Application of a cyclic (D-leucine-L-proline) dipeptide and its plant antibacterial agent

Abstract

The present invention relates to an application of a cyclic (D-leucine-L-proline) dipeptide and a plant antibacterial agent thereof, wherein the application is as a plant antibacterial agent. The application of a cyclic (D-leucine-L-proline) dipeptide described in the present invention is the first to discover that the cyclic (D-leucine-L-proline) dipeptide has a significant inhibitory effect on Ralstonia solanacearum and is applied to a plant antibacterial agent, providing a new prevention and control method for diseases caused by Ralstonia solanacearum in plants, which is beneficial to ensuring the normal growth of plants.

Description

Technical Field

[0001] The invention relates to the field of plant disease prevention and control, and in particular to the application of a cyclic (D-leucine-L-proline) dipeptide and a plant antibacterial agent. Background Art

[0002] Morphological characteristics of Ralstonia solanacearum The bacterial morphology is oblong, nearly rod-shaped, and a Gram-negative bacterium. It is a devastating soil-borne disease that can cause bacterial wilt in many plants, specifically involving more than 300 species of plants in 44 families, among which Solanaceae crops are the most seriously affected. Ralstonia solanacearum is mainly distributed in tropical, subtropical and temperate regions, and has serious occurrences in various provinces and cities in southern my country. When the disease occurs, the plant vascular bundle is destroyed, the stems and leaves wilt vertically or even die completely, and the host mortality rate is extremely high. In severe cases, large-scale crop deaths or even crop failures can be caused, resulting in reduced yields and quality, which seriously affects the healthy development of the crop industry. At present, the prevention and control of bacterial wilt is still a difficult problem, and it is necessary to find a safer and more effective compound to inhibit Ralstonia solanacearum and thus prevent and treat bacterial wilt.

[0003] According to existing research, cyclo(D-leucine-L-proline) dipeptide can be isolated from the endophytic fungus GT20036029 of the semi-mangrove medicinal plant Hibiscus tiliaceus. There is no report on the antibacterial effect of cyclo(D-leucine-L-proline) dipeptide on plants. Summary of the invention

[0004] Based on this, the purpose of the present invention is to provide an application of a cyclic (D-leucine-L-proline) dipeptide and a plant antibacterial agent thereof. The present invention is the first to apply a cyclic (D-leucine-L-proline) dipeptide to a plant antibacterial agent, which is beneficial to ensure the normal growth of plants.

[0005] The invention discloses an application of a cyclic (D-leucine-L-proline) dipeptide as a plant antibacterial agent.

[0006] The application of a cyclic (D-leucine-L-proline) dipeptide described in the present invention is the first to discover that the cyclic (D-leucine-L-proline) dipeptide has a significant inhibitory effect on Ralstonia solanacearum and applies it to plant antibacterial agents, providing a new prevention and control method for plant diseases caused by Ralstonia solanacearum, which is beneficial to ensuring the normal growth of plants.

[0007] Furthermore, the application method comprises the following steps: soaking the plants in the cyclo(L-leucine-D-proline) dipeptide solution before planting.

[0008] Furthermore, the application method comprises the following steps: after planting, the cyclo(L-leucine-D-proline) dipeptide solution is injected into the root system of the plant.

[0009] Furthermore, the concentration of the cyclo(L-leucine-D-proline) dipeptide solution is 10 mg / L.

[0010] Furthermore, the plants include crops of the Solanaceae family.

[0011] Furthermore, the frequency of the perfusion is 18-20 days / time.

[0012] Furthermore, the total number of perfusions is 3 times.

[0013] Furthermore, the bacteria is Ralstonia solanacearum.

[0014] The invention also provides a plant antibacterial agent, comprising cyclo(D-leucine-L-proline) dipeptide and a solvent.

[0015] Furthermore, the solvent includes methanol, and the content of the cyclo(D-leucine-L-proline) dipeptide is 10 mg / L.

[0016] For better understanding and implementation, the present invention is further described in detail below in conjunction with specific examples and drawings. The following examples are only used to illustrate the present invention and are not used to limit the present invention. The experimental methods used in the following examples are generally conventional conditions unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is a schematic diagram of the structural formula of cyclo(D-leucine-L-proline) dipeptide;

[0018] Figure 2 Schematic diagram of the chromatography results of cyclo(D-leucine-L-proline) dipeptide;

[0019] Figure 3 Schematic diagram of ESI-MS results of cyclo(D-leucine-L-proline) dipeptide;

[0020] Figure 4 This is a schematic diagram of the antibacterial activity experimental results of Ralstonia solanacearum;

[0021] Among them, CK: methanol; P: Amp; DL: cyclo(D-leucine-L-proline) dipeptide; DD: cyclo(D-leucine-D-proline) dipeptide; LL: cyclo(L-leucine-L-proline) dipeptide; LD: cyclo(L-leucine-D-proline) dipeptide. DETAILED DESCRIPTION

[0022] The cyclo(D-leucine-L-proline) dipeptide of the present invention is obtained by chemical synthesis, and its structural formula is as follows: Figure 1 As shown, the crude cyclic (D-leucine-L-proline) dipeptide synthesized was purified and identified by HPLC, and the purified cyclic (D-leucine-L-proline) dipeptide was analyzed by ESI-MS.

[0023] The process and results of HPLC purification and identification are as follows: the crude product of chemically synthesized cyclo(D-leucine-L-proline) dipeptide was subjected to HPLC chromatography, wherein the solvent phase A was 0.1% (v / v) trifluoroacetic acid, and the solvent was chromatographically pure water; the solvent phase B was trifluoroacetic acid, and the solvent was chromatographically pure acetonitrile; the elution procedure was 15% B phase elution for 0.01 min, then 40% B phase elution for 25 min, then 100% B phase elution for 40 min, and finally 15% B phase elution for 50 min, and the whole process was 1 mL / min. Among them, the chromatographic results of cyclo(D-leucine-L-proline) dipeptide are as follows Figure 2 As shown, there is a single peak at 12-13 min, and the purity of cyclo(D-leucine-L-proline) dipeptide reaches 98.393%.

[0024] The process and results of ESI-MS analysis are as follows: the purified cyclo(D-leucine-L-proline) dipeptide was dissolved in a mixed solution of 5% acetic acid + 8% acetonitrile + 87% pure water and filtered, and then subjected to ESI-MS analysis. The analysis results are as follows Figure 3 As shown, the relative molecular mass of cyclo(D-leucine-L-proline) dipeptide is 210.20, which is consistent with the theory.

[0025] Example 1

[0026] This example provides an application example of cyclo(D-leucine-L-proline) dipeptide as a plant antibacterial agent in the process of tomato cultivation.

[0027] The implementation location of this embodiment is a greenhouse facility at the experimental teaching base of South China Agricultural University in Zengcheng District, Guangzhou City, Guangdong Province. The tomatoes and peppers planted in the greenhouse are seriously affected by bacterial wilt caused by Ralstonia solanacearum, and the mortality rate of tomatoes and peppers is about 30%. This embodiment selects 5 rows of land for application.

[0028] The planting method is as follows: 50 tomato seedlings are planted per row. Before planting, each tomato seedling needs to dip its roots in a 10 mg / mL cyclo(D-leucine-L-proline) dipeptide dilution for half a minute, and then plant it in the soil. After planting, 50 ml of cyclo(D-leucine-L-proline) dipeptide dilution is drip-irrigated into the root system. After that, every 18 days, 10 mg / mL of cyclo(D-leucine-L-proline) dipeptide dilution is drip-irrigated into the root system with the same specification, and the dilution is injected three times in total. The 10 mg / mL cyclo(D-leucine-L-proline) dipeptide dilution is prepared by cyclo(D-leucine-L-proline) dipeptide and methanol.

[0029] After the tomato seedlings grow to the peak fruit-bearing stage, the number of surviving plants and the survival rate are calculated, where the calculation formula for the survival rate is survival rate = number of surviving plants / total number of plants × 100%.

[0030] After the 250 tomato seedlings of this embodiment grew to the peak fruit-bearing stage, the survival rate was 96.2%.

[0031] Example 2

[0032] This example provides an application example of cyclic (D-leucine-L-proline) dipeptide as a plant antibacterial agent in the process of pepper cultivation.

[0033] The implementation location of this embodiment is a greenhouse facility at the experimental teaching base of South China Agricultural University in Zengcheng District, Guangzhou City, Guangdong Province. The tomatoes and peppers planted in the greenhouse are seriously affected by bacterial wilt caused by Ralstonia solanacearum, and the mortality rate of tomatoes and peppers is about 30%. This embodiment selects 5 rows of land for application.

[0034] The planting method is as follows: 50 pepper seedlings are planted per row. Before planting, each pepper seedling needs to dip its roots in a 10 mg / mL cyclo(D-leucine-L-proline) dipeptide dilution for half a minute, and then plant it in the soil. After planting, 50 ml of cyclo(D-leucine-L-proline) dipeptide dilution is drip-irrigated into the root system. After that, every 20 days, 10 mg / mL of cyclo(D-leucine-L-proline) dipeptide dilution is drip-irrigated into the root system with the same specification, and the dilution is injected three times in total. The 10 mg / mL cyclo(D-leucine-L-proline) dipeptide dilution is prepared by cyclo(D-leucine-L-proline) dipeptide and methanol.

[0035] After the pepper seedlings grow to the peak fruit-bearing stage, the number of surviving plants and the survival rate are calculated, where the survival rate is calculated using the formula: survival rate = number of surviving plants / total number of plants × 100%.

[0036] After the 250 pepper seedlings of this embodiment grew to the peak fruit-bearing stage, the survival rate was 95.3%.

[0037] Comparative Example 1

[0038] This embodiment provides an example of tomato planting without using plant antibacterial agents.

[0039] The implementation location of this embodiment is a greenhouse facility at the experimental teaching base of South China Agricultural University in Zengcheng District, Guangzhou City, Guangdong Province. The tomatoes and peppers planted in the greenhouse are seriously affected by bacterial wilt caused by Ralstonia solanacearum, and the mortality rate of tomatoes and peppers is about 30%. This embodiment selects 5 rows of land for application.

[0040] The planting method is: plant 50 tomato seedlings per row. Before planting, each tomato seedling needs to dip its roots in water for half a minute, and then plant them in the soil. After planting, drip irrigation is used to irrigate the roots at a rate of 50 ml per row. After that, every 18 days, the same rate of water needs to be used to irrigate the roots through drip irrigation, for a total of three times.

[0041] After the tomato seedlings grow to the peak fruit-bearing stage, the number of surviving plants and the survival rate are calculated, where the calculation formula for the survival rate is survival rate = number of surviving plants / total number of plants × 100%.

[0042] After the 250 tomato seedlings of this embodiment grew to the peak fruit-bearing stage, the survival rate was 70.4%.

[0043] Comparative Example 2

[0044] This embodiment provides an example of pepper cultivation without using plant antibacterial agents.

[0045] The implementation location of this embodiment is a greenhouse facility at the experimental teaching base of South China Agricultural University in Zengcheng District, Guangzhou City, Guangdong Province. The tomatoes and peppers planted in the greenhouse are seriously affected by bacterial wilt caused by Ralstonia solanacearum, and the mortality rate of tomatoes and peppers is about 30%. This embodiment selects 5 rows of land for application.

[0046] The planting method is: plant 50 pepper seedlings per row. Before planting, each pepper seedling needs to dip its roots in water for half a minute, and then plant them in the soil. After planting, drip irrigation is used to irrigate the roots at a rate of 50 ml per row. After that, every 20 days, the same rate of water needs to be used to irrigate the roots through drip irrigation, for a total of three times.

[0047] After the pepper seedlings grow to the peak fruit-bearing stage, the number of surviving plants and the survival rate are calculated, where the survival rate is calculated using the formula: survival rate = number of surviving plants / total number of plants × 100%.

[0048] After the 250 pepper seedlings of this embodiment grew to the peak fruit-bearing stage, the survival rate was 73.6%.

[0049] Comparative Example 3

[0050] In this example, the antibacterial activity of Ralstonia solanacearum was tested on sterile methanol, Amp (ampicillin) solution, cyclo(D-leucine-L-proline) dipeptide, cyclo(D-leucine-D-proline) dipeptide, cyclo(L-leucine-L-proline) dipeptide and cyclo(L-leucine-D-proline) dipeptide.

[0051] The experimental method is:

[0052] Dissolve cyclo(D-leucine-L-proline) dipeptide, cyclo(D-leucine-D-proline) dipeptide, cyclo(L-leucine-L-proline) dipeptide and cyclo(L-leucine-D-proline) dipeptide in methanol to prepare a 10 mg / L cyclodipeptide methanol solution, and filter through a 0.22 μm filter membrane for sterilization;

[0053] Ralstonia solanacearum was cultured to OD 600 =1.5, the Ralstonia solanacearum solution was spread on the LB medium, and 6 filter paper pieces (diameter 6mm) were placed at equal distances in the middle of the medium to form 6 spot areas. The LB liquid medium formula is: 10g tryptone, 5g yeast powder, 7g sodium chloride, and distilled water to make 1L; solid LB medium is added with 1.5% agar.

[0054] Add 10 μL of sterile methanol, 10 mg / mL Ampicillin solution, 10 mg / L cyclic (D-leucine-L-proline) dipeptide, 10 mg / L cyclic (D-leucine-D-proline) dipeptide, 10 mg / L cyclic (L-leucine-L-proline) dipeptide, and 10 mg / L cyclic (L-leucine-D-proline) dipeptide to the 6 spot areas respectively; place in a 30°C incubator for 1 day, observe, and measure the diameter of the inhibition zone.

[0055] The results of the antibacterial activity test of Ralstonia solanacearum are as follows Figure 4As shown, the substance corresponding to CK is methanol; the substance corresponding to P is 10 mg / mLAmp (ampicillin), and the diameter of the inhibition zone is 23.1 mm; the substance corresponding to DL is 10 mg / L cyclic (D-leucine-L-proline) dipeptide, and the diameter of the inhibition zone is 21.6 mm; the substance corresponding to DD is 10 mg / L cyclic (D-leucine-D-proline) dipeptide, and the diameter of the inhibition zone is 9.2 mm; the substance corresponding to LL is 10 mg / L cyclic (L-leucine-L-proline) dipeptide, and there is no inhibition zone; the substance corresponding to LD is 10 mg / L cyclic (D-leucine-L-proline) dipeptide, and the diameter of the inhibition zone is 14.7 mm. It is not difficult to see that cyclic (L-leucine-D-proline) dipeptide has a significant antibacterial effect on Ralstonia solanacearum, and its inhibition zone diameter is 21.6 mm, which is equivalent to the effect of the traditional antibiotic ampicillin (Amp). Therefore, cyclic (D-leucine-L-proline) dipeptide can be used to prevent the growth of Ralstonia solanacearum in the process of plant cultivation, prevent plant wilt disease, avoid affecting plant yields, and increase planting costs.

[0056] By comparing the results of Example 1 and Comparative Example 1, and Example 2 and Comparative Example 2, it can be seen that the use of cyclo(D-leucine-L-proline) dipeptide greatly improves the survival rate of tomatoes and peppers and reduces the mortality rate of tomatoes and peppers.

[0057] The present invention first discovered that the cyclic (D-leucine-L-proline) dipeptide has a significant inhibitory effect on Ralstonia solanacearum and applies it to plant antibacterial agents. Specifically, it has a significant inhibitory effect on bacterial wilt of tomatoes and peppers, providing a new prevention and control method for diseases caused by Ralstonia solanacearum in plants, which is beneficial to ensure the normal growth of plants. In addition, the cyclic (D-leucine-L-proline) dipeptide of the present invention can be isolated from Bacillus B1032 derived from mangroves, which allows the cyclic (D-leucine-L-proline) dipeptide of the present invention to be extracted from natural plants, without pollution to the environment and harmless to humans and animals. Among them, Bacillus B1032 is preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO: M 2020498.

[0058] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, and the present invention is also intended to include these modifications and modifications.

Claims

1. An application of a cyclic (D-leucine-L-proline) dipeptide, characterized in that: The application is as a plant antibacterial agent, the plants include Solanaceae crops, the bacteria are Ralstonia solanacearum, and the application method comprises the following steps: before planting, the plants are immersed in the cyclic (D-leucine-L-proline) dipeptide solution, and after planting, the cyclic (D-leucine-L-proline) dipeptide solution is perfused into the root system of the plants, the concentration of the cyclic (D-leucine-L-proline) dipeptide solution is 10 mg / L, and the frequency of perfusion is 18-20 days / time.

2. The use of a cyclic (D-leucine-L-proline) dipeptide according to claim 1, characterized in that: The total number of perfusions was 3 times.

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