Leptinotarsa decemlineata nucleic acid interferon liquid formulation and use thereof

Abstract

The present application relates to a Leptinotarsa decemlineata nucleic acid interferon liquid formulation. The liquid formulation is prepared by taking a Leptinotarsa decemlineata nucleic acid interferon as an active ingredient and adding an auxiliary agent, the content of the Leptinotarsa decemlineata nucleic acid interferon is 0.8-30% in percentage by mass, and the Leptinotarsa decemlineata nucleic acid interferon is selected from dsLdSrp54k, dsATPaseE, dsATPaseA, and cact::snap dsRNA. Provided is a use of the Leptinotarsa decemlineata nucleic acid interferon liquid formulation in prevention and treatment of Leptinotarsa decemlineata. The Leptinotarsa decemlineata nucleic acid interferon liquid formulation is prepared by taking the Leptinotarsa decemlineata nucleic acid interferon as an active ingredient and adding a suitable auxiliary agent, thereby effectively improving the stability of the nucleic acid interferon, and having important significance for improving the efficacy of nucleic acid interferon in prevention and treatment of Leptinotarsa decemlineata.

Description

A liquid preparation of interferon nucleic acid for potato beetle and its application Technical Field

[0001] The invention belongs to the technical field of biological pesticides, and in particular relates to a liquid preparation of nucleic acid interferon for potato beetles and application thereof. Background Art

[0002] The potato beetle is an internationally recognized destructive quarantine pest and a major invasive alien species in my country. Since invading northern Xinjiang in the early 1990s, the beetle has caused severe losses to potato and other Solanaceae crops. The beetle's strong dispersal ability, high reproductive capacity, and nearly perfect biological learning and survival strategies, including facultative diapause, increase the risk of its further eastward spread into major potato-producing areas such as Gansu and Inner Mongolia. Furthermore, in Northeast my country, the beetle has already spread into provinces such as Heilongjiang and Jilin, increasing the risk of further spread.

[0003] dsRNA, also known as potato beetle interferon, is highly selective against potato beetles and has excellent control efficacy against them. It has no effect on beneficial organisms such as earthworms and bees, or on beneficial microorganisms in the soil, such as photosynthetic bacteria and nitrogen-fixing bacteria. Interferon is environmentally friendly and has demonstrated excellent cost-effectiveness and applicability. However, the nitrogen-glycosidic or phosphodiester bonds between the base and the pentose sugar in potato beetle interferon are susceptible to hydrolysis under physical, chemical, and biological factors, leading to RNA strand breakage. During storage, the actual decomposition rate of the active ingredient is generally greater than 20%. Potato beetle interferon formulations are subject to prolonged high temperatures and large diurnal temperature swings during long-distance and cross-sea transportation, making their decomposition a particularly challenging issue. Furthermore, after application in the field, ambient oxygen, moisture, ultraviolet light, and temperature can easily cause their decomposition, thus compromising their efficacy.

[0004] Summary of the Invention

[0005] To address the poor stability of interferon against potato beetles, the applicant disclosed in CN116267920A a compound emulsifier for interferon formulations against potato beetles. Building on this foundation, the inventors conducted extensive and in-depth research on liquid formulations of interferon against potato beetles. The results revealed that even when the thermal storage temperature (54°C, 14 days) was increased to 70°C, the formulation maintained excellent stability. This discovery is of great significance to the research on interferon pesticide formulations.

[0006] Therefore, a first aspect of the present invention provides a liquid preparation of potato beetle interferon nucleic acid, which is prepared by using potato beetle interferon nucleic acid as an active ingredient and adjuvants, wherein the content of the potato beetle interferon nucleic acid is 0.8%-30% by mass, and the potato beetle interferon nucleic acid is selected from: dsLdSrp54k, dsATPaseE, dsATPaseA, and cact::snap dsRNA.

[0007] According to the present invention, the dosage form of the liquid preparation is a soluble solution, an aqueous emulsion, a microemulsion, a suspoemulsion, a suspension or a dispersible oil suspension.

[0008] According to the present invention, the auxiliary agent is selected from one or more of: a dispersant, a wetting agent, an emulsifier, a stabilizer, a pH regulator, a defoaming agent, a preservative, an antifreeze agent, a thickener, and a solvent.

[0009] Furthermore, the dispersant is selected from one or more of alkylnaphthalene sulfonates, polyoxyethylene polyoxypropylene block copolymers, aromatic phenol polyoxyethylene succinate sulfonates, polycarboxylates, lignin sulfonic acid sodium salts, alkylphenol polyoxyethylene ether formaldehyde condensate sulfates, alkylbenzene sulfonic acid calcium salts, naphthalenesulfonic acid formaldehyde condensate sodium salts, phenethylphenol polyoxyethylene ether phosphates and fatty acid polyoxyethylene esters.

[0010] Furthermore, the wetting agent is selected from one or more of sodium lauryl sulfate, sodium dodecylbenzenesulfonate, alkyl aryl sulfonate, sodium lauryl sulfate, soapberry powder, tea saponin and sodium dioctyl sulfosuccinate.

[0011] Furthermore, the emulsifier is selected from one or more of alkyl glycosides, alkyl betaines, fatty alcohol polyoxyethylene ethers, oleic acid polyoxyethylene ethers, cardanol polyoxyethylene ethers, castor oil polyoxyethylene ethers, polyarylphenol polyoxyethylene ethers, and EO / PO block copolymers.

[0012] Furthermore, the stabilizer is a combination of tea polyphenols, inositol and N,N-bicine, and the mass ratio of the three is tea polyphenols: inositol: N,N-bicine = 3:1:1.

[0013] Furthermore, the pH adjuster is selected from the group consisting of lactic acid, citric acid, phytic acid, DL-malic acid, oxalic acid, triethanolamine, sodium carbonate, and sodium bicarbonate.

[0014] Furthermore, the defoaming agent is selected from one or more of polydimethylsiloxane, isomeric alcohol ether, dimethyl silicone oil and polyether-modified silicone oil.

[0015] Furthermore, the preservative is selected from: kasone, potassium sorbate, 1,2-benzisothiazolin-3-one and sodium benzoate.

[0016] Furthermore, the antifreeze agent is selected from: urea, 1,2-propylene glycol and glycerol.

[0017] Furthermore, the thickener is selected from the group consisting of: gum arabic, gelatin, attapulgite, magnesium aluminum silicate, organic bentonite and xanthan gum.

[0018] Furthermore, the solvent is selected from one or more of: N,N-dimethylacetamide, soybean oil, rosin-based vegetable oil, polyol mixed ester, N,N-dimethylacetamide, olive oil, castor oil and deionized water.

[0019] The second aspect of the present invention provides the use of the liquid preparation of interferon nucleic acid against potato beetles for controlling potato beetles.

[0020] The third aspect of the present invention provides a stabilizer for a liquid preparation of interferon nucleic acid for potato beetles, wherein the stabilizer is a combination of tea polyphenols, inositol and N,N-dihydroxyethylglycine, and the mass ratio of the three is tea polyphenols: inositol: N,N-dihydroxyethylglycine = 3:1:1.

[0021] The present invention has the following beneficial effects:

[0022] 1. The present invention uses interferon nucleic acid from potato beetles as an active ingredient and combines it with suitable adjuvants to prepare a liquid preparation of interferon nucleic acid from potato beetles, thereby effectively improving the stability of interferon nucleic acid, which is of great significance for improving the effect of interferon nucleic acid in controlling potato beetles.

[0023] 2. The preparation process of the potato beetle interferon nucleic acid liquid preparation of the present invention is simple and can be industrialized, which is of great significance for promoting the industrialization of interferon nucleic acid pesticide preparations.

[0024] 3. The potato beetle interferon liquid preparation of the present invention is environmentally friendly and has strong selectivity. It has no effect on beneficial organisms such as earthworms and bees and beneficial microorganisms such as photosynthetic bacteria and nitrogen-fixing bacteria in the soil.

[0025] 4. The potato beetle interferon nucleic acid liquid preparation of the present invention has no dust hazard, has excellent diffusion in water, is convenient to use, is easy to measure, and is safe for the operator. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG1 shows the electrophoresis detection results of different nucleic acid interferon samples after hot storage, wherein: M corresponds to DL 5000 Marker; lanes 1-3 correspond to Examples 1-3 respectively; lanes 4-6 correspond to Examples 1-3 respectively. DETAILED DESCRIPTION

[0027] The present invention will be further described in detail below with reference to specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0028] The interferon cact::snap dsRNA used in the following examples has been disclosed in CN116042620A, and dsLdSrp54k, dsATPaseE, and dsATPaseA have been disclosed in CN109907068A.

[0029] Example 1: Preparation of 0.8% potato beetle interferon soluble solution

[0030] A 0.8% interferon-1a soluble solution of potato beetle nucleic acid was prepared according to the components and proportions shown in Table 1 below:

[0031] Table 1: 0.8% potato beetle interferon soluble solution

[0032] According to the formula in Table 1 above, alkyl glycoside and alkyl betaine, tea polyphenols, inositol, N,N-dihydroxyethylglycine, 1,2-benzisothiazolin-3-one, oxalic acid, and 1,2-propylene glycol were thoroughly mixed with deionized water in sequence. After dissolution, potato beetle interferon cact::snap dsRNA was added, and after complete dissolution, it was filtered to obtain a 0.8% potato beetle interferon soluble solution.

[0033] Example 2: Preparation of 1.6% potato beetle interferon aqueous emulsion

[0034] A 1.6% potato beetle interferon aqueous emulsion was prepared according to the components and proportions shown in Table 2 below:

[0035] Table 2: 1.6% potato beetle interferon aqueous emulsion

[0036] According to the formulation in Table 2 above, tea polyphenols, inositol, N,N-dihydroxyethylglycine, 1,2-benzisothiazolin-3-one, lactic acid, 1,2-propylene glycol, and potato beetle interferon cact::snap dsRNA were dissolved in deionized water in sequence to form an aqueous phase; castor oil polyoxyethylene ether, fatty alcohol polyoxyethylene ether, and EO / PO block copolymer were dissolved in olive oil in sequence to form an oil phase; finally, the oil phase was slowly added to the aqueous phase under high-speed homogenization emulsification conditions to obtain a 1.6% potato beetle interferon aqueous emulsion.

[0037] Example 3: Preparation of 3.2% potato beetle interferon microemulsion

[0038] A 3.2% potato beetle interferon microemulsion was prepared according to the components and ratios shown in Table 3 below:

[0039] Table 3: 3.2% potato beetle interferon microemulsion

[0040] According to the formulation in Table 3 above, tea polyphenols, inositol, N,N-dihydroxyethylglycine, 1,2-benzisothiazolin-3-one, phytic acid, 1,2-propylene glycol, and potato beetle interferon cact::snap dsRNA were dissolved in deionized water to form an aqueous phase; polyarylphenol polyoxyethylene ether, cardanol polyoxyethylene ether, and EO / PO block copolymer were dissolved in a polyol mixed ester to form an oil phase; finally, the aqueous phase was slowly added to the oil phase under stirring to obtain a 3.2% potato beetle interferon microemulsion.

[0041] Example 4: Preparation of 5% potato beetle interferon suspoemulsion

[0042] A 5% interferon-nucleic acid suspoemulsion of potato beetle was prepared according to the components and proportions shown in Table 4 below:

[0043] Table 4: 5% potato beetle interferon suspensoid emulsion

[0044] According to the above-mentioned formula, polyoxyethylene-polyoxypropylene block copolymer, cardanol polyoxyethylene ether, oleic acid polyoxyethylene ether, EO / PO block copolymer, polyether-modified silicone oil, tea polyphenols, inositol, N,N-dihydroxyethylglycine, potassium sorbate, DL-malic acid, glycerol, olive oil, xanthan gum, potato beetle interferon nucleic acid dsLdSrp54k and deionized water are mixed in sequence, sheared and homogenized, and then wet-grinded to obtain a 5% potato beetle interferon nucleic acid suspoemulsion.

[0045] Example 5: Preparation of 10% Colorado potato beetle interferon suspension

[0046] A 10% interferon suspension concentrate of potato beetle nucleic acid was prepared according to the components and proportions shown in Table 5 below:

[0047] Table 5: 10% potato beetle interferon suspension

[0048] According to the formula in Table 5 above, polycarboxylates, alkylnaphthalene sulfonates, sodium dioctyl sulfosuccinate, isomeric alcohol ethers, tea polyphenols, inositol, N,N-dihydroxyethylglycine, sodium benzoate, triethanolamine, urea, magnesium aluminum silicate, xanthan gum, potato beetle interferon dsATPaseE and deionized water were mixed in sequence, sheared and homogenized, and then wet-grinded to obtain a 10% potato beetle interferon suspension.

[0049] Example 6: Preparation of 30% potato beetle interferon dispersible oil suspension

[0050] A 30% dispersible oil suspension concentrate of interferon nucleic acid against potato beetles was prepared according to the components and proportions shown in Table 6 below:

[0051] Table 6: 30% potato beetle interferon dispersible oil suspension concentrate

[0052] According to the formula in Table 6 above, phenylethylphenol polyoxyethylene ether phosphate, emulsifier castor oil polyoxyethylene ether, cardanol polyoxyethylene ether, EO / PO block copolymer, tea polyphenols, inositol, N,N-dihydroxyethylglycine, kasone, sodium carbonate, organobentonite, potato beetle interferon nucleic acid dsATPaseA and soybean oil were mixed in sequence, sheared and homogenized, and then wet-grinded to obtain a 30% potato beetle interferon nucleic acid dispersible oil suspension concentrate.

[0053] Comparative Example 1

[0054] This comparative example follows the formulation and preparation process of Example 1, except for the ratio of stabilizers, as shown in Table 7 below:

[0055] Table 7: 0.8% potato beetle interferon soluble solution

[0056] Comparative Example 2

[0057] This comparative example follows the formulation and preparation process of Example 2, except for the ratio of stabilizers, as shown in Table 8 below:

[0058] Table 8: 1.6% potato beetle interferon aqueous emulsion

[0059] Comparative Example 3

[0060] This comparative example follows the components and preparation process of Example 3, except for the ratio of stabilizers, as shown in Table 9 below:

[0061] Table 9: 3.2% potato beetle interferon microemulsion

[0062] Comparative Example 4

[0063] This comparative example follows the formulation and preparation process of Example 1, except that the mass percentage of the potato beetle interferon nucleic acid is 0.5%, as shown in the following Table 10:

[0064] Table 10: 0.5% potato beetle interferon soluble solution

[0065] Comparative Example 5

[0066] This comparative example follows the formulation and preparation process of Example 6, except that the mass percentage of the potato beetle interferon nucleic acid is 40%, as shown in the following Table 11:

[0067] Table 11: 40% potato beetle interferon dispersible oil suspension concentrate

[0068] Example 7, stability test

[0069] The samples from Examples 1-6 and Comparative Examples 1-3 were sealed in glass bottles and stored in a thermostat at 70°C ± 2°C for 14 days. The samples were then removed, placed in a desiccator, and cooled to room temperature. The active ingredient mass fraction and other required parameters were determined within 24 hours. The test results are shown in Table 12.

[0070] Table 12: Stability test of liquid formulation of interferon nucleic acid against potato beetle

[0071] As shown in Table 12, the interferon nucleic acid liquid formulations of Examples 1-6 all exhibited good stability, with decomposition rates of less than 5% after 14 days of storage at 70°C. The interferon nucleic acid liquid formulations of Comparative Examples 1-3 achieved a maximum decomposition rate of 37.21% during heat storage, demonstrating the crucial importance of the stabilizer ratio in the formulation of the interferon nucleic acid liquid formulation.

[0072] Example 8, agarose gel electrophoresis detection

[0073] Heat storage samples (70° C., 14 days) of Examples 1 to 3 and Comparative Examples 1 to 3 were respectively taken, and then the effective components (dsRNA fragment length was 516 bp) were detected by agarose gel electrophoresis.

[0074] In this example, samples (70°C, 14 days) from Examples 1-3 and Comparative Examples 1-3 were diluted proportionally to the same concentration. A 1.5% agarose concentration was used, and 10 μL of each sample was loaded. Electrophoresis was performed at 150 V for 20 minutes, followed by imaging using a gel imaging system. The results are shown in Figure 1.

[0075] As shown in Figure 1 , the nucleic acid bands in Examples 1-3 are primarily located at 500 bp of the DL5000 marker and show no degradation. In contrast, in Comparative Examples 1-3, in addition to the nucleic acid band at 500 bp of the DL5000 marker, bands are also observed between 100 and 250 bp. This indicates that the target dsRNA in the formulations of Comparative Examples 1-3 degrades after 14 days of heat storage at 70°C. This suggests that changes in the stabilizer ratio are detrimental to the stability of the potato beetle interferon.

[0076] Example 9, field control effect

[0077] This application example is used to verify the field application effect of the liquid preparation of the present invention on potato beetles, and the field application effects of the potato beetle nucleic acid interferon liquid preparations prepared in Test Examples 1 to 6 and the potato beetle nucleic acid interferon liquid preparations prepared in Comparative Examples 1 to 5 on potato beetles.

[0078] This experiment was conducted in potato fields in Muli County, Changji Hui Autonomous Prefecture, Xinjiang, where potato beetles were severely infested. Using water as a blank control, the recommended dosage of the formulation (45 kg of water per mu) was applied evenly to the leaves during the early larval stage of the potato beetles. The base population and plant damage level in each plot were investigated before, 2 days after, 7 days after, and 14 days after application. Sampling methods included sampling at five locations per plot, or in parallel, with 10 clumps of signs posted at each location. Larvae were counted on the plants at the designated locations and control efficacy was calculated. Plant damage was also visually assessed (damage was graded according to the following method), with sampling avoiding edge rows. Plant survival efficacy was calculated, and the results are shown in Table 13.

[0079] Plant damage grade classification:

[0080] Level 0: No symptoms of victimization;

[0081] Level 1: The leaves of the entire clump are sporadically damaged;

[0082] Level 3: less than 1 / 3 of the leaves in the entire clump are damaged;

[0083] Level 5: 1 / 3 to 1 / 2 of the leaves in the entire clump are damaged;

[0084] Level 7: 1 / 2 to 2 / 3 of the leaves in the entire clump are damaged;

[0085] Level 9: More than 2 / 3 of the leaves in the entire cluster are damaged.

[0086] Prevention and control effect:

[0087] Plant protection effect:

[0088] Table 13: Field test results of liquid interferon preparation against potato beetles

[0089] The field test results in Table 13 show that the interferon nucleic acid formulations prepared in Examples 1-6 all achieved control efficacy against potato beetles exceeding 80% 7 days after application and exceeding 90% 14 days after application, significantly exceeding the efficacy of the products prepared in Comparative Examples 1-5. Comparative Example 4, when applied in the field at a dosage of 650 ml / mu, caused phytotoxicity to potato leaves after spraying. Symptoms included acute water-soaked spots 2 hours after application, burns 4 hours after application, and perforations 24 hours after application. Newly grown leaves showed little effect. The formulation in Comparative Example 5 exhibited poor dispersibility in water and remained poorly dispersed even after 10 minutes of stirring, resulting in unsatisfactory control of potato beetles.

[0090] Field test results demonstrate that the interferon nucleic acid liquid formulations of Examples 1-6 can still exert good efficacy despite field conditions such as rain and ultraviolet light, effectively protecting potato plants. This reduces yield losses caused by potato beetle damage, resulting in direct economic benefits for farmers.

Claims

1. A liquid preparation of interferon nucleic acid for potato beetle, characterized in that: The liquid preparation is prepared by using potato beetle interferon nucleic acid as an active ingredient and adjuvants, wherein the content of potato beetle interferon nucleic acid is 0.8%-30% by mass percentage, and the potato beetle interferon nucleic acid is selected from: dsLdSrp54k, dsATPaseE, dsATPaseA, and cact::snap dsRNA.

2. The liquid preparation of interferon nucleic acid against potato beetles according to claim 1, characterized in that: The dosage form of the liquid preparation is a soluble solution, an aqueous emulsion, a microemulsion, a suspoemulsion, a suspension or a dispersible oil suspension.

3. The liquid preparation of interferon nucleic acid against potato beetles according to claim 1, characterized in that: The auxiliary agent is selected from: one or more of a dispersant, a wetting agent, an emulsifier, a stabilizer, a pH regulator, a defoamer, a preservative, an antifreeze agent, a thickener, and a solvent.

4. The liquid preparation of interferon nucleic acid against potato beetles according to claim 3, characterized in that: The dispersant is selected from: one or more of alkylnaphthalene sulfonates, polyoxyethylene polyoxypropylene block copolymers, aromatic phenol polyoxyethylene succinate sulfonates, polycarboxylates, lignin sulfonate sodium salts, alkylphenol polyoxyethylene ether formaldehyde condensate sulfates, alkylbenzene sulfonate calcium salts, naphthalenesulfonic acid formaldehyde condensate sodium salts, phenethylphenol polyoxyethylene ether phosphates and fatty acid polyoxyethylene esters.

5. The liquid preparation of interferon nucleic acid against potato beetles according to claim 3, characterized in that: The wetting agent is selected from one or more of sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, alkyl aryl sulfonate, sodium lauryl sulfate, soapberry powder, tea saponin and sodium dioctyl sulfonate succinate.

6. The liquid preparation of interferon nucleic acid against potato beetles according to claim 3, characterized in that: The emulsifier is selected from one or more of alkyl glycoside, alkyl betaine, fatty alcohol polyoxyethylene ether, oleic acid polyoxyethylene ether, cardanol polyoxyethylene ether, castor oil polyoxyethylene ether, polyarylphenol polyoxyethylene ether, and EO / PO block copolymer.

7. The liquid preparation of interferon nucleic acid against potato beetles according to claim 3, characterized in that: The stabilizer is a combination of tea polyphenols, inositol and N,N-dihydroxyethylglycine, and the mass ratio of the three is tea polyphenols: inositol: N,N-dihydroxyethylglycine = 3:1:

1.

8. The liquid preparation of interferon nucleic acid against potato beetles according to claim 3, characterized in that: The pH adjuster is selected from the group consisting of lactic acid, citric acid, phytic acid, DL-malic acid, oxalic acid, triethanolamine, sodium carbonate, and sodium bicarbonate.

9. The liquid preparation of interferon nucleic acid against potato beetles according to claim 3, characterized in that: The defoaming agent is selected from one or more of polydimethylsiloxane, isomeric alcohol ether, dimethyl silicone oil and polyether modified silicone oil.

10. The liquid preparation of interferon nucleic acid against potato beetles according to claim 3, characterized in that: The preservative is selected from the group consisting of: kasone, potassium sorbate, 1,2-benzisothiazolin-3-one and sodium benzoate.

11. The liquid preparation of interferon nucleic acid against potato beetle according to claim 3, characterized in that: The antifreeze agent is selected from the group consisting of urea, 1,2-propylene glycol and glycerol.

12. The liquid preparation of interferon nucleic acid against potato beetles according to claim 3, characterized in that: The thickener is selected from the group consisting of gum arabic, gelatin, attapulgite, magnesium aluminum silicate, organic bentonite and xanthan gum.

13. The liquid preparation of interferon nucleic acid against potato beetle according to claim 3, characterized in that: The solvent is selected from one or more of: N,N-dimethylacetamide, soybean oil, rosin-based vegetable oil, polyol mixed ester, N,N-dimethyl linalool, olive oil, castor oil and deionized water.

14. The use of the liquid preparation of interferon nucleic acid against potato beetles according to any one of claims 1 to 13, characterized in that: Used to control potato beetles.

15. A stabilizer for a liquid preparation of interferon nucleic acid against potato beetles, characterized in that: The stabilizer is a combination of tea polyphenols, inositol and N,N-dihydroxyethylglycine, and the mass ratio of the three is tea polyphenols: inositol: N,N-dihydroxyethylglycine = 3:1:1.

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