provided. The present invention will be described in further detail below with reference to the drawings.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 is a schematic representation of the scorched spots of tobacco leaves treated with 5 μ M p-coumaroylhomoserine lactone pharmacological solution in example 1 and a control group;
FIG. 2 is a graph showing the results of TMV-GFP measurements at the tips of Nicotiana benthamiana leaves in example 2 and a control group;
FIG. 3 is a graph showing the results of detecting TMV in tobacco system leaves by Dot blot in example 3 and a control group;
FIG. 4 is a graph showing the results of the tobacco system lamina TMV detection using qRT-PCR in example 3 and the control group;
FIG. 5 is a graph showing the results of qRT-PCR detection of expression level of the plant disease resistance related gene PR1 of example 3 and a control group after TMV virus inoculation;
FIG. 6 is a graph showing the results of expressed quantity of the plant disease resistance related gene PR10 after TMV virus inoculation in example 3 and the control group detected by qRT-PCR.
FIG. 7 is a schematic diagram of the results of qRT-PCR detection on the expression level of the disease resistance related gene WRKY8 of the plants in example 3 and the control group after TMV virus inoculation.
Detailed Description
The embodiments of the invention will be described in detail below with reference to the accompanying drawings, but the invention can be embodied in many different forms, which are defined and covered by the following description.
The embodiment provides application of p-coumaroyl homoserine lactone in prevention and treatment of tobacco mosaic virus diseases.
p-Coumaroyl-homoserine lactone (p-Coumaroyl) -L-homoserine lactone, pC-HSL), a quorum sensing hormone produced by Rhodopseudomonas palustris, is a novel acyl homoserine lactone signal molecule, and has the following structural formula:
plants are able to sense signals transmitted between microbial populations, quorum sensing hormones, over the course of long-term evolution. The invention discovers that p-coumaroyl homoserine lactone as a quorum sensing hormone generated by rhodopseudomonas palustris can promote the expression of genes PR1, PR10 and WRKY8 related to tobacco disease resistance, stimulate a tobacco system to obtain resistance, thereby enhancing the immunity of tobacco and resisting the infection of tobacco mosaic virus, so that the tobacco mosaic virus can be prevented by spraying a p-coumaroyl homoserine lactone medicament solution on the leaf surface of a tobacco plant in advance. The p-coumaroyl homoserine lactone does not act on tobacco mosaic virus, does not cause the resistance of virus and the like, is different from the traditional chemical pesticide and biological pesticide control, and meets the maximum requirements of agricultural sustainable development and environmental protection in China.
In this embodiment, the application method of p-coumaroyl homoserine lactone for preventing and treating tobacco mosaic virus disease is as follows: p-coumaroyl homoserine lactone is used as a raw material, a solvent is added for dissolving to prepare a mother solution, then the mother solution is diluted by sterile water to obtain a p-coumaroyl homoserine lactone medicament solution, and the p-coumaroyl homoserine lactone medicament solution is sprayed to leaf surfaces of tobacco seedlings. Preferably, the solvent is one of acetone or methanol.
Spraying the solution of p-coumaroyl homoserine lactone on the leaf surface of a tobacco seedling plant to induce the expression of tobacco disease-resistant related genes PR1, PR10 and WRKY8, so that the p-coumaroyl homoserine lactone is prepared into the solution firstly, and then the tobacco plant is treated by adopting a leaf surface spraying mode. P-coumaroyl homoserine lactone is a novel acyl homoserine lactone signal molecule, is insoluble in water, is dissolved by being added into a solvent, has high solubility to p-coumaroyl homoserine lactone due to both acetone and methanol, is dissolved as the solvent of p-coumaroyl homoserine lactone, is low in dosage and non-toxic, and has relatively small influence on plants.
In this example, the molar concentration of p-coumaroyl homoserine lactone in the mother liquor obtained after dissolving p-coumaroyl homoserine lactone is: 40 to 60 mM. The concentration can completely dissolve p-coumaroyl homoserine lactone, and the waste of the solvent can not be caused, thereby reducing the influence of the solvent on the growth of tobacco plants as much as possible and ensuring the quality of tobacco.
Preferably, the concentration of p-coumaroyl homoserine lactone in the p-coumaroyl homoserine lactone medicament solution is 1-10 mu M. Experiments prove that when the concentration of p-coumaroyl homoserine lactone is 1-10 mu M, tobacco can generate resistance to tobacco mosaic virus diseases, and the effect of preventing and treating the tobacco mosaic virus diseases is achieved. Further preferably, the concentration of p-coumaroyl homoserine lactone in the p-coumaroyl homoserine lactone drug solution is 4-8 μ M. Still more preferably, the concentration of p-coumaroyl homoserine lactone in the solution of p-coumaroyl homoserine lactone is 5 μ M. The p-coumaroyl homoserine lactone medicament solution with the concentration is sprayed on the leaf surfaces of tobacco, so that the prevention and treatment effect on tobacco mosaic virus diseases is optimal.
In the embodiment, the tobacco seedlings grow for 4-8 weeks. Seedlings at this stage are in the early growth stage of tobacco and are easily infected by tobacco mosaic virus, and-coumaroyl homoserine lactone reagent solution is sprayed on leaf surfaces at this stage to stimulate the expression of relevant disease-resistant genes of a tobacco system, so that the tobacco mosaic virus disease can be prevented.
In this example, the foliar application amount of the p-coumaroyl homoserine lactone insecticide solution was to foliar apply the tobacco seedling until the foliar insecticide solution started dripping. The mechanism of p-coumaroyl homoserine lactone for preventing and treating tobacco mosaic virus diseases is to promote the expression of tobacco disease-resistant related genes PR1, PR10 and WRKY8, the application is too little to promote the expression of the disease-resistant genes, the cost is too high when the application is too much, the spraying amount can achieve the effect of preventing and treating the tobacco mosaic virus diseases, and the prevention and treatment cost cannot be obviously increased.
The following example is used to further explain the induction of TMV resistance of different tobacco varieties by coumaroylhomoserine lactone, and only two tobacco varieties of heart-leaf tobacco and Nicotiana benthamiana are selected in the following example, but it can be understood that the method is also applicable to the prevention and treatment of tobacco mosaic virus diseases by other varieties of tobacco.
Preparing p-coumaroyl homoserine lactone solution: p-coumaroyl homoserine lactone is weighed as a raw drug, and then analytically pure acetone or methanol is added into the raw drug for dissolution to prepare 40mM mother liquor.
The above mother solution was diluted with sterile water to concentrations of 1. mu.M, 4. mu.M, 5. mu.M, 8. mu.M, and 10. mu.M, respectively, to prepare p-coumaroyl homoserine lactone drug solutions.
Example 1
TMV resistance test was induced on P-coumaroylhomoserine lactone-induced leaf tobacco.
And (3) after transplanting the heart-leaf tobacco for 4-8 weeks, selecting leaf surfaces of heart-leaf tobacco plants with uniform growth vigor, spraying the prepared p-coumaroyl homoserine lactone medicament solutions with different concentrations, adopting sterile water (added with equivalent acetone or methanol) as a contrast, not adding p-coumaroyl homoserine lactone to a contrast group, and keeping the other conditions the same. Spraying tobacco seedlings on the leaf surfaces of the coumaroyl homoserine lactone medicament solution until the leaf surface medicament solution begins to drip; after 24h, the third and fourth leaves were inoculated with TMV virions. Spreading carborundum on leaf surface, wherein each leaf is 25 μ L, TMV concentration is 0.74mg/ml, washing bottle and washing after TMV inoculation. The culture was carried out at 25 ℃ under light, and the treatment was repeated three times for each 10 strains. After 72h, the number of dead spots was counted.
Table 1 shows the average number of dry spots of the heart-leaf tobacco treated with p-coumaroyl homoserine lactone solutions of different concentrations, followed by TMV, and fig. 1 is a schematic diagram of dry spots of the heart-leaf tobacco leaves treated with p-coumaroyl homoserine lactone solutions of 5 μ M and the control group.
TABLE 1 average number of lesions after TMV cut tobacco
Referring to table 1 and fig. 1, TMV resistance test data of P-coumaroyl homoserine lactone-induced tobacco leaf smoke shows that after the plants of the P-coumaroyl homoserine lactone treatment group are inoculated for 3 days, the number of dead spots on the leaf surface of the tobacco leaf smoke is obviously lower than that of a control group, which indicates that P-coumaroyl homoserine lactone with the concentration of 1-10 mu M has an effect on preventing and treating tobacco mosaic virus diseases, and P-coumaroyl homoserine lactone with the concentration of 4-8 mu M has a relatively high effect on preventing and treating the tobacco mosaic virus diseases.
Example 2
Accumulation test of TMV in Nicotiana benthamiana on p-coumaroylhomoserine lactone.
After the Shigella benthamiana is transplanted for 4-5 weeks, leaf surfaces of the Shigella benthamiana plants with uniform growth vigor are selected and sprayed with 5 mu M p-coumaroyl homoserine lactone agent solution, sterile water (added with equivalent acetone or methanol) serves as a control, p-coumaroyl homoserine lactone is not added to the control group, and other conditions are the same. Spraying tobacco seedlings on the leaf surfaces of the coumaroyl homoserine lactone medicament solution until the leaf surface medicament solution begins to drip; after 24h, the third and fourth leaves were rubbed to receive GFP-tagged infectious cloned TMV virions. Spreading carborundum on leaf surface, wherein each leaf is 25 μ L of TMV, the concentration of TMV is 0.74mg/ml, washing bottle and washing after TMV inoculation. The culture was carried out at 25 ℃ under light, and the treatment was repeated three times for each 10 strains. After 7d, the GFP status at the leaf top was observed.
FIG. 2 is a graph showing the results of TMV-GFP measurements at the tips of Nicotiana benthamiana leaves in example 2 and a control group.
Referring to FIG. 2, GFP results under UV light after 7d showed that TMV accumulation was lower in apical system leaf treated with p-coumaroylhomoserine lactone at a concentration of 5. mu.M, compared to the control group.
Example 3
P-coumaroylhomoserine lactone-induced Nicotiana benthamiana anti-TMV test
After the Shigella benthamiana is transplanted for 4-5 weeks, leaf surfaces of the Shigella benthamiana plants with uniform growth vigor are selected and sprayed with 5 mu M p-coumaroyl homoserine lactone agent solution, sterile water (added with equivalent acetone or methanol) serves as a control, p-coumaroyl homoserine lactone is not added to the control group, and other conditions are the same. Spraying tobacco seedlings on the leaf surfaces of the coumaroylhomoserine lactone agent solution until the leaf surface agent solution begins to drip; after 24h, TMV is connected to the third and fourth pieces. Spreading carborundum on leaf surface, wherein each leaf is 25 μ L of TMV, the concentration of TMV is 0.74mg/ml, washing bottle and washing after TMV inoculation. The culture was incubated at 25 ℃ with light, and the treatment was repeated three times for each 20 strains. At 72h after TMV virus exposure, Dot blot detects the TMV condition of the system blade; detecting the TMV condition of the leaf blade by using qRT-PCR at 48h, 72h and 96 h; detecting the expression conditions of disease-resistant related genes PR1, PR10 and WRKY8 at 0h, 12h, 24h and 48 h. Fig. 3 is a schematic diagram of the results of the Dot blot detection system of the leaf TMV in example 3 and the control group, fig. 4 is a schematic diagram of the results of the Dot blot detection system of the leaf TMV in example 3 and the control group, fig. 5 is a schematic diagram of the expression levels of the plant disease-resistance related gene PR1 in example 3 and the control group after TMV inoculation, fig. 6 is an expression level of the plant disease-resistance related gene PR10 in example 3 and the control group after qRT-PCR detection after TMV inoculation, and fig. 7 is an expression level of the plant disease-resistance related gene WRKY8 in example 3 and the control group after qRT-PCR detection after TMV inoculation (it indicates that the difference is significant at a confidence level of 95%).
Referring to fig. 3, data of a test for inducing ben's smoke resistance to coumaroyl homoserine lactone show that after plants treated by coumaroyl homoserine lactone are inoculated for 72 hours, the result of the content of TMV in leaves of a Dot blot detection system shows that the content of TMV is significantly lower than that of a control group; as can be seen from fig. 4, the content of TMV in the 48h, 72h and 96h system leaves detected by qRT-PCR shows that the relative expression amount of TMV in the plants treated with p-coumaroyl homoserine lactone is significantly lower than that in the control group at 48h and 72h, so that it can be obtained that p-coumaroyl homoserine lactone inhibits the multiplication of tobacco mosaic virus at the initial stage of TMV infection, and the difference between the p-coumaroyl homoserine lactone and the control group is not large at 96h, which indicates that the onset time of p-coumaroyl homoserine lactone is about 72 h; as can be seen from fig. 6 and 7 in combination with fig. 5, qRT-PCR detection shows that the expression levels of disease-resistance related genes PR1, PR10 and WRKY8 of plants treated with p-coumaroylhomoserine lactone are significantly higher than those of a control group after TMV inoculation for 24 hours, and thus it can be found that: the coumaroyl homoserine lactone is used for inducing Nicotiana benthamiana to resist TMV, and the expression of disease-resistant related genes of tobacco is induced, so that the multiplication of tobacco mosaic virus is inhibited in the early stage of TMV infection, the disease time is delayed, and the disease rate is reduced; p-coumaroylhomoserine lactone has an onset time of about 72 hours, so that it can be applied every 72 hours (every three days) at the tobacco seedling stage until the whole seedling stage is passed in field operation.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. An application of p-coumaroyl homoserine lactone in preventing and treating tobacco mosaic virus diseases is characterized in that,
the application method of p-coumaroyl homoserine lactone for preventing and treating tobacco mosaic virus diseases comprises the following steps: dissolving the p-coumaroyl homoserine lactone serving as a raw material in a solvent to prepare a mother solution, diluting the mother solution with sterile water to obtain a p-coumaroyl homoserine lactone medicament solution, and spraying the p-coumaroyl homoserine lactone medicament solution to tobacco seedlings on leaf surfaces;
in the p-coumaroyl homoserine lactone medicament solution, the concentration of the p-coumaroyl homoserine lactone is 1-10 mu M.
2. The use of p-coumaroyl homoserine lactone for the control of tobacco mosaic virus disease according to claim 1,
the solvent is one of acetone or methanol.
3. The use of p-coumaroyl homoserine lactone for the control of tobacco mosaic virus disease according to claim 2,
the molar concentration of p-coumaroyl homoserine lactone in the mother liquor obtained after dissolving p-coumaroyl homoserine lactone is as follows: 40 to 60 mM.
4. The use of p-coumaroyl homoserine lactone for the control of tobacco mosaic virus disease according to claim 1,
in the p-coumaroyl homoserine lactone medicament solution, the concentration of the p-coumaroyl homoserine lactone is 4-8 mu M.
5. The use of p-coumaroyl homoserine lactone for the control of tobacco mosaic virus disease according to claim 4,
in the p-coumaroyl homoserine lactone drug solution, the concentration of the p-coumaroyl homoserine lactone is 5 μ M.
6. The use of p-coumaroyl homoserine lactone according to any one of claims 3 to 5, for controlling tobacco mosaic virus disease,
the tobacco seedlings are grown for 4-8 weeks.
7. The use of p-coumaroyl homoserine lactone for the control of tobacco mosaic virus disease according to claim 6,
the foliage spraying amount of the p-coumaroyl homoserine lactone insecticide solution is that tobacco seedlings are sprayed on the foliage until the insecticide solution on the foliage begins to drip.
8. The use of p-coumaroyl homoserine lactone according to any one of claims 1 to 5, for preventing and treating tobacco mosaic virus diseases,
the p-coumaroylhomoserine lactone is a quorum sensing hormone produced by rhodopseudomonas palustris.
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