Immunomodulator for improving canker resistance of poplar as well as preparation method and application of immunomodulator

Abstract

The invention relates to the technical field of plant disease control, in particular to an immunomodulator for improving poplar canker resistance and a preparation method and application thereof. The invention provides application of salicylic acid in preparation of a preparation for preventing and treating poplar canker. Salicylic acid, absolute ethyl alcohol and Tween 80 cooperate to prepare the immunomodulator, so that a plant ethylene signal channel is activated, and scab expansion caused by botryosphaeria dothidea is remarkably inhibited. Transcriptome analysis proves that the disease resistance is improved by enhancing the gene expression quantity of an ethylene signal channel, and the gene has the advantages of high efficiency, environmental protection, broad spectrum and the like, and is suitable for green prevention and control of poplar canker.

Description

Technical Field

[0001] This invention relates to the field of plant disease control technology, and in particular to an immunomodulator for improving the resistance of poplar to canker disease, its preparation method and application. Background Technology

[0002] Poplar, as an important fast-growing timber species, plays a vital role in forestry production and ecological protection. However, poplar canker is an extremely serious fungal disease that damages poplar cultivation, and its pathogen is mainly *Botrytis cinerea* (…). Botryosphaeria dothidea This disease mainly infects the trunks and branches of poplar trees, causing cankers and bark rot on the trunks. In severe cases, it can lead to dieback or even the death of the entire tree, causing huge economic losses to forestry production.

[0003] Currently, the control of poplar canker mainly relies on chemical spraying. However, long-term use of chemical agents can easily lead to drug resistance in pathogens, reducing the control effect. At the same time, chemical residues can cause environmental pollution such as soil and water bodies, disrupting the ecological balance and contradicting the concept of green forestry development. Therefore, developing eco-friendly, efficient, and sustainable disease control technologies has become a research hotspot in the field of forestry disease control.

[0004] Plants possess their own immune system, and immunomodulatory technologies that enhance disease resistance by activating the plant's own defense responses have attracted widespread attention due to their advantages such as being environmentally friendly and less likely to induce drug resistance. Salicylic acid (SA), as an important endogenous signaling molecule in plants, can induce systemically acquired resistance (SAR), thereby enhancing resistance to various pathogens. However, current research has found that when salicylic acid is used alone, its absorption efficiency within plants is limited, resulting in less than ideal disease resistance and failing to meet the control needs of practical production.

[0005] Based on the above problems, this invention optimizes the formulation of immunomodulators by screening suitable solvents, surfactants and salicylic acid, aiming to improve the absorption and translocation efficiency of salicylic acid in poplar trees, further activate the plant's own disease resistance signaling pathways, significantly enhance the resistance of poplar trees to canker, and provide a new technical means for the green control of poplar canker. Summary of the Invention

[0006] The purpose of this invention is to provide an immunomodulator for enhancing the resistance of poplar trees to canker, its preparation method, and its application. 。

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of 0.05~0.5mM salicylic acid in the preparation of formulations for the prevention and treatment of poplar canker.

[0008] The present invention also provides the application of 0.05~0.5mM salicylic acid in the preparation of formulations to improve the disease resistance of poplar trees.

[0009] Preferably, the disease is ulcer disease.

[0010] The present invention also provides an immunomodulatory agent comprising, in water as a solvent, components at the following concentrations: Salicylic acid 1.38~6.91mg / 100mL, anhydrous ethanol 15~25ml / 100mL, Tween 80 1.5~2.5ml / 100mL.

[0011] Preferably, the pH of the immunomodulator is 5.5 to 6.5.

[0012] This invention also provides a method for preparing an immunomodulator, comprising the following steps: (1) Weigh each component according to the concentration of the immunomodulator described above; (2) Mix salicylic acid, anhydrous ethanol, Tween 80 and water to obtain a mixture, adjust the pH of the mixture to obtain an immunomodulator.

[0013] The present invention also provides the application of the immunomodulator described herein or the immunomodulator prepared by the method described herein in the preparation of formulations that enhance the resistance of poplar to canker disease.

[0014] The present invention also provides the application of the immunomodulator described herein or the immunomodulator prepared by the method described herein in the preparation of formulations for the prevention and treatment of poplar canker.

[0015] The present invention also provides a method for improving the resistance of poplar trees to canker, comprising the following steps: spraying poplar trees with a diluted solution of the immunomodulator prepared by the above-described method or the immunomodulator prepared by the above-described method; The dilution factor is 200 to 10,000 times.

[0016] The present invention also provides a method for preventing and controlling poplar canker, comprising the following steps: spraying poplar trees with a diluted solution of the immunomodulator prepared by the above-described method or the immunomodulator prepared by the above-described method; The dilution factor is 200 to 10,000 times.

[0017] The present invention has the following advantages: The immunomodulator of this invention, through the synergistic effect of salicylic acid, anhydrous ethanol, and Tween 80, not only improves the absorption efficiency of salicylic acid in poplar trees but also specifically activates the upregulation of genes related to the ethylene signaling pathway, constructing a highly efficient plant disease resistance system and significantly enhancing the resistance of poplar trees to canker. Compared with existing technologies, this immunomodulator has the following outstanding beneficial effects: (1) Significant disease resistance: The area of ​​lesion spread was reduced by 54.98% compared with the control, and the proportion of lesions that did not expand in the field reached 46.15%, which effectively reduced the damage of poplar canker; (2) Ecological and environmental protection: All components are biodegradable, with no residue or pollution, and will not damage the soil microbial community and ecological environment, which meets the requirements of green forestry development; (3) Wide range of applications: It is applicable to black poplar and various poplar varieties. It has a control effect on canker caused by Gracilaria pulmonale and similar stem fungal diseases. Its application prospects are broad. (4) Simple preparation and application: The process is simple and the cost is low. It can be produced on a large scale. It is applied by spraying, which is convenient to operate and suitable for large-scale field promotion. (5) Strong stability: The formulation has a stable pH value, a long storage period, and is not easily affected by environmental factors during use, resulting in stable and reliable control effects; In summary, the immunomodulator of this invention solves the problems of drug resistance and environmental pollution caused by traditional chemical control, as well as the limited control effect of salicylic acid alone. It provides a new technical solution for the green and efficient control of poplar canker, and has important economic and ecological significance. Attached Figure Description

[0018] Figure 1 The effect of different concentrations of immunomodulators on the area and length of lesions in Populus tomentosa after inoculation with Staphylococcus aureus (A is a visual diagram of each group after inoculation; B is a statistical result diagram, with the left graph of the statistical result diagram showing the lesion area and the right graph showing the lesion length). Figure 2 The MAPK signaling pathway in the response of black poplar to salicylic acid (red boxes indicate upregulated gene expression, blue boxes indicate downregulated gene expression); Figure 3 A heatmap showing the expression of major upregulated and downregulated genes in the MAPK signaling pathway during the response of poplar to salicylic acid. Detailed Implementation

[0019] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0020] Example 1

[0021] Dissolve 1.38 mg of salicylic acid in 20 mL of anhydrous ethanol, add 2 mL of Tween 80 and stir well. Then slowly add deionized water to make up to 100 mL and adjust the pH to 6.0 to obtain a transparent liquid, which is an immunomodulatory agent with a salicylic acid concentration of 100 mM.

[0022] Example 2

[0023] Following the method of Example 1, Example 2 was prepared by diluting the obtained 100mM salicylic acid immunomodulator with water by 2000 times to obtain an immunomodulator with a salicylic acid concentration of 0.05mM.

[0024] Example 3

[0025] Following the method of Example 1, Example 2 was prepared by diluting the obtained 100mM salicylic acid immunomodulator with water by 200 times to obtain an immunomodulator with a salicylic acid concentration of 0.5mM.

[0026] Experimental Example 1

[0027] Field trial: Two-year-old poplar seedlings were selected and sprayed (100 ml / seedling) with immunomodulators of different concentrations and compositions, namely 0.05 mM immunomodulator (0.05 mM SA) from Example 2, 0.5 mM immunomodulator (0.5 mM SA) from Example 3, and 0.05 g / L alginate (AO) from Example 3. Three days later, the seedlings were inoculated with *Botrytis cinerea* (strain number: CXY001, similar to the strain described in Li Yongxia's article "Poplar and Canker Pathogens"). Botryosphaeria dothidea (The interaction mechanism is consistent with CXY001 in "Different Expression of Metabolites and Proteins in Poplar after Infection with Canker Bacteria"). The control group (CK) was inoculated with sterile PDA medium, and the lesion area was measured after 7 days. The optimal immunomodulatory component was screened based on the lesion spread. The results are as follows: Figure 1 As shown.

[0028] Figure 1 The results showed no difference in lesion area and length after treatment with 0.5 mM SA and 0.05 mM SA. Therefore, 0.05 mM SA, i.e., the 0.05 mM immunomodulator in Example 2, was selected as the optimal concentration. After treatment with 0.05 mM immunomodulator for Populus tomentosa inoculated with Staphylococcus aureus, the lesion spread area was reduced by 54.98% compared with the CXY001 group (the control group without treatment).

[0029] After treating black poplar with 0.05mM immunomodulator for 48h and 72h, stems were collected and sent to Shanghai Meiji Biotechnology Co., Ltd. for sequencing and transcriptomics analysis of gene expression. The results are as follows: Figures 2-3 As shown.

[0030] Transcriptomic analysis showed that after treatment with immunomodulators, the expression levels of genes involved in the plant hormone ethylene signaling pathway, including ethylene receptor (ETR), mitogen-activated protein kinase 9 (MKK9), EIN3-binding F-box protein (EBF), and ethylene response transcription factor (ERF1), were significantly upregulated (p<0.01), indicating that the ethylene signaling regulatory network involved in the resistance response was activated. This suggests that immunomodulators enhance disease resistance and inhibit pathogen spread by increasing the upregulated expression of genes involved in the ethylene signaling pathway.

[0031] Experiment Example 2

[0032] A field efficacy test of pesticides was conducted on poplar seedlings in Youfang Forest Farm, Youyu County, Shanxi Province, where poplar canker was prevalent. The experiment included treatment groups: salicylic acid (SA) (0.05 mM immunomodulator from Example 2), oligosaccharide (AO, 5% diluted 1000 times), and a mixture of SA+AO (0.05 mM immunomodulator + 5% oligosaccharide diluted 1000 times). Different pesticides were applied to different areas (Table 1). After 45 days, the length and width of the lesions in the poplar were recorded. The control effect was determined based on indicators such as lesion length, the percentage of lesion length changes, and the percentage of lesions that did not enlarge.

[0033] Table 1. Changes in the severity of poplar canker treated with different agents.

[0034] Table 1 shows that salicylic acid has the best prevention and control effect, with 46.15% of the lesions not expanding.

[0035] As demonstrated by the above embodiments, the immunomodulator of the present invention activates the ethylene signaling pathway in plants through the synergistic effect of salicylic acid, anhydrous ethanol, and Tween 80, significantly inhibiting the spread of lesions caused by *Botrytis cinerea*. Transcriptome analysis proves that it enhances disease resistance by increasing the expression of genes in the ethylene signaling pathway, possessing advantages such as high efficiency, environmental friendliness, and broad spectrum, making it suitable for the green control of poplar canker.

[0036] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

Application of 0.05~0.5mM salicylic acid in the preparation of formulations for the prevention and treatment of poplar canker. Application of 0.05~0.5mM salicylic acid in the preparation of formulations to improve the disease resistance of poplar trees.

3. Use according to claim 2, characterized in that, The disease in question is ulcer disease.

4. An immunomodulator, characterized by, The components using water as a solvent include those with the following concentrations: Salicylic acid 1.38~6.91mg / 100mL, anhydrous ethanol 15~25ml / 100mL, Tween 80 1.5~2.5ml / 100mL.

5. The immunomodulator of claim 4, wherein, The pH of the immunomodulator is 5.5-6.

5.

6. A method of preparing an immunomodulator, characterized by, Includes the following steps: (1) Weigh each component according to the concentration of the immunomodulator as described in claim 4 or 5; (2) Mix salicylic acid, anhydrous ethanol, Tween 80 and water to obtain a mixture, adjust the pH of the mixture to obtain an immunomodulator.

7. The use of the immunomodulator according to any one of claims 4 to 5 or the immunomodulator prepared by the preparation method according to claim 6 in the preparation of formulations to improve the resistance of poplar to canker disease.

8. The use of the immunomodulator according to any one of claims 4 to 5 or the immunomodulator prepared by the preparation method according to claim 6 in the preparation of a formulation for preventing and treating poplar canker.

9. A method for increasing the ability of a poplar to resist canker disease, characterized by, The process includes the following steps: spraying poplar trees with a diluted solution of the immunomodulator prepared by any one of the immunomodulators described in claims 4-5 or by the preparation method described in claim 6; The dilution factor is 200 to 10,000 times.

10. A method for controlling poplar canker disease, characterized by, The process includes the following steps: spraying poplar trees with a diluted solution of the immunomodulator prepared by any one of the immunomodulators described in claims 4-5 or by the preparation method described in claim 6; The dilution factor is 200 to 10,000 times.

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