Application of Vitamin K3 in the Preparation of Plant Pathogen Inhibitors

Abstract

The present invention relates to the use of vitamin K3 in the preparation of plant pathogen inhibitors. The vitamin K3 of the present invention can inhibit the growth of various plant pathogens, including tobacco bacterial wilt pathogen, citrus canker pathogen, rice bacterial leaf streak, cucumber Rhizoctonia solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium graminearum, cucumber Botrytis cinerea, Aspergillus niger, Verticillium foetida, Colletotrichum gloeosporioides, Corynespora multilocus, and Pseudomonas aeruginosa. The vitamin K3 can be used in the development of biopesticides and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of plant disease prevention and control, and particularly relates to the application of vitamin K3 in the preparation of plant pathogen inhibitors. Background Art

[0002] There are over 100,000 species of fungi in nature, of which over 8,000 are harmful pathogens. The diseases they cause not only account for 80% of all plant diseases but are also extremely harmful. Some fungi can cause mold and spoilage in agricultural products, wood, and food; some parasitize humans and animals, causing skin diseases; and some even produce toxic substances such as aflatoxin, posing a health risk to both humans and animals. Furthermore, fungi reproduce rapidly and spread rapidly, making them difficult to eliminate. Currently, the primary control method for plant pathogens remains chemical pesticides. However, due to the various drawbacks of chemical pesticide use and the need for large-scale production of pollution-free agricultural products, there is an urgent need for low-toxicity, low-residue, pollution-free, and environmentally friendly plant inhibitors. Summary of the Invention

[0003] In view of this, the object of the present invention is to provide a new use of vitamin K3.

[0004] The novel use of vitamin K3 provided by the present invention is the use of vitamin K3 in the preparation of plant pathogen inhibitors.

[0005] The plant pathogens of the present invention include plant fungi or bacteria.

[0006] The plant pathogens include but are not limited to bacterial wilt, canker species, bacterial leaf streak, Rhizoctonia solani, Alternaria, Fusarium, Fusarium, Botrytis cinerea, Aspergillus niger, Verticillium wilt, Colletotrichum gloeosporioides, Polymyxa spp., and Polytrichosporium truncatum.

[0007] The plants described in the present invention are crops, including food crops, cash crops (oil crops, vegetable crops, flowers, grasses, trees, fruits), industrial raw material crops, feed crops, medicinal crops, etc.; the food crops include but are not limited to rice, corn, beans, potatoes, barley, broad beans, wheat, cereals, etc.; the oil crops include but are not limited to tobacco, oilseeds, turnips, mustard, peanuts, sesame, hemp, sunflower, etc.; the vegetable crops include but are not limited to eggplant, radish, cabbage, celery, leek, garlic, onion, carrot, melon, lotus, Jerusalem artichoke, sword bean, coriander, lettuce, daylily, pepper, cucumber, tomato, coriander, etc.; the fruits include but are not limited to citrus, melons, mango, grapes, bananas, etc.; the medicinal crops include but are not limited to ginseng, angelica, honeysuckle, mint, mugwort, etc.

[0008] In the present invention, the plants include but are not limited to tobacco, citrus, rice, melons, cereals, eggplant, mango, grapes, etc.

[0009] The plant pathogenic bacteria of the present invention is selected from at least one of tobacco bacterial wilt pathogen, citrus canker pathogen, rice bacterial leaf streak, cucumber Rhizoctonia solani, Alternaria alternata, Fusarium solani, Fusarium oxysporum, Fusarium graminearum, cucumber botrytis cinerea, Aspergillus niger, Verticillium wilt, Colletotrichum gloeosporioides, Polymyxa sporeans, and Pseudomonas aeruginosa.

[0010] The dosage form of the plant pathogen inhibitor of the present invention is at least one selected from the group consisting of dust, wettable powder, granule, water-dispersible granule, suspension, emulsifiable concentrate, microemulsion and aqueous solution.

[0011] Furthermore, the plant pathogen inhibitor is in the form of a suspension concentrate.

[0012] The effective concentration of the vitamin K3 when applied in the present invention is 10 to 500 ppm; further 50 to 150 ppm; further, the effective concentration of the vitamin K3 is 100 ppm.

[0013] The vitamin K3 of the present invention can also be used in combination with other agricultural fungicides, including but not limited to Jinggangmycin, Hymexazol, and Coronin.

[0014] The present invention also provides an agricultural fungicide composition, the components of which include but are not limited to the effective active ingredient vitamin K3, excipients, and solvents;

[0015] Further, it includes 0.1-20 wt% vitamin K3; further 0.5-5 wt%;

[0016] In the present invention, the auxiliary materials include but are not limited to dispersants, further include 0.5 to 30 wt% of dispersants, and further include 2 to 10 wt% of dispersants.

[0017] Furthermore, in the present invention, the agricultural fungicide composition also includes at least one component selected from the group consisting of validamycin, oxadixyl, and coronatine; and further, validamycin and / or oxadixyl.

[0018] The dispersant includes at least one of carboxylates, sulfonates, and phosphates.

[0019] The beneficial effects of the present invention are as follows: the vitamin K3 of the present invention can be eaten as a common medicine. Compared with common chemical pesticides, when used as a plant pathogen inhibitor, the vitamin K3 of the present invention has little impact on the environment and is less toxic, and is not likely to cause phytotoxicity to crops. The vitamin K3 of the present invention has a low dosage when applied and is not likely to produce resistance. The effective concentration of general chemical pesticides is about 1000 ppm, while the antibacterial effective concentration of the vitamin K3 of the present invention is only about 100 ppm. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The antibacterial effect diagram of the present invention and other antibacterial agents, A, B, C, D are respectively Mycorrhizal fungus, Colletotrichum gloeosporioides, Pseudomonas aeruginosa, and Fusarium oxysporum. In each figure, from left to right, they represent CK, solvent control, 1% vitamin K3 suspension, and 98% oxamectin. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0023] It should be understood that the experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0024] As used herein, the terms "comprises," "including," "having," "containing" or any other variations thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, or article that comprises a listed element is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, or article.

[0025] The 98% hydroxychloroquine soluble powder used in the present invention (Tianjin Lvheng Chemical Co., Ltd., pesticide registration certificate number: PD20190146, production date: 20211008); zhongshengmycin (effective content 3%, Fujian Kaili Biological Products Co., Ltd., pesticide registration certificate number: PD20190224; production batch number: 20210315); jinggangmycin (effective content 13%, Zhejiang Tonglu Huifeng Biotechnology Co., Ltd., pesticide registration certificate number: PD20130887; production batch number: 20220708).

[0026] Example 1

[0027] An agricultural fungicide composition comprises the following components: 1 wt% vitamin K3, 4 wt% dispersant, and the remainder water, wherein the dispersant comprises one of carboxylates, sulfonates, and phosphates.

[0028] The dispersant used in the present invention will not affect the application effect of the preparation.

[0029] Preparation method: Grind the active ingredient vitamin K3 and the dispersant, stir and add water to mix evenly to obtain a suspension.

[0030] Example 2

[0031] Antibacterial test of 1% vitamin K3 prepared in Example 1 against bacterial pathogens

[0032] Method: The activity test was carried out using OD method.

[0033] Prepare medium B: 1 g / L yeast extract, 10 g / L bacterial peptone, and 1 g / L casamino acids; dissolve in 1000 mL of water (add 15 g / L agar to the solid medium), divide into portions, and sterilize by moist heat at 121°C for 30 min.

[0034] (1) Activate the low-temperature preserved tobacco bacterial wilt pathogen under a sterile environment. Inoculate the frozen strain into B medium on a clean bench and culture at 30°C in a constant temperature shaker at 180 rpm / min until OD600 = 1.0. Streak the B medium plate and incubate in a constant temperature incubator at 30°C for 24 h to obtain a single colony. Inoculate the plate into PDA medium and culture at 25°C for 3-5 days. Detect the growth characteristics of the strain.

[0035] (2) Prepare the drug under a sterile environment. The entire experimental process should be performed aseptically. All consumables must be sterilized at high temperature and high pressure and not reused. Weigh the drug according to the set concentration and add it to a sterilized measuring cylinder. Add 30 mL of medium B to the sterilized measuring cylinder. Add 300 μL of the test drug to each bottle of medium and shake well for later use.

[0036] (3) Take a sterilized conical flask and add 20 mL of liquid medium B. Pick a well-growing colony (pink colony with a wide white band at the center and strong fluidity) from the cultured single colony plate and place it in the medium. Incubate in a constant temperature shaker at 30°C, 180 rpm / min until OD600 = 1.0 to prepare the inoculum stock solution of the bacterial solution. Add the inoculum solution at a ratio of 200:1 of drug-containing culture medium: inoculum solution and incubate in a constant temperature shaker at 180 rpm / min at 30°C.

[0037] (4) Culture at constant temperature for 24 h, measure the OD600 of the bacterial solution using a UV spectrophotometer, and calculate the growth inhibition rate of the drug.

[0038] The formula for calculating the antibacterial rate is: I = [(D0-Dt) / (D0)] × 100%

[0039] I: Growth inhibition rate

[0040] D0: Blank bacterial solution OD600

[0041] Dt: OD600 of bacterial solution treated with chemical

[0042] Table 1 The preventive effect of 1% vitamin K3 suspension of the present invention on bacterial pathogens

[0043]

[0044] Example 3

[0045] The antibacterial ability of the prepared 1% vitamin K3 suspension was tested

[0046] Method: The activity test was carried out using the growth rate method. The specific steps are as follows:

[0047] (1) Fungal strains stored at -80°C were activated under a sterile environment. Botrytis cinerea Pers.ex Fr. cucumber leaves, Rhizoctonia solani, Colletotrichum gloeosporioides and other fungi were inoculated into PDA culture medium and cultured at 25°C for 3 to 5 days. The strains were then tested based on their growth characteristics.

[0048] (2) Prepare the drug under a sterile environment: The entire process of preparing the pyridone ethanol ammonium salt suspension and the existing fungicides oxadipate and myclobutanil is performed under sterile conditions. All consumables must be sterilized at high temperature and high pressure and not reused. Weigh the drug according to the set concentration, add it to a sterilized centrifuge tube, add sterile water to make up to 10 mL, shake well and set aside.

[0049] (3) Heat and melt the prepared rye culture medium and PDA culture medium, cool to 45℃~50℃, take 1mL of each prepared sample into a culture dish, add 9mL of culture medium, shake gently, mark, and place horizontally to cool.

[0050] (4) Use a hole puncher to make concentric holes in the activated culture medium. Then, inoculate the holes onto the culture medium containing the drug and incubate the plate upside down at 25°C. When the blank mycelium has grown to cover 2 / 3 of the plate or the two bacterial circles have grown to almost touch each other, use the cross-hatch method to measure the diameter of the colony, calculate the average diameter, and calculate the control effect of the drug.

[0051] The formula for calculating the prevention effect is: I = [(D0-Dt) / (D0-4)] * 100%

[0052] I: mycelial growth inhibition rate;

[0053] D0: blank colony diameter (mm);

[0054] Dt: Diameter of drug-treated colonies in mm.

[0055] The antifungal effects of the suspension concentrate of Example 1 and the existing chemical fungicide oxazolidinone on fungi were measured, where CK represents clean water treatment. 84.82 75.22 62.97

[0056] Table 2 Antibacterial effect of vitamin K3 suspension of the present invention (%)

[0057]

[0058]

[0059] Among them, carbendazim is a common chemical control for fungal diseases in agriculture. It is widely used and has a broad spectrum of targets. A 2000-fold dilution is the recommended concentration in the product manual.

[0060] As can be seen from the above results, according to the results of the indoor plate antibacterial test, compared with commonly used chemical controls, the 1% vitamin K3 suspension concentrate obtained by the present invention can inhibit a variety of pathogens such as Rhizoctonia solani, Colletotrichum gloeosporium, and Botrytis cinerea, with an antibacterial effect close to that of existing fungicides. At the same time, it has no significant inhibitory effect on some Fusarium graminearum, Fusarium oxysporum, and Fusarium solani of cucurbits. The synthesis method of the 1% vitamin K3 preparation of the present invention has the advantages of mild conditions and simple operation, and is suitable for industrial large-scale production requirements.

[0061] Example 4

[0062] Antibacterial experiment on 1% vitamin K3 suspension prepared in Example 1

[0063] Method: Indoor pot experiment was used to determine the efficacy of the pesticide on tobacco bacterial wilt.

[0064] (1) Tobacco plant cultivation: Tobacco seeds were sown in peat substrate and transplanted into pots when they were 40 days old. When the tobacco plants were 50 days old, seedlings with uniform growth were selected for testing.

[0065] (2) Prepare the agents according to the experimental design concentration, measure each agent according to the calculated amount, and then dilute to the required volume with sterilized water, shake well and set aside. Take a 100mL beaker for root irrigation, irrigate the roots of each plant with 100mL, and wait for the next day to inoculate the bacteria. According to the current application technology of allicin for preventing and treating bacterial wilt, allicin and Alliclin-2 are both configured into a 1% microemulsion form (according to 10% anhydrous ethanol, 5% dimethyl sulfoxide, 15% castor oil polyoxyethylene ether, 5% glycerol, 1% garlic extract / Allicin-2 mixture, water is supplemented to 100%, for configuration), so as to better promote and improve the application value of the invention.

[0066] (3) 10 h before inoculation, activate the R. solanacearum strain of tobacco and take 200 mL of medium B. Pick a single activated colony for propagation. When the R. solanacearum grows to OD600 = 1.0 (concentration of 1 × 109 cfu / mL), dilute it 10-fold with sterile water. Take a 10 mL centrifuge tube and inoculate 10 mL of the strain to each plant's root. Keep the tobacco plant substrate moist during the disease period. The greenhouse temperature is set at 30°C, the humidity is 85%, and the light period is 16 h and the dark period is 8 h.

[0067] (4) After inoculation, observe the disease condition of tobacco plants every day. When tobacco plants begin to develop disease, investigate and record the disease condition. Calculate the disease condition according to the formula: disease incidence = (number of diseased plants / total number of plants investigated) × 100%. Calculate the disease index according to the formula: disease index = ∑ (disease level of diseased plants × number of plants with the disease level) / (total number of plants investigated × highest disease level) × 100%. Calculate the relative prevention effect according to the formula: relative prevention effect (%) = (disease index of control group - disease index of treatment group) / disease index of control group × 100%. The disease grade is based on the indoor tobacco bacterial wilt disease grading standard (in units of plants):

[0068] Level 0: No symptoms of disease in tobacco seedlings; Level 1: 1 to 2 leaves are half-wilted, or the chlorotic streaks on the stem are less than one-third of the plant height; Level 2: 2 to 3 leaves are wilted, or the chlorotic streaks on the stem are between one-third and one-half of the plant height; Level 3: 1 to 2 healthy leaves, or the chlorotic streaks on the stem are between one-half and two-thirds of the plant height; Level 4: The tobacco plant has no healthy leaves and is basically dead, or the chlorotic streaks on the stem exceed two-thirds of the plant height.

[0069] Table 3 The preventive effect of 1% vitamin K3 suspension of the present invention on tobacco bacterial wilt potted plants

[0070]

[0071] Note: 3% Zhongshengmycin diluted 500 times is the recommended concentration in the product manual.

[0072] Example 5

[0073] Antibacterial experiment on 1% vitamin K3 suspension prepared in Example 1

[0074] Method: Indoor pot experiment was used to determine the efficacy of the pesticide on cucumber damping-off disease.

[0075] (1) Culture: Transfer the cucumber damping-off pathogen to a PDA plate for activation for 3 days, then transfer to PDB liquid culture medium, transfer 6 plates per bottle, and culture at 25°C, 200 rpm, for 3 days;

[0076] (2) Inoculation: Crush the cultured mycelium with a mycelium crusher for 5 minutes to form a mycelium suspension. Mix the treated mycelium suspensions with a certain amount of planting soil (worm castings + vermiculite). Plant cucumber seeds of uniform size that have been germinated in advance. Add 15 mL of the diluted solution to each treatment according to the test dosage. The cucumber seeds are coated with the 11% methylpyrimidine suspension seed coating agent, a control agent for cucumber damping-off, in advance according to the required concentration of the seed coating agent. Cover the seeds with soil and culture normally. On the 4th day after the first treatment, add 15 mL of the solution to each treatment according to the test requirements. Add 15 mL of the control agent, 11% methylpyrimidine suspension seed coating agent, at a 1500-fold dilution. After the seedlings of each treatment are fully emerged, keep the seeds moist and culture until the disease investigation results are obtained.

[0077] (3) After the blank control is fully diseased, conduct a disease classification survey and calculate the disease index and control effect. Grading standards for severity of cucumber wilt, root rot and damping-off:

[0078] Level 0, no symptoms;

[0079] Level 1, mild symptoms on stems and leaves;

[0080] Level 2: The plant is slightly wilted, necrotic spots appear on the stem, and the leaves are yellowing;

[0081] Level 3, the plant is moderately wilted, and the leaves are drooping and yellowing;

[0082] Level 4: The plants wilt severely, fall over and die.

[0083] Disease index = ∑ (disease grade of diseased plants × number of plants with that grade) / (total number of plants surveyed × highest disease grade) × 100% to calculate the disease index;

[0084] Relative protective effect (%) = (disease index of control group - disease index of treated group) / disease index of control group × 100% to calculate the relative protective effect.

[0085] Table 4 Control effect of different formulas on cucumber damping-off disease

[0086]

[0087] Example 6

[0088] Based on the experimental results of vitamin K3's antibacterial activity against various pathogens, the present invention also tested the antibacterial activity of other vitamin substances, using the method described in Example 3. All B vitamins are water-soluble and can be dissolved in water to prepare a 1% formulation. Vitamin E, which is insoluble in water, was prepared using the same method as vitamin K3, as described in Example 1. Solvent controls (for vitamin K3 and vitamin E) were also included in this experiment to eliminate solvent effects and potential errors. The results are shown in Table 5.

[0089] Table 5 Antibacterial effect of other vitamins of the present invention (%)

[0090]

[0091]

[0092] Note: Negative values ​​of the control effect in the table mean that the fungus growth is promoted but not inhibited.

[0093] Based on the above test results, it is proved that the 1% vitamin K3 of the present invention has the advantages of strong antibacterial activity and a broader antibacterial spectrum, and can be used to prepare antibacterial agents. Other vitamins, such as vitamin B1, vitamin B2, vitamin B3, vitamin B4, vitamin B5, vitamin B6, etc., do not have an antibacterial effect on plant pathogens, indicating that the antibacterial effect of vitamin K3 is specific. At the same time, the synthesis method of the present invention has the advantages of mild conditions and simple operation. Compared with the currently common fungicide 98% oxadiazon, which has an effective range of 1000ppm to 500ppm, the preparation of the present invention has an effective range of 100ppm, a lower usage concentration, and reduces the resistance of crops to the compound. At the same time, the active ingredients of the preparation of the present invention are edible ingredients, which can reduce environmental pollution and pesticide residues.

[0094] Example 7

[0095] Referring to Example 5, an indoor potted cucumber damping-off efficacy test was conducted. A 1% vitamin K3 suspension concentrate was formulated with the common fungicides 98% oxadiazon and 13% jinggangmycin, respectively. The test results are shown in Table 6. (Note: Because the initial efficacy of 1% vitamin K3 diluted 100-fold and 98% oxadiazon diluted 1000-fold was higher, to more clearly verify the synergistic effect of the combined formulations, 1% vitamin K3 was diluted 200-fold and 98% oxadiazon diluted 1500-fold.)

[0096] Table 6 Control effect of different formulas on cucumber damping-off disease

[0097]

[0098] 98% oxadiazol and 13% jinggangmycin are both common control agents for damping-off disease. The results of this test show that when 98% oxadiazol diluted 1500 times and 13% jinggangmycin diluted 1500 times are used alone, the protective efficacy is low, at 45.5% and 40.3%, respectively. After being used in combination with 1% vitamin K3 of the present invention, the protective efficacy can be increased by about 40%, indicating that the preparation of the present invention has a significant synergistic effect after being used in combination with oxadiazol and jinggangmycin, and has great application potential.

[0099] The foregoing examples are merely illustrative and serve to illustrate some of the features of the method of the present invention. The appended claims are intended to claim the widest possible scope that can be envisioned, and the embodiments presented herein are merely illustrative of selected implementations according to a combination of all possible embodiments. Therefore, it is the applicant's intention that the appended claims are not limited by the selection of examples illustrating the features of the present invention. Some numerical ranges used in the claims also include subranges therein, and variations in these ranges should also be interpreted as being covered by the appended claims where possible.

Claims

1. Use of vitamin K3 in the preparation of plant pathogen inhibitors, characterized in that: The plant pathogen is selected from at least one of Alternaria alternata, Fusarium oxysporum, Fusarium graminearum, Aspergillus niger, Verticillium foetida, Colletotrichum gloeosporioides, and Polytrichomonas gloeosporioides.

2. The use according to claim 1, characterized in that The plant is selected from at least one of tobacco, citrus, rice, melons, cereals, eggplant, mango, and grapes.

3. The use according to claim 1, characterized in that The dosage form of the plant pathogen inhibitor is at least one selected from the group consisting of dust, wettable powder, granule, water-dispersible granule, suspension, emulsifiable concentrate, microemulsion and aqueous solution.

4. The use according to claim 3, characterized in that The dosage form of the plant pathogen inhibitor is a suspension concentrate.

5. The use according to claim 1, characterized in that The effective concentration of the vitamin K3 is 10 to 500 ppm.

6. The use according to claim 5, characterized in that The effective concentration of the vitamin K3 is 50 to 150 ppm.

7. An agricultural fungicide composition, characterized in that Its components include effective active ingredient vitamin K3, excipients, and solvents; Including 0.1-20wt% vitamin K3; The auxiliary material includes a dispersant, which includes 0.5 to 30 wt% of the dispersant.

8. The agricultural fungicide composition according to claim 7, characterized in that The dispersant includes at least one of carboxylates, sulfonates, and phosphates.

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