Application of ningnanmycin salt in prevention and treatment of tobacco mosaic virus
By developing salt derivatives of ningnanmycin, the problem of unstable control effect of ningnanmycin was solved, and the control effect against tobacco mosaic virus was significantly improved, achieving a control effect comparable to that of commonly used agents.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-31
- Publication Date
- 2026-03-27
AI Technical Summary
The efficacy of ningnanmycin in controlling tobacco mosaic virus disease is unstable, and high-concentration application may cause physiological stress to plants, limiting its large-scale application.
Develop salt derivatives of ningnanmycin, such as acetate, lactate, and phosphate, prepare ningnanmycin salts through specific processes, and apply them to the prevention and control of tobacco mosaic virus to improve the control effect.
Ningnanmycin salt significantly improved the control effect against tobacco mosaic virus, reaching or even exceeding the control effect of commonly used agent morpholine guanidine hydrochloride, showing good application prospects.
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Figure CN121730306A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to the application of ningnanmycin salt in the prevention and control of tobacco mosaic virus disease. Background Technology
[0002] Tobacco mosaic virus (TMV) is a highly destructive plant virus in agricultural production, widely affecting the quality and yield of various crops such as tobacco, vegetables, and fruits. TMV spreads rapidly and infects a wide range of plants, and common pesticides have limited effectiveness. Therefore, developing efficient and environmentally friendly biological control methods has become one of the important directions in current agricultural research.
[0003] Ningnanmycin, as a novel biological pesticide, has gained increasing attention in recent years due to its good control efficacy and environmental friendliness. However, although ningnanmycin has shown certain control effects in laboratory and field trials, its efficacy is not very stable, which limits its application under different environmental conditions. Furthermore, high concentrations of ningnanmycin may cause physiological stress to plants, affecting their normal growth. These issues pose challenges to the large-scale application of ningnanmycin, necessitating further improvement and optimization.
[0004] Therefore, developing new derivatives may lead to new mechanisms of action or enhance existing antiviral effects, thereby more effectively controlling tobacco mosaic virus. Summary of the Invention
[0005] To address the shortcomings of existing technologies, this invention provides the application of ningnanmycin salt in the prevention and control of tobacco mosaic virus (TMV) disease.
[0006] This invention provides the use of ningnanmycin salt in the preparation of antiviral agents for the prevention and / or treatment of tobacco mosaic virus.
[0007] Preferably, the salt comprises at least one of the following: acetate, lactate, phosphate, sulfate, citrate, benzoate, oxalate, hydrochloride, and nitrate.
[0008] Preferably, the salt comprises at least one of the following: acetate, lactate, phosphate, sulfate, citrate, and benzoate.
[0009] Preferably, the salt is selected from at least one of monosalt, disalt, trisalt, and tetrasalt.
[0010] Preferably, the ningnanmycin salt is prepared by ningnanmycin free base and acid, and the molar ratio of ningnanmycin to acid anion is ≤3:1.
[0011] Preferably, the acid is selected from at least one of acetic acid, lactic acid, phosphoric acid, sulfuric acid, citric acid, benzoic acid, oxalic acid, hydrochloric acid, and nitric acid.
[0012] Preferably, when the salt is an acetate, the molar ratio of ningnanmycin to acetate is ≤1:1; When the salt is a lactate, the molar ratio of ningnanmycin to lactate is ≤1:1; When the salt is a phosphate, the molar ratio of ningnanmycin to phosphate is ≤3:1; When the salt is a sulfate, the molar ratio of ningnanmycin to sulfate is ≤2:1; When the salt is citrate, the molar ratio of ningnanmycin to citrate ion is ≤1:1; When the salt is a benzoate, the molar ratio of ningnanmycin to benzoate is ≤1:1; When the salt is oxalate, the molar ratio of ningnanmycin to oxalate is ≤2:1; When the salt is a hydrochloride salt, the molar ratio of ningnanmycin to hydrochloride ion is ≤1:1; When the salt is a nitrate, the molar ratio of ningnanmycin to nitrate is ≤1:1.
[0013] Preferably, the ningnanmycin salt is prepared according to the following steps: Step 1: Adsorb the Ningnanmycin fermentation broth onto a cation exchange resin or activated carbon; Step 2: Elute with an alkaline solution and collect the eluent; Step 3: Add acid to the eluent and mix.
[0014] Preferably, the ningnanmycin content in the ningnanmycin fermentation broth is 0.01-100 g / L; And / or, the alkaline solution is selected from ammonia water or sodium hydroxide aqueous solution with a volume fraction of 0.01%-10%; And / or, in step 2, before elution, the resin is washed with water until the light transmittance is ≥40%; And / or, in step 2, elute until the ningnanmycin content is ≤0.5g / L; And / or, in step 3, the mixing is modulated until the pH change is ≤0.1; And / or, when the salt is an acetate, in step 3, the mixing is adjusted to pH ≤ 6.0; When the salt is lactate, in step 3, the mixing is adjusted to pH ≤ 6.0; When the salt is a phosphate, in step 3, the mixing is adjusted to pH ≤ 6.5; When the salt is a sulfate, in step 3, the mixture is adjusted to pH ≤ 6.5; When the salt is citrate, in step 3, the mixing is adjusted to a pH ≤ 6.0. When the salt is a benzoate, in step 3, the mixing is adjusted to pH ≤ 6.0; When the salt is oxalate, in step 3, the mixing is adjusted to pH ≤ 6.0; When the salt is a hydrochloride salt, in step 3, the mixing is adjusted to pH ≤ 6.5; When the salt is a nitrate, in step 3, the mixture is adjusted to pH ≤ 6.5.
[0015] Preferably, in step 3, the mixture is adjusted to pH ≤ 6.0.
[0016] Preferably, in step 2, the water is deionized water with a conductivity ≤10μS / cm.
[0017] Preferably, the amount of ningnanmycin in the dose of the ningnanmycin salt is 1-500 mg / L.
[0018] Preferably, the amount of ningnanmycin in the dose of the ningnanmycin salt is 100-500 mg / L.
[0019] Preferably, the ningnanmycin salt is prepared by ningnanmycin free base and acid, and the molar ratio of ningnanmycin to acid anion is ≤3:1.
[0020] Preferably, the molar ratio of ningnanmycin to acid radical is ≤1:1.
[0021] This invention provides an antiviral agent, characterized in that it is prepared from ningnanmycin salt.
[0022] This invention develops derivatives of ningnanmycin salts, including ningnanmycin acetate, ningnanmycin lactate, ningnanmycin phosphate, ningnanmycin sulfate, ningnanmycin citrate, ningnanmycin benzoate, ningnanmycin oxalate, ningnanmycin hydrochloride, and ningnanmycin nitrate. These ningnanmycin salts were applied to the control of tobacco mosaic virus. Experiments showed that, compared with ningnanmycin, ningnanmycin salts significantly improved the control effect against tobacco mosaic virus, achieving or even exceeding the control effect of the commonly used agent morpholine guanidine hydrochloride, demonstrating unexpected results and promising application prospects.
[0023] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.
[0024] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description
[0025] Figure 1 The X-ray powder diffraction (XRPD) pattern of ningnanmycin acetate is shown.
[0026] Figure 2 The image shows the X-ray powder diffraction (XRPD) pattern of ningnanmycin lactate.
[0027] Figure 3 The X-ray powder diffraction (XRPD) pattern of ningnanmycin phosphate is shown.
[0028] Figure 4 The X-ray powder diffraction (XRPD) pattern of ningnanmycin sulfate is shown.
[0029] Figure 5 The image shows the X-ray powder diffraction (XRPD) pattern of ningnanmycin citric acid.
[0030] Figure 6 The image shows the X-ray powder diffraction (XRPD) pattern of ningnanmycin benzoate.
[0031] Figure 7 The image shows the X-ray powder diffraction (XRPD) pattern of ningnanmycin oxalate.
[0032] Figure 8 The image shows the X-ray powder diffraction (XRPD) pattern of ningnanmycin hydrochloride.
[0033] Figure 9 The X-ray powder diffraction (XRPD) pattern of ningnanmycin nitrate is shown. Detailed Implementation
[0034] Unless otherwise specified, all reagents and materials used in the following examples and experimental cases are commercially available.
[0035] 1. Test materials Test plants: common tobacco ( Nicotiana Tabacum L.) is the host of the TMV system and is sown in sterilized soil.
[0036] The tested virus source was Tobacco mosaic virus (TMV), provided by the College of Plant Protection, Northwest A&F University, and isolated from single spots three times from heart-leaf tobacco.
[0037] Preparation method of TMV: Weigh about 0.5g of diseased tobacco leaves and place them in a mortar. Add 25mL of phosphate buffer solution (0.01mol / mL, pH7.0), grind, and filter through four layers of gauze. Place the filtrate at 0℃ for later use.
[0038] The preparation method of fermentation broth containing ningnanmycin is based on the fermentation method in the reference "A new agricultural antibiotic - ningnanmycin. Acta Microbiologica Sinica, 35(5): 368-374, 1995".
[0039] Example 1: Ningnanmycin acetate for the prevention and control of tobacco mosaic virus 1. The ningnanmycin salt used in this embodiment is ningnanmycin acetate, and its preparation method is as follows: (1) The fermentation broth containing 5 g / L of ningnanmycin was adsorbed onto a 724 type cation exchange resin using conventional techniques; (2) Clean the resin with deionized water (conductivity ≤10μS / cm), the transmittance of the effluent is 54%; (3) Elute with 0.4% sodium hydroxide until the concentration reaches 0.5 g / L, then stop and collect the eluent; (4) Add acetic acid to the eluent to make the molar ratio of ningnanmycin to acetic acid 1:1.9. After mixing and preparing, take two samples for testing. The pH value was 5.9 in both cases. (5) Ningnanmycin acetate was obtained by spray drying at a temperature of 95°C inside the tower.
[0040] Ningnanmycin acetate is an amorphous substance, and its X-ray powder diffraction (XRPD) pattern is shown below. Figure 1 As shown.
[0041] Ningnanmycin acetate was dissolved in 0.1% Tween-80 aqueous solution to prepare 100 mg / L and 500 mg / mL ningnanmycin solutions.
[0042] In other embodiments, the ningnanmycin content in the fermentation broth in step (1) can be adjusted within the range of 0.01-100 g / L, and the adsorption resin can be selected as cation exchange resin, activated carbon or macroporous resin; the alkaline solution in step (3) can be adjusted to 0.01%-10% ammonia water and sodium hydroxide solution, and eluted to a content ≤0.5 g / L; the acetic acid in step (4) can be adjusted to a 5%-34% acetic acid solution, so that the molar concentration of ningnanmycin to acetic acid is ≤1:1; the drying temperature in the tower in step (5) can be adjusted within the range of 80-150℃.
[0043] 2. Experimental Methods (1) Control necrotic spot method: Select healthy tobacco seedlings with 5-6 leaves, uniform growth, and vigorous growth. After treating with Ningnanmycin acetate solution for 48 h, inoculate them by friction inoculation. The inoculation concentration is 1:50 (w / v). Four plants are treated per treatment, and the treatment is repeated 3 times. The disease incidence is checked 5 days after inoculation, and the number of necrotic spots and control effect are calculated.
[0044] (2) Two tobacco leaves were surveyed for each potted plant and the survey was conducted in stages.
[0045] Disease severity grading method: a) Level 0: The entire plant is disease-free; b) Grade 1: Clear veins in the heart leaves or slight electrical activity in the flowers; no obvious dwarfing in diseased plants; c) Level 3: 1 / 3 of the leaves are mosaic but not deformed, or the diseased plant is stunted to more than 3 / 4 of its normal height; d) Level 5: 1 / 3 to 1 / 2 of the leaves are mosaic or a few leaves are deformed, or the main vein turns black, or the diseased plant is stunted to 2 / 3 to 3 / 4 of the normal plant height; e) Level 7: 1 / 2 to 2 / 3 of the leaves are mosaic, deformed, or have necrosis of the main and lateral veins, or the diseased forest is dwarfed to 1 / 2 to 2 / 3 of the normal plant height; f) Level 9: All leaves of the plant are mottled, severely deformed or necrotic, or the diseased forest is dwarfed to more than 1 / 2 of the normal plant height.
[0046] (3) Calculation method Leaf damage index = Adult insect control efficacy (%) = 3. Effects of controlling tobacco mosaic virus In this embodiment, ningnanmycin acetate showed a control efficacy of 47.01% against tobacco mosaic virus at a dosage of 100 mg / L ningnanmycin and 74.28% at a dosage of 500 mg / L ningnanmycin, which is far superior to the control efficacy of ningnanmycin and morpholine guanidine hydrochloride.
[0047] Example 2: Ningnanmycin lactate for the prevention and control of tobacco mosaic virus 1. The method is the same as in Example 1, except for the preparation method of ningnanmycin salt.
[0048] The ningnanmycin salt used in this embodiment is ningnanmycin lactate, and its preparation method is as follows: (1) The fermentation broth containing 5 g / L of ningnanmycin was adsorbed onto 732 resin using conventional techniques; (2) Clean the resin with deionized water (conductivity ≤10μS / cm), the transmittance of the effluent is 40%; (3) Elute with 2% ammonia water until the content is 0.5 g / L, and collect the eluent; (4) Add lactic acid to the eluent to make the molar concentration of ningnanmycin to lactic acid 1:1.5. After mixing and preparation, the pH was measured twice and both times it was 5.8. (5) Ningnanmycin lactate was obtained by spray drying at a temperature of 100°C inside the tower.
[0049] Ningnanmycin lactate is an amorphous substance, and its XRPD spectrum is shown below. Figure 2 As shown.
[0050] In other embodiments, the lactic acid in step (4) can also be adjusted to a lactic acid solution with a concentration of 5%-88%.
[0051] 2. Effects on the prevention and control of tobacco mosaic virus: In this embodiment, ningnanmycin lactate showed a control efficacy of 44.93% against tobacco mosaic virus at a dosage of 100 mg / L ningnanmycin and 65.79% at a dosage of 500 mg / L ningnanmycin, which was significantly higher than the control efficacy of ningnanmycin and reached a control efficacy comparable to that of morpholine guanidine hydrochloride.
[0052] Example 3: Ningnanmycin phosphate for the prevention and control of tobacco mosaic virus 1. The method is the same as in Example 1, except for the preparation method of ningnanmycin salt.
[0053] The ningnanmycin salt used in this embodiment is ningnanmycin phosphate, and its preparation method is as follows: (1) The fermentation broth containing 0.01 g / L of ningnanmycin was adsorbed onto macroporous 110 resin using conventional techniques; (2) Clean the resin with deionized water (conductivity ≤10μS / cm), and the transmittance of the effluent is 55%; (3) Elute with 0.5% sodium hydroxide solution until the concentration is 0.4 g / L, and collect the eluent; (4) Add phosphoric acid to the eluent to make the molar ratio of ningnanmycin to phosphoric acid 1:1, mix well, and the pH of the two samples is 6.1. (5) Ningnanmycin phosphate was obtained by spray drying at a temperature of 110°C inside the tower.
[0054] Ningnanmycin phosphate is an amorphous substance, and its XRPD spectrum is shown below. Figure 3 As shown.
[0055] 2. Effects on the prevention and control of tobacco mosaic virus: In this embodiment, ningnanmycin phosphate showed a control efficacy of 47.01% against tobacco mosaic virus at a dosage of 100 mg / L and 64.13% at a dosage of 500 mg / L, which was significantly higher than the control efficacy of ningnanmycin and reached a control efficacy comparable to that of morpholine guanidine hydrochloride.
[0056] Example 4: Ningnanmycin sulfate for the prevention and control of tobacco mosaic virus 1. The method is the same as in Example 1, except for the preparation method of ningnanmycin salt.
[0057] The ningnanmycin salt used in this embodiment is ningnanmycin sulfate, and its preparation method is as follows: (1) The fermentation broth containing 100 g / L of ningnanmycin was adsorbed onto 732 cation exchange resin using conventional techniques; (2) Clean the resin with deionized water (conductivity ≤10μS / cm), the light transmittance is 49%; (3) Elute with 2% ammonia water until the content is 0.5 g / L, and collect the eluent; (4) Add sulfuric acid to the eluent to make the molar ratio of ningnanmycin to sulfuric acid 1:0.70. Add the sulfuric acid while stirring and mixing evenly. After preparation, the pH was measured twice and both times it was 6.0. (5) Ningnanmycin sulfate was obtained by spray drying at a temperature of 85°C inside the tower.
[0058] Ningnanmycin sulfate is an amorphous substance, and its XRPD spectrum is shown below. Figure 4 As shown.
[0059] 2. Effects on the prevention and control of tobacco mosaic virus: In this embodiment, ningnanmycin sulfate showed a control efficacy of 44.23% against tobacco mosaic virus at a dosage of 100 mg / L ningnanmycin and 64.13% at a dosage of 500 mg / L ningnanmycin, which was significantly higher than the control efficacy of ningnanmycin alone and reached a control efficacy comparable to that of morpholine guanidine hydrochloride.
[0060] Example 5: Ningnanmycin citrate for the prevention and control of tobacco mosaic virus 1. The method is the same as in Example 1, except for the preparation method of ningnanmycin salt.
[0061] The ningnanmycin salt used in this embodiment is ningnanmycin citrate, and its preparation method is as follows: (1) The fermentation broth containing 50 g / L of ningnanmycin was adsorbed onto cation exchange resin 732 using conventional techniques; (2) Clean the resin with deionized water (conductivity ≤10μS / cm), the transmittance of the effluent is 40%; (3) Elute with 3% ammonia water until the content is 0.2 g / L, and collect the eluent; (4) Add citric acid to the eluent to make the molar ratio of ningnanmycin to citric acid 1:1.9, stir and mix evenly, and the pH was 5.9 in two tests after preparation; (5) Ningnanmycin citrate was obtained by spray drying at a temperature of 95°C inside the tower.
[0062] Ningnanmycin citrate is an amorphous substance, and its XRPD spectrum is shown below. Figure 5 As shown.
[0063] In other embodiments, in step (4), the citric acid can be adjusted to a lemon solution with a concentration of 5-60%, so that the molar concentration of ningnanmycin to citric acid is ≤1:1.
[0064] 2. Effects on the prevention and control of tobacco mosaic virus: In this embodiment, ningnanmycin citrate, at a dosage of 500 mg / L of ningnanmycin, showed a control efficacy of 61.60% against tobacco mosaic virus, which was significantly higher than that of ningnanmycin alone, and reached a control efficacy comparable to that of morpholine guanidine hydrochloride.
[0065] Example 6: Ningnanmycin benzoate for the prevention and control of tobacco mosaic virus 1. The method is the same as in Example 1, except for the preparation method of ningnanmycin salt.
[0066] The ningnanmycin salt used in this embodiment is ningnanmycin benzoate, and its preparation method is as follows: (1) The fermentation broth containing 35 g / L of ningnanmycin was adsorbed onto activated carbon using conventional techniques; (2) Clean the resin with deionized water (conductivity ≤10μS / cm), the transmittance of the effluent is 48%; (3) Elute with 1% ammonia water until the content is 0.5 g / L, and collect the eluent; (4) Add benzoic acid to the eluent to make the molar ratio of ningnanmycin to benzoic acid 1:2, mix and adjust evenly, and the pH is 5.6 in two tests after adjustment.
[0067] (5) Ningnanmycin benzoate was obtained by spray drying at a temperature of 125°C inside the tower.
[0068] Ningnanmycin benzoate is an amorphous substance, and its XRPD spectrum is as follows: Figure 6 As shown.
[0069] In other embodiments, in step (4), benzoic acid can also be adjusted to a benzoic acid solution with a concentration of 0.1%-0.3%, and the molar ratio of ningnanmycin to benzoic acid is ≤1:1.
[0070] 2. Effects on the prevention and control of tobacco mosaic virus: In this embodiment, ningnanmycin benzoate, at a dosage of 500 mg / L of ningnanmycin, showed a control efficacy of 60.74% against tobacco mosaic virus, which was significantly higher than that of ningnanmycin alone, and reached a control efficacy comparable to that of morpholine guanidine hydrochloride.
[0071] Example 7: Ningnanmycin oxalate for the prevention and control of tobacco mosaic virus 1. The method is the same as in Example 1, except for the preparation method of ningnanmycin salt.
[0072] The ningnanmycin salt used in this embodiment is ningnanmycin oxalate, and its preparation method is as follows: (1) The fermentation broth containing 35 g / L of ningnanmycin was adsorbed onto a 732 cation exchanger using conventional techniques; (2) The resin was cleaned with deionized water (conductivity ≤10μS / cm), and the transmittance of the effluent was 61%. (3) Elute with 3% ammonia water until the content is 0.5 g / L, and collect the eluent; (4) Add oxalic acid to the eluent to make the molar ratio of ningnanmycin to oxalic acid 1:0.8, mix and prepare evenly, and the pH is 6.0 in two tests after preparation; (5) Ningnanmycin oxalate was obtained by spray drying at a temperature of 95°C inside the tower.
[0073] Ningnanmycin oxalate is an amorphous substance, and its XRPD spectrum is shown below. Figure 7 As shown.
[0074] In other embodiments, in step (4), oxalic acid can also be adjusted to a 1%-10% oxalic acid solution so that the molar ratio of ningnanmycin to oxalic acid is ≤2:1.
[0075] 2. Effects on the prevention and control of tobacco mosaic virus: In this embodiment, ningnanmycin benzoate, at a dosage of 500 mg / L of ningnanmycin, showed a control efficacy of 48.83% against tobacco mosaic virus, which was significantly higher than that of ningnanmycin alone.
[0076] Example 8: Ningnanmycin hydrochloride for the prevention and control of tobacco mosaic virus 1. The method is the same as in Example 1, except for the preparation method of ningnanmycin salt.
[0077] The ningnanmycin salt used in this embodiment is ningnanmycin hydrochloride, and its preparation method is as follows: (1) The fermentation broth containing 100 g / L of ningnanmycin was adsorbed onto 732 cation exchange resin using conventional techniques; (2) Clean the resin with deionized water (conductivity ≤10μS / cm), the transmittance of the effluent is 42%; (3) Elute with 2% ammonia water until the content is 0.4 g / L, and collect the eluent; (4) Add hydrochloric acid to the eluent to make the molar ratio of ningnanmycin to hydrochloric acid 1:1.5, mix and prepare evenly, and the pH value is 6.2 in two tests after preparation; (5) Ningnanmycin hydrochloride was obtained by spray drying at a temperature of 105°C inside the tower.
[0078] Ningnanmycin hydrochloride is an amorphous substance, and its XRPD spectrum is as follows: Figure 8 As shown.
[0079] 2. Effects on the prevention and control of tobacco mosaic virus: In this embodiment, ningnanmycin hydrochloride, at a dosage of 500 mg / L of ningnanmycin, showed a control efficacy of 47.25% against tobacco mosaic virus, which was significantly higher than that of ningnanmycin alone.
[0080] Example 9: Ningnanmycin nitrate for the prevention and control of tobacco mosaic virus. The method is the same as in Example 1, except that the preparation method of ningnanmycin salt is different. The ningnanmycin salt used in this embodiment is ningnanmycin nitrate, and its preparation method is as follows: (1) The fermentation broth containing 10 g / L of ningnanmycin was adsorbed onto activated carbon using conventional techniques; (2) Clean the resin with deionized water (conductivity ≤10μS / cm), and the transmittance of the effluent is 50%; (3) Elute with 0.4% sodium hydroxide solution until the content is ≤0.5g / L, and collect the eluent; (4) Add nitric acid to the eluent to make the molar ratio of ningnanmycin to nitric acid 1:1.5, mix and prepare evenly, and the pH is 6.3 in two tests after preparation; (5) Ningnanmycin nitrate was obtained by spray drying at a temperature of 90°C inside the tower.
[0081] Ningnanmycin nitrate is an amorphous substance, and its XRPD spectrum is shown below. Figure 9 As shown.
[0082] 2. Effects on the prevention and control of tobacco mosaic virus: In this embodiment, ningnanmycin nitrate, at a dosage of 500 mg / L of ningnanmycin, showed a control efficacy of 47.17% against tobacco mosaic virus, which was significantly higher than that of ningnanmycin alone.
[0083] The technical solution of the present invention will be further explained through experiments below.
[0084] Experiment 1: Different Ningnanmycin Salts Used to Control Tobacco Mosaic Virus I. Experimental Methods 1. Materials and Methods 1.1 Test Materials Test plants: common tobacco ( Nicotiana Tabacum L.) is the host of the TMV system and is sown in sterilized soil.
[0085] The tested virus source was Tobacco mosaic virus (TMV), provided by the College of Plant Protection, Northwest A&F University, and isolated from single spots three times from heart-leaf tobacco.
[0086] 1.2 Test Methods 1.2.1 Preparation of TMV Weigh about 0.5g of diseased tobacco leaves and place them in a mortar. Add 25mL of phosphate buffer solution (0.01mol / mL, pH 7.0), grind them, and filter them through four layers of gauze. Store the filtrate at 0℃ for later use.
[0087] 2 Experimental Design 2.1 Test reagents Table 1. Names and concentrations of the test reagents 2.2 Control reagent The 96% morpholine guanidine hydrochloride technical grade was purchased from Shaanxi Yingxin Biotechnology Co., Ltd. Moroxyline guanidine hydrochloride, also known as "virazole," is a broad-spectrum, low-toxicity antiviral agent.
[0088] 2.3 Processing 2.3.1 Dosage setting Ten different drugs and the control drug morpholine guanidine hydrochloride were prepared into solutions of 100 mg / mL and 500 mg / mL using 0.1% Tween-80 aqueous solution, respectively.
[0089] 2.3.2 Experimental Repetition The experiment was repeated in 3 replicates.
[0090] 2.4 Test Methods The control necrotic spot method was used: healthy tobacco seedlings with uniform growth at the 5-6 leaf stage were selected and inoculated by friction inoculation at a concentration of 1:50 (w / v). For indoor protective testing, inoculation was performed 48 hours after uniform spraying with 100 mg / ml and 500 mg / ml of the agent, with 4 plants per treatment and 3 replicates. Disease incidence was checked on the 5th day after inoculation, and the number of necrotic spots and control efficacy were calculated. 3. Survey Two tobacco leaves were examined from each potted plant, and the survey was conducted at different levels.
[0091] Disease severity grading method: a) Level 0: The entire plant is disease-free; b) Grade 1: Clear veins in the heart leaves or slight electrical activity in the flowers; no obvious dwarfing in diseased plants; c) Level 3: 1 / 3 of the leaves are mosaic but not deformed, or the diseased plant is stunted to more than 3 / 4 of its normal height; d) Level 5: 1 / 3 to 1 / 2 of the leaves are mosaic or a few leaves are deformed, or the main vein turns black, or the diseased plant is stunted to 2 / 3 to 3 / 4 of the normal plant height; e) Level 7: 1 / 2 to 2 / 3 of the leaves are mosaic, deformed, or have necrosis of the main and lateral veins, or the diseased forest is dwarfed to 1 / 2 to 2 / 3 of the normal plant height; f) Level 9: All leaves of the plant are mottled, severely deformed or necrotic, or the diseased forest is dwarfed to more than 1 / 2 of the normal plant height.
[0092] 4. Calculation Method Leaf damage index = Adult insect control efficacy (%) = 5. Significance of differences and interpretation of their markers The DMRT test was used to compare the differences in means among multiple treatment groups, and the significance of the differences was expressed using significance markers. At the same significance level, if two groups have the same letter marker (e.g., both are "a" or both are "A"), the difference is not significant; if the letters are different (e.g., "a" and "b" or "A" and "B"), the difference is significant. Lowercase letters correspond to the 0.05 level, and uppercase letters correspond to the 0.01 level; both are used independently.
[0093] II. Experimental Results The experimental results are shown in Table 2.
[0094] Table 2. Results of the survey on the indoor control efficacy of 10 pesticides against tobacco mosaic virus (TMV) disease. As shown in Table 1, the indoor control efficacy of the 10 pesticides and the control pesticide at concentrations of 100 and 500 mg / L against tobacco mosaic virus (TMV) was 44.93%, 65.79%, 30.71%, 60.74%, 30.71%, 61.60%, 30.71%, 48.83%, 47.01%, 74.28%, 30.71%, 47.25%, 47.01%, 64.13%, 44.23%, 64.13%, 30.71%, 47.17%, 31.71%, 32.13%, 47.01%, and 64.13%, respectively.
[0095] The DMRT test results showed that, at the 0.05 and 0.01 levels, the control efficacy of the treatments of pesticides 1 (100 mg / L), 4 (500 mg / L), 5 (100 mg / L), 6 (500 mg / L), 7 (100 mg / L), 8 (100 mg / L), and 9 (500 mg / L) was not significantly different from that of the control treatment of 96% morpholine guanidine hydrochloride (100 mg / L). All of these treatments were superior to those of pesticides 2 (100 mg / L), 3 (100 mg / L), 4 (100 mg / L), 6 (100 mg / L), 9 (100 mg / L), 10 (100 mg / L), and 10 (500 mg / L). The treatment efficacy is not as good as that of the treatments using agent 1 (500 mg / L), agent 2 (500 mg / L), agent 3 (500 mg / L), agent 5 (500 mg / L), agent 7 (500 mg / L), agent 8 (500 mg / L), and 96% morpholine guanidine hydrochloride (500 mg / L).
[0096] The efficacy of pesticide 10 aqueous solution at 500 mg / L was significantly lower than that of the control pesticide and pesticides 1-9.
[0097] In summary, the control effect of the ningnanmycin salts of this invention against tobacco mosaic virus is significantly better than that of ningnanmycin. Specifically, the control effects of agent 1 (ningnanmycin lactate) aqueous solution 500 mg / L, agent 2 (ningnanmycin benzoate) aqueous solution 500 mg / L, agent 3 (ningnanmycin citrate) aqueous solution 500 mg / L, agent 7 (ningnanmycin phosphate) aqueous solution 500 mg / L, and agent 8 (ningnanmycin sulfate) aqueous solution 500 mg / L are close to or reach the control effect of 96% morpholine guanidine hydrochloride 500 mg / L treatment. In particular, the control effect of agent 5 (ningnanmycin acetate) aqueous solution 500 mg / L exceeds that of 96% morpholine guanidine hydrochloride 500 mg / L.
[0098] As can be seen from the above embodiments and experimental examples, this invention develops derivatives of ningnanmycin salts, including ningnanmycin acetate, ningnanmycin lactate, ningnanmycin phosphate, ningnanmycin sulfate, ningnanmycin citrate, ningnanmycin benzoate, ningnanmycin oxalate, ningnanmycin hydrochloride, and ningnanmycin nitrate. When these ningnanmycin salts were applied to control tobacco mosaic virus, experiments showed that compared with ningnanmycin, the ningnanmycin salts significantly improved the control effect against tobacco mosaic virus, achieving or even exceeding the control effect of morpholine guanidine hydrochloride, achieving unexpected results and showing good application prospects.
Claims
1. Use of ningnanmycin salt in the preparation of antiviral agents for the prevention and / or treatment of tobacco mosaic virus.
2. The use according to claim 1, characterized in that, The salts include at least one of the following: acetate, lactate, phosphate, sulfate, citrate, benzoate, oxalate, hydrochloride, and nitrate.
3. The use according to claim 1, characterized in that, The salt includes at least one of the following: acetate, lactate, phosphate, sulfate, citrate, and benzoate.
4. The use according to claim 1, characterized in that, The salt is selected from at least one of monosalt, disalt, trisalt, and tetrasalt.
5. The use according to claim 1, characterized in that, The ningnanmycin salt is prepared by ningnanmycin free base and acid, and the molar ratio of ningnanmycin to acid anion is ≤3:
1.
6. The use according to claim 5, characterized in that, When the salt is an acetate, the molar ratio of ningnanmycin to acetate is ≤1:1; When the salt is a lactate, the molar ratio of ningnanmycin to lactate is ≤1:1; When the salt is a phosphate, the molar ratio of ningnanmycin to phosphate is ≤3:1; When the salt is a sulfate, the molar ratio of ningnanmycin to sulfate is ≤2:1; When the salt is citrate, the molar ratio of ningnanmycin to citrate ion is ≤1:1; When the salt is a benzoate, the molar ratio of ningnanmycin to benzoate is ≤1:1; When the salt is oxalate, the molar ratio of ningnanmycin to oxalate is ≤2:1; When the salt is a hydrochloride salt, the molar ratio of ningnanmycin to hydrochloride ion is ≤1:1; When the salt is a nitrate, the molar ratio of ningnanmycin to nitrate is ≤1:
1.
7. The use according to claim 5 or 6, characterized in that, The ningnanmycin salt was prepared according to the following steps: Step 1: Adsorb the Ningnanmycin fermentation broth onto a cation exchange resin or activated carbon; Step 2: Elute with an alkaline solution and collect the eluent; Step 3: Add acid to the eluent and mix.
8. The use according to claim 7, characterized in that: The ningnanmycin content in the fermentation broth ranges from 0.01 to 100 g / L; And / or, the alkaline solution is selected from ammonia water or sodium hydroxide aqueous solution with a volume fraction of 0.01%-10%; And / or, in step 2, before elution, the resin is washed with water until the light transmittance is ≥40%; And / or, in step 2, elute until the ningnanmycin content is ≤0.5g / L; And / or, in step 3, the mixing is modulated until the pH change is ≤0.1; And / or, when the salt is an acetate, in step 3, the mixing is adjusted to pH ≤ 6.0; When the salt is lactate, in step 3, the mixing is adjusted to pH ≤ 6.0; When the salt is a phosphate, in step 3, the mixing is adjusted to pH ≤ 6.5; When the salt is a sulfate, in step 3, the mixture is adjusted to pH ≤ 6.5; When the salt is citrate, in step 3, the mixing is adjusted to a pH ≤ 6.
0. When the salt is a benzoate, in step 3, the mixing is adjusted to pH ≤ 6.0; When the salt is oxalate, in step 3, the mixing is adjusted to pH ≤ 6.0; When the salt is a hydrochloride salt, in step 3, the mixing is adjusted to pH ≤ 6.5; When the salt is a nitrate, in step 3, the mixture is adjusted to pH ≤ 6.
5.
9. The use according to claim 1, characterized in that: The dosage of the ningnanmycin salt contains 1-500 mg / L of ningnanmycin.
10. An antiviral agent, characterized in that: It is prepared from ningnanmycin salt.
11. The antiviral agent according to claim 10, characterized in that: The ningnanmycin salt is prepared by ningnanmycin free base and acid, and the molar ratio of ningnanmycin to acid anion is ≤3:1.