Application of polymyxin B in weed control and use method

Abstract

The invention discloses application of polymyxin B in control of various weeds, and belongs to the technical field of agricultural biology. On one hand, polymyxin B can effectively inhibit growth of roots of various weeds; on the other hand, the plant leaves can be whitened or withered by spraying the fertilizer on the leaves. Experiments show that the root growth inhibition effect of weeds such as arabidopsis thaliana and barnyard grass is enhanced along with increase of the concentration of the polymyxin B, when the concentration of the polymyxin B in a plant culture medium is 10 [mu] M, the arabidopsis thaliana has a stress phenomenon, and when the concentration of the polymyxin B is increased to 50 [mu] M, leaves of the arabidopsis thaliana have a whitening phenomenon and plants die. If a leaf spraying mode is adopted, spraying of polymyxin B with the concentration of 500 [mu] M can make barnyard grass leaves whitened, and alfalfa leaves can be damaged. And through experiments, polymyxin B can whiten various weeds such as ryegrass and amaranthus retroflexus, and the scheme has high efficiency and environmental friendliness.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, and specifically relates to the application and usage method of polymyxin B in weed control. Background Art

[0002] In recent years, due to the enhanced herbicide resistance of weeds, their threat to agricultural production has become increasingly serious, often resulting in crop yield reduction. Currently, the main methods for controlling plant diseases and weeds are the use of chemical pesticides. However, the long-term use of chemical pesticides can lead to enhanced herbicide resistance of weeds and may also cause harm to the environment and non-target organisms. Therefore, the development of new, highly efficient, low-toxic, and environmentally friendly pesticides has become an urgent need in the current agricultural field.

[0003] Polymyxin B is a known antibacterial peptide used in medicine, mainly for treating infections caused by Gram-negative bacteria. Polymyxin B is a compound with antimicrobial activity produced by chemical synthesis or microbial fermentation, and its chemical structure is known and publicly available. However, its application in the agricultural field, especially as a pesticide for controlling weeds, has not been reported. The present invention for the first time discovers that polymyxin B has an inhibitory effect on various plants, especially weeds, and under specific conditions, causes albinism in weeds (such as Arabidopsis thaliana (L.) Heynh. and barnyard grass (provide English name)), which provides a new direction for its application in the agricultural field. Summary of the Invention

[0004] The object of the present invention is to provide a new application of polymyxin B in controlling weeds, specifically an innovative application of polymyxin B (Polymyxin B) from a medical source as a new biological pesticide in the albinism or damage of Arabidopsis thaliana, alfalfa, barnyard grass, and tomato leaves, so as to solve the problems existing in the prior art.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] The first part: The use of polymyxin B for the albinism of Arabidopsis thaliana

[0007] The present invention discovers that when wild-type Arabidopsis thaliana is cultured in a 1 / 2MS medium containing polymyxin B at a concentration of 0.5 μM - 1 mM, albinism appears in the leaves or other green tissues of Arabidopsis thaliana. This albinism phenomenon should be that polymyxin B interferes with the synthesis of chlorophyll, resulting in the affected absorption and conversion of light energy by plants. The concentration of polymyxin B is preferably 500 μM, 250 μM, 125 μM, 62.5 μM, 50 μM, 10 μM, 0.5 μM.

[0008] The second part: The use of polymyxin B for the albinism and stress growth of tomato leaves

[0009] The present invention discovers that when tomatoes are cultured in a 1 / 2MS medium containing polymyxin B at a concentration of 0.5 μM - 1 mM, the tomato leaves will show albinism, and as the concentration of polymyxin B in the culture dish increases, the inhibitory effect on tomato growth becomes more obvious. The concentration of polymyxin B is preferably 62.5 μM, 125 μM, or 250 μM.

[0010] Part III: Use of polymyxin B in damaging alfalfa leaves

[0011] The present invention discovers that when alfalfa is sprayed with a solution containing polymyxin B, the alfalfa leaves are damaged. The concentration of the polymyxin B solution is 500 μM.

[0012] Part IV: Use of polymyxin B in causing barnyard grass albinism

[0013] The present invention discovers that when barnyard grass is sprayed with a solution containing polymyxin B, the barnyard grass leaves show albinism. The concentration of the polymyxin B solution is 500 μM.

[0014] Advantages of the present invention:

[0015] High efficiency and broad spectrum: Polymyxin B causes leaf albinism or damage to barnyard grass of the Gramineae family, tomatoes of the Solanaceae family, Arabidopsis thaliana of the Brassicaceae family, alfalfa of the Leguminosae family, etc., such as Arabidopsis thaliana leaves, tomato leaves, alfalfa leaves, barnyard grass leaves, and various weeds such as ryegrass and redroot amaranth. \n

[0016] Environmentally friendly: Compared with traditional pesticides, it is a natural product and meets the requirements of green agriculture. Description of the drawings

[0017] Figure 1 Schematic diagram of the inhibitory effect of polymyxin B and mesotrione on 7-day-old Arabidopsis thaliana.

[0018] Figure 2 Schematic diagram of the albino effect of polymyxin B and mesotrione on 14-day-old Arabidopsis thaliana.

[0019] Figures 3 - 6 Schematic diagram of the time (24 - 168 h) dependence of the albino effect of polymyxin B and mesotrione on 7-day-old Arabidopsis thaliana.

[0020] Figure 7 Bar graph of the root length inhibition rate of polymyxin B and mesotrione on 7-day-old Arabidopsis thaliana.

[0021] Figure 8 Schematic diagram of the inhibition of root hairs of polymyxin B on 7-day-old Arabidopsis thaliana.

[0022] Figure 9Schematic diagram of the inhibitory effect of polymyxin B and mesotrione on 7-day-old alfalfa.

[0023] Figure 10 Schematic diagram of the albino effect of polymyxin B and mesotrione on 7-day-old barnyardgrass.

[0024] Figure 11 Schematic diagram of the inhibitory effect of polymyxin B on 7-day-old tomatoes. Detailed implementation manners

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below. The described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0026] For reagents or instruments whose manufacturers are not specified, they are all conventional products that can be purchased through regular channels. Unless otherwise specified, all raw materials used are commercially available products.

[0027] Example 1:

[0028] 1.1 Experimental materials

[0029] Wild-type Arabidopsis thaliana (Arabidopsis thaliana (L.) Heynh.) was selected and cultured under laboratory conditions (25°C, 70% Rh, photoperiod 12 / 12 (light / dark)).

[0030] Culture medium and culture dishes: All the culture media used in this experiment were half-strength MS medium. The culture dishes were 9-cm-diameter round culture dishes and 13*13-cm square culture dishes.

[0031] 1.2 Experimental methods

[0032] 1.2.1 Preparation of the culture medium:

[0033] Weigh 1.659 g of MS powder (without agar powder and sucrose) and 7 g of sucrose, dissolve them with distilled water and adjust the pH to 5.7 - 5.8. Then weigh 5.6 g of agar powder and add it to the flask, sterilize it at 121°C for 21 min, and pour the plate for later use.

[0034] 1.2.2 Preparation of the peptide solution

[0035] Dissolve the weighed polymyxin B in sterile water, and then prepare a stock solution with a concentration of 1 mM using sterile water. Aliquot the prepared polymyxin B solution into 2 mL EP tubes and store at -20°C. When using it later, dilute the stock solution with sterile water to the target concentration (such as 250 μM, 125 μM, 62.5 μM, 50 μM, 10 μM, 1 μM, 0.5 μM) solution.

[0036] 1.2.3 Preparation of experimental plants

[0037] Place the wild-type Arabidopsis thaliana (L.) Heynh. seeds stored at 4°C in an EP tube. Then add 75% alcohol to the EP tube until the seeds are submerged. Shake the EP tube for 1 min and then remove the alcohol. Repeat this operation twice. Add 12.5% sodium hypochlorite solution to the EP tube containing the seeds so that the 12.5% sodium hypochlorite covers the seeds, and place it on a shaker. Set the shaker to 25°C, 220 rpm, and the time to 20 min. Spread the disinfected seeds evenly on a petri dish containing 1 / 2 MS medium, and place the petri dish in an incubator at 25°C, 70% Rh, and a photoperiod of 12 / 12 (light / dark) for cultivation. Use the plants when the second leaf grows for subsequent experiments.

[0038] 1.2.4 Study on the growth inhibition phenomenon of Arabidopsis thaliana caused by polymyxin B

[0039] Plant the Arabidopsis thaliana in step 1.1 using the cultivation method in step 1.2. Select Arabidopsis thaliana (L.) Heynh. at 7-day-old or 14-day-old with similar sizes, take it out of the petri dish, place it in a 1 / 2 MS medium with the drug, and cultivate it in an incubator at 25°C, 70% Rh, and a photoperiod of 12 / 12 (light / dark). Take pictures and record, and calculate the root length of the plant and its inhibition rate. Figure 1 It can be clearly observed that after 7 days of treatment with polymyxin B and mesotrione at the same concentration, the polymyxin B group hardly grew at concentrations of 250 μM, 125 μM, and 62.5 μM. The mesotrione group hardly grew at concentrations of 250 μM and 125 μM but grew at a concentration of 62.5 μM. Figure 2 It was observed that 14-day-old Arabidopsis thaliana showed albino phenomenon earlier in the polymyxin B group when treated with the same concentration of polymyxin B and mesotrione at 250 μM, 125 μM, and 62.5 μM. Figures 3 - 6 It can be observed that Arabidopsis thaliana showed albino phenomenon at the 24th hour when treated with polymyxin B at a concentration of 50 μM, and as time passed, the plants hardly grew. Figure 3 For 24 - 48 h, Figure 4 For 60 - 84 h,Figure 5 It was 96 - 120 h, Figure 6 and it was 132 - 168 h, indicating that the albino phenomenon and growth inhibition phenomenon of polymyxin B were better than those of mesotrione at low concentrations. Figure 7 It can be observed that there was almost no difference in the root length inhibition rate of Arabidopsis thaliana under the treatments of 1 μM, 10 μM, and 50 μM of mesotrione. However, in the polymyxin B group, the root length inhibition rate of Arabidopsis thaliana increased with the increase in concentration, and the root length inhibition rate of the polymyxin B group was greater than that of the mesotrione group at the same concentration. Figure 8 It can be observed that after 7 days of treatment at 7 days old, compared with the control group, with the increase in the concentration of polymyxin B in the culture medium, the root length and root hair length of Arabidopsis thaliana decreased significantly, indicating that polymyxin B inhibited the growth of Arabidopsis thaliana root hairs.

[0040] Inhibition rate of Arabidopsis thaliana root length: Root length inhibition rate (%) = (Control - Treatment) / Control × 100%.

[0041] Example 2:

[0042] 2.1 Experimental materials

[0043] Wild - type alfalfa (Medicago sativa L.) was selected and cultured in a greenhouse at a temperature of 22°C and a humidity of 60%.

[0044] 2.2 Experimental methods

[0045] 2.2.1 Preparation of peptide solution

[0046] The weighed polymyxin B was dissolved in sterile water, and then a stock solution with a concentration of 1 mM was prepared with sterile water. The prepared polymyxin B solution was dispensed into 2 - mL EP tubes and stored at - 20°C. When used later, the stock solution was diluted to a solution of 500 μM with sterile water.

[0047] 2.2.2 Preparation of experimental plants

[0048] After filling the flowerpots with substrate soil, 15 wild - type alfalfa (Medicago sativa L.) seeds stored at 4°C were evenly sown in each pot and covered with substrate soil to a depth of about 1 cm. Subsequently, water was added until saturation, and the next experiment was started when they grew to 1 - week - old.

[0049] 2.2.3 Study on the leaf damage phenomenon of alfalfa caused by polymyxin B

[0050] The alfalfa (Medicago sativa L.) in step 2.1 was planted using the cultivation method in step 2.2.2. Alfalfa (Medicago sativa L.) at 7 - day - old with similar sizes was selected and sprayed once every 24 h, 0.4 mL each time, for a total of 7 days, and the photos were taken for recording. SeeFigure 9 , after 7 days of treatment with the 500 μM mesotrione and polymyxin B group, albino phenomenon occurred in the mesotrione group, and damaged leaves appeared in the polymyxin B group of plants.

[0051] Example 3:

[0052] 3.1 Experimental materials

[0053] Wild type barnyardgrass (Echinochloa crus-galli (L.) P. Beauv.) was selected and cultivated in a greenhouse at a temperature of 22 °C and a humidity of 60%.

[0054] 3.2 Experimental methods

[0055] 3.2.1 Preparation of peptide solution

[0056] The weighed polymyxin B was dissolved in sterile water, and then a stock solution with a concentration of 1 mM was prepared with sterile water. The prepared polymyxin B solution was dispensed into 2 mL EP tubes and stored at -20 °C. When used later, the stock solution was diluted to a solution with the target concentration with sterile water. The concentration of the polymyxin B solution was 500 μM.

[0057] 3.2.2 Preparation of experimental plants

[0058] After filling the substrate soil into pots, 15 seeds of wild type barnyardgrass (Echinochloa crus-galli (L.) P. Beauv.) stored at 4 °C were evenly sown in each pot and covered with the substrate soil to a depth of about 2 cm. Subsequently, water was added until saturation, and the next experiment was started when it grew to 1-week-old.

[0059] 3.2.3 Study on the albino phenomenon of barnyardgrass caused by polymyxin B

[0060] The wild type barnyardgrass (Echinochloa crus-galli (L.) P. Beauv.) in step 3.1 was planted using the cultivation method in step 3.2.2. Seven-day-old wild type barnyardgrass (Echinochloa crus-galli (L.) P. Beauv.) with similar sizes was selected, sprayed once every 24 h, 0.4 mL each time, for a total of 7 days, and photographed and recorded. See Figure 10 , after 7 days of treatment with the 500 μM mesotrione and polymyxin B group, albino phenomenon occurred in both the polymyxin B group and the mesotrione group.

[0061] Example 4:

[0062] 4.1 Experimental materials

[0063] The tomato (Solanum lycopersicum L.) variety "Little Tom" was selected as the experimental subject and cultured under laboratory conditions (25°C, 70% Rh, photoperiod 12 / 12 (light / dark)) after vernalization. Medium and Petri dish: All the media used in this experiment were 1 / 2MS media.

[0064] 4.2 Experimental methods

[0065] 4.2.1 Preparation of the medium:

[0066] Weigh 1.659 g of MS powder (without agar powder and sucrose) and 7 g of sucrose, dissolve them in distilled water and adjust the pH to 5.7 - 5.8. Then weigh 5.6 g of agar powder and add it to the flask, autoclave at 121°C for 21 minutes, and pour the plates for later use.

[0067] 4.2.2 Preparation of the peptide solution

[0068] Dissolve the weighed polymyxin B in sterile water, and then prepare a stock solution with a concentration of 1 mM using sterile water. Aliquot the prepared polymyxin B solution into 2 mL EP tubes and store it at -20°C. When using it later, dilute the stock solution to the target concentration (such as 250 μM, 125 μM, 62.5 μM) with sterile water.

[0069] 4.2.3 Preparation of experimental plants

[0070] Place the tomato (Solanum lycopersicum L.) seeds stored at 4°C in an EP tube, then add 75% alcohol to the EP tube until the seeds are submerged. Shake the EP tube for 1 minute to remove the alcohol, and repeat this operation twice. Add 12.5% sodium hypochlorite solution to the EP tube containing the seeds so that the seeds are covered by the 12.5% sodium hypochlorite. Place the seeds on a shaker set at 25°C, 220 rpm for 20 minutes. Spread the disinfected seeds evenly on a Petri dish containing 1 / 2MS medium, and place the Petri dish in an incubator at 25°C, 70% Rh, photoperiod 12 / 12 (light / dark) for cultivation. Use the germinated seeds for subsequent experiments.

[0071] 4.2.4 Study on the growth inhibition phenomenon of tomato caused by polymyxin B

[0072] Plant the tomatoes in step 4.1 using the cultivation method in step 4.2.3. Select 7-day-old tomatoes (Solanum lycopersicum L.) of similar size, take them out of the Petri dish, place them on the 1 / 2MS medium with the drug, and cultivate them in an incubator at 25°C, 70% Rh, photoperiod 12 / 12 (light / dark), and take pictures for recording. See Figure 11, it can be observed that after 14 days of treatment with 62.5 μM, the tomato leaves will show albinism, and the root length is inhibited. As the concentration of polymyxin B in the culture medium increases, the growth inhibition effect of tomatoes becomes stronger.

[0073] Finally, it should be stated again that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of polymyxin B as a herbicide, characterized in that, Polymyxin B can cause albinism or leaf damage in plants.

2. The method of using polymyxin B as a herbicide according to claim 1, characterized in that Add 0.5 μM - 1 mM low-concentration polymyxin B to 1 / 2 MS medium to make a polypeptide culture dish, and transplant the plants into the culture dish for cultivation and growth.

3. The usage method according to claim 2, wherein, The concentrations of the polymyxin B are 500 μM, 250 μM, 125 μM, 62.5 μM, 50 μM, 10 μM, 0.5 μM.

4. The usage method according to claim 3, characterized in that, The plants include Arabidopsis thaliana.

5. The usage method according to claim 2, characterized in that, The concentrations of the polymyxin B are 62.5 μM, 125 μM, 250 μM.

6. The usage method according to claim 5, characterized in that, The plants include tomatoes.

7. The method of using polymyxin B as claimed in claim 1 for herbicidal application, characterized in that, Configure polymyxin B into a solution and spray it on the plants for treatment.

8. The usage method according to claim 7, characterized in that, The concentration of the solution is 500 μM.

9. The method of use according to claim 7 or 8, characterized in that The plants include alfalfa and barnyard grass.

Images