Use of spermidine in preventing and / or treating apple tree canker

Abstract

This invention relates to the field of plant disease control technology, specifically to the application of spermidine in the prevention and / or treatment of apple tree canker. This invention screens and confirms the effectiveness of spermidine against apple tree canker pathogens. Cytospora pyri Direct antibacterial activity; its dose-response relationship and effective concentration range were clarified through treatment with different concentrations; based on EC... 50 Based on the high-concentration antibacterial results, the appropriate application concentration was determined; spermidine was prepared into a control agent suitable for orchard use; it was applied to lesions or susceptible parts of apple tree branches and trunks through spraying, smearing, etc., thereby achieving control of apple tree rot pathogens. Cytospora pyri Effective suppression and green control of apple tree rot disease.

Description

Technical Field

[0001] This invention relates to the field of plant disease control technology, specifically to the application of spermidine in the prevention and / or treatment of apple tree canker. Background Technology

[0002] apple( Domestic apple Borkh. is a representative species of temperate fruit trees, with important economic and nutritional value. It occupies a core position in the industry system and shows a development trend of "slightly decreasing area and stable yield", reflecting that the apple industry is transforming from extensive expansion to quality improvement and efficiency enhancement, and the industrial structure is constantly being optimized.

[0003] However, with the extension of orchard planting years and the increase in planting density, apple tree rot is showing a rapid expansion trend, becoming a key factor restricting the sustainable development of the industry. Apple tree rot is mainly caused by fungi of the genus *Cyclophorus* (…). Cytospore Caused by (spp.), this is a typical devastating disease affecting branches and trunks. After infection, the lesions rapidly expand along the bark, damaging the vascular tissue, hindering the transport of water and nutrients within the tree, and ultimately causing the death of the entire tree.

[0004] Currently, the control of apple tree canker mainly relies on chemical fungicides, such as triazoles (tebuconazole, propiconazole, flusilazole, difenoconazole), benzimidazoles (thiophanate-methyl), and organosulfur compounds (lime sulfur, mancozeb). However, the long-term and excessive use of chemical fungicides has led to the continuous enhancement of pathogen resistance, resulting in a year-by-year decline in control effectiveness. It has also brought about a series of problems, including excessive pesticide residues, environmental pollution, and disruption of the orchard's ecological balance, making it difficult to meet the requirements of modern green agriculture and food safety.

[0005] Therefore, developing safe, efficient, and environmentally friendly biological control technologies and bio-based fungicides has become an urgent technical problem to be solved in the field of apple tree rot disease control. Summary of the Invention

[0006] To address the above problems, this invention provides the application of spermidine in the prevention and / or treatment of apple tree rot.

[0007] This invention is achieved through the following technical solution: Application of spermidine in the prevention and / or treatment of apple tree rot.

[0008] Application of spermidine in the preparation of biological agents for the prevention and / or treatment of apple tree rot.

[0009] Preferably, the pathogen causing the apple tree rot disease is... Cytospora pyri .

[0010] Preferably, the dosage form of the biological agent is an aqueous solution, a soluble liquid, a topical application, or a compound biological control agent. Preferably, the concentration of spermidine in the biological agent is 0.1 mmol / L to 2.5 mmol / L.

[0011] Preferably, the concentration of spermidine in the biological agent is 1.6 mmol / L to 2.5 mmol / L.

[0012] Preferably, the biological agent is applied by smearing, spraying, or local penetration treatment.

[0013] Preferably, the biological agent is applied to the lesions and / or wounds on the apple tree branches.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. Achieves significant inhibitory effect on apple tree rot pathogens. This invention is the first to discover the effect of spermidine on apple tree rot pathogens. Cytospora pyri It exhibits direct antibacterial activity and demonstrates a significant concentration-dependent inhibitory effect. The inhibition rate reaches over 86% at concentrations above 2.0 mmol / L, and 100% inhibition is achieved at 2.5 mmol / L, significantly outperforming traditional control strategies.

[0015] 2. The effective concentration and dose-response relationship were clarified. EC was obtained through system experiments. 50 The optimal effective range for use was determined to be 1.6 mmol / L, and the range of 1.6 mmol / L to 2.5 mmol / L was established, providing a scientific basis for practical application and improving the feasibility and stability of the technology.

[0016] 3. Provide green and safe prevention and control technology solutions. Using spermidine, a small biomolecule, to replace or reduce the use of chemical fungicides can effectively reduce pesticide residues, environmental pollution, and the risk of pesticide resistance, which meets the requirements of green agricultural development.

[0017] 4. Highly applicable and flexible in application. It can be prepared as an aqueous solution, a topical agent, or a compound formulation, and applied to different scenarios in orchards through various methods such as spraying, applying, and wound treatment, to meet actual production needs.

[0018] 5. Possesses promising prospects for promotion and application. This invention establishes a complete technical system from antibacterial mechanism and dosage parameters to application methods, which can be used for large-scale green prevention and control of apple tree canker, with significant economic and ecological benefits. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This invention relates to different concentrations of spermidine treatment solutions for the effects of apple tree rot pathogens (…). Cytospora pyri The results of the inhibition are shown in the following figures: a) is the result of the control group; b) is the result of the treatment with 0.1 mmol / L spermidine; c) is the result of the treatment with 0.5 mmol / L spermidine; d) is the result of the treatment with 1.0 mmol / L spermidine; e) is the result of the treatment with 1.5 mmol / L spermidine; f) is the result of the treatment with 2.0 mmol / L spermidine; and g) is the result of the treatment with 2.5 mmol / L spermidine.

[0021] Figure 2 The diagram shows the experimental results of detached branches of the present invention; where a is a diagram of the expansion of lesions on detached apple tree branches; and b is a statistical diagram of lesion length. Detailed Implementation

[0022] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0023] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention and in its specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0024] The inventive concept of this invention is as follows: Currently, the control of apple tree canker mainly relies on chemical fungicides, such as triazoles (tebuconazole, propiconazole, flusilazole, difenoconazole), benzimidazoles (thiophanate-methyl), and organosulfur compounds (lime sulfur, mancozeb). However, the long-term and excessive use of chemical fungicides has led to the continuous enhancement of pathogen resistance, resulting in a year-on-year decline in control efficacy. It has also brought a series of problems, including excessive pesticide residues, environmental pollution, and disruption of the orchard's ecological balance, making it difficult to meet the requirements of modern green agriculture and food safety. Therefore, developing safe, efficient, and environmentally friendly biological control technologies and bio-based fungicides has become an urgent technical problem to be solved in the field of apple tree canker control.

[0025] Polyamines are a class of small-molecule, multi-cationic compounds widely found in organisms, mainly including putrescine, spermidine, and spermine, which play important roles in cell growth, membrane stability regulation, signal transduction, and stress response. Existing research indicates that exogenous polyamines can participate in plant disease resistance responses through multiple pathways, including disrupting the cell membrane structure of pathogenic fungi, inducing reactive oxygen species accumulation to trigger oxidative stress, and impairing mitochondrial function to interfere with energy metabolism. However, current research mainly focuses on the role of polyamines as endogenous signaling molecules in inducing host disease resistance. Research on the direct effects of polyamines as microbial metabolites on plant pathogens remains relatively limited, especially regarding the effects of spermidine on *Pseudomonas aeruginosa*, the causal agent of apple tree canker. Cytospora pyri There are no reports of direct inhibitory effects, nor are there any technical solutions for applying spermidine to the prevention and control of apple tree canker.

[0026] In response to the above situation, this invention proposes for the first time to use spermidine as an inhibitor. Cytospora pyri Based on the concentration-dependent direct antibacterial activity of its core active ingredient against the pathogen, a bio-based antibacterial technology solution suitable for the green control of apple tree canker was established. The basic idea is to systematically determine the inhibitory effect of different concentrations of spermidine on the mycelial growth of *C. pyri*, clarifying its effective inhibitory concentration range and median effective concentration (EC50). 50 This invention provides a scientific basis for the subsequent development of spermidine single-agent, compound formulations, and supporting orchard application technologies. At the application level, spermidine can be prepared into aqueous solutions, soluble liquids, topical agents, or compound biological control agents for the prevention and treatment of apple tree canker. Given that this disease mainly occurs on branches, pruning cuts, grafting points, and mechanically damaged areas, the application methods are flexible and diverse, including spraying, smearing, brushing, wound sealing, or application after scraping away lesions. Specifically: for existing lesions, necrotic tissue can be removed first, and then a spermidine-containing treatment solution can be applied to the lesion and its boundary with healthy tissue to inhibit further colonization and spread of the pathogen; for susceptible sites such as pruning cuts, grafting points, and mechanically damaged areas, spermidine treatment solution can be applied promptly after wound formation to effectively block early pathogen infection; during peak disease periods or epidemic seasons, it can also be combined with conventional orchard management practices for spraying protection on branches or targeted treatment of key areas.

[0027] In summary, this invention is the first to screen and confirm spermidine's effect on... Cytospora pyri The direct antibacterial activity was observed; its dose-response relationship and effective concentration range were clarified through multi-concentration gradient treatment; based on EC... 50 EC 70The results of complete inhibition at high concentrations determined the appropriate application concentration; spermidine was developed into a control agent suitable for orchard application; and finally, it was applied to lesions or susceptible parts of apple tree branches and trunks through spraying, smearing, and wound protection to achieve effective inhibition of C. pyri and green control of apple tree canker.

[0028] The beneficial effects of the present invention will be illustrated below through specific embodiments.

[0029] The apple tree rot pathogen of this invention is Cytospora pyri The specific strain used is *Pseudomonas aeruginosa*, the pathogen causing apple tree rot. Cytospora pyri CXTA 329, this strain has been disclosed in the literature "Identification of Fungal Species Associated with Apple Canker in Tarim Basin, China".

[0030] Spermine was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. Example 1 First, spermidine treatment solutions of different concentrations (0.1 mmol / L, 0.5 mmol / L, 1.0 mmol / L, 1.5 mmol / L, 2.0 mmol / L, and 2.5 mmol / L) were prepared with water and applied to apple tree canker pathogens isolated from apple tree canker tissue exhibiting typical symptoms from the Tarim Basin in Xinjiang, China. Cytospora pyri CXTA 329: Observe and measure the growth of pathogenic mycelia, and calculate the inhibition rate using colony diameter or mycelial growth as indicators.

[0031] The inhibitory effect of different concentrations of spermidine on the mycelial growth of CXTA 329 was determined by adding spermidine treatment solutions to PDA medium and using the mycelial growth rate method. PDA medium without spermidine served as the control group (CK). Each treatment was inoculated with CXTA 329 mycelial cakes with a diameter of 5 mm, and three groups were repeatedly cultured at 25℃ for 3 days. The colony diameter was measured using the cross-cross method, and the mycelial growth inhibition rate was calculated. Regression analysis was performed with spermidine concentration as the independent variable and the inhibition rate as the dependent variable to calculate the half-maximal effective concentration (EC50) of spermidine for CXTA 329. The EC50 concentration was used as the core treatment concentration in subsequent mechanistic experiments.

[0032] Experimental results are as follows Figure 1 As shown, spermidine has the effect of Cytospora pyri CXTA 329 exhibited significant inhibitory activity, and its inhibitory effect increased with increasing spermidine concentration. At a spermidine concentration of 0.1 mmol / L, it significantly reduced the inhibitory effect on spermidine. Cytospore old manThe antibacterial rate of CXTA 329 was 8.50%; when the concentration was increased to 0.5 mmol / L, the antibacterial rate was 13.75%; when the concentration was 1.0 mmol / L, the antibacterial rate reached 22.78%; when the concentration was 1.5 mmol / L, the antibacterial rate increased to 47.06%; when the concentration was 2.0 mmol / L, the antibacterial rate significantly increased to 86.27%; when the concentration was further increased to 2.5 mmol / L, the antibacterial rate was significantly reduced. Cytospore old man CXTA 329 has an antibacterial rate of 100%.

[0033] The above results indicate that spermidine can effectively inhibit apple tree rot pathogens. Cytospora pyri The growth of the organism exhibited a significant dose-response relationship.

[0034] Example 2 The spermidine concentration gradient inhibition rate data used in this embodiment were obtained from the same batch of experiments as those in Example 1 of the aforementioned virulence determination. Specifically, the final concentrations of spermidine were set to 0 mmol / L, 0.01 mmol / L, 0.5 mmol / L, 1.0 mmol / L, 1.5 mmol / L, 2.0 mmol / L, 2.5 mmol / L, and 3.0 mmol / L, respectively, and equal volumes of culture medium were inoculated using the poisoning medium method. Cytospora pyri After CXTA 329 mycelial cakes were cultured at 25 ℃ for 3 days, the colony diameter was measured using the cross-sectional method and calculated according to the formula.

[0035] .

[0036] The inhibition rate at each concentration was calculated (three biological replicates for each concentration). A dose-response curve was plotted with spermidine concentration on the x-axis and inhibition rate on the y-axis. Probit regression analysis was performed using SPSS 26.0 software to fit the dose-response equation and calculate the effective inhibitory concentration. Statistical analysis revealed the inhibitory effect of spermidine on... Cytospora pyri EC 50 The concentration was 1.6 mmol / L, and the MIC was 2.5 mmol / L (the lowest concentration to completely inhibit visible growth); curve fitting and multiple comparisons both reached a significant level. P <0.05).

[0037] Therefore, it can be determined that spermidine at approximately 1.6 mmol / L can effectively target... Cytospora pyriIt produces more than half of the inhibitory effects, achieving a high level of inhibition at approximately 1.8 mmol / L, and strong antibacterial activity at concentrations above 2.0 mmol / L. Therefore, the present invention preferably sets the effective concentration of spermidine to 1.6 mmol / L to 2.5 mmol / L, more preferably 1.774 mmol / L to 2.5 mmol / L; when a higher control effect is required, a spermidine treatment concentration of 2.0 mmol / L to 2.5 mmol / L is preferred.

[0038] Example 3 Healthy, one-year-old apple branches with uniform growth were selected and tested on the tree. After surface disinfection of the inoculation site, holes were uniformly punched using a 5mm diameter sterile punch. Holes were uniformly punched in the middle of the branch using a 5mm diameter sterile punch.

[0039] The following treatments were set up: (1) Blank control (PDA): the wound was directly inoculated with blank PDA culture medium bacterial cakes; (2) Pathogen control (CK): the wound was inoculated with... Cytospora pyri (3) Preventive treatment: Apply a 2.0 mmol / L spermidine aqueous solution to the wound first, and inoculate 24 h later. Cytospora pyri (4) Treatment: Inoculate the wound first. Cytospora pyri CXTA 329 mycelial cake was applied, and after 24 hours, a spermidine aqueous solution of the same concentration and volume as the preventive treatment group was applied. In each group... Cytospora pyri The inoculation amount of the fungal cake was exactly the same. After inoculation, the wound was wrapped with a moisturizing material, and the longitudinal length of the lesions on the branches was measured regularly. At least three biological replicates were set up for each treatment. The spermidine solution was prepared in the same way as in Example 1.

[0040] Results of the detached branch experiment are as follows Figure 2 As shown, spermidine aqueous solution significantly inhibited lesion expansion under both preventive and therapeutic treatments: the average lesion length in the pathogen control (CK) group was approximately 2.57 cm, while the lesion length in the preventive group was reduced to approximately 0.73 cm, and in the treatment group it was reduced to approximately 1.43 cm, showing a significant difference compared to the CK group. P <0.05), where the preventive effect is better than the therapeutic effect, indicating that spermidine has both protective and therapeutic potential.

[0041] It should be noted that, at the application level, this invention uses spermidine as the sole or main active ingredient to design a control system suitable for fruit tree branch and trunk diseases, including but not limited to wound application, branch and trunk spraying, or local penetration treatment, enabling it to directly act on apple tree canker lesions and infected tissues, achieving control... Cytospora pyri In situ inhibition.

[0042] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0043] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this invention should be determined by the appended claims.

Claims

1. Application of spermidine in the prevention and / or treatment of apple tree rot.

2. Application of spermidine in the preparation of biological agents for the prevention and / or treatment of apple tree rot.

3. The application according to claim 1 or 2, characterized in that, The pathogen causing the apple tree rot disease is... Cytospora pyri .

4. The application according to claim 2, characterized in that, The biological agent is available in the form of an aqueous solution, a soluble liquid, a topical application, or a compound biological control agent. According to claim 2, the concentration of spermidine in the biological agent is 0.1 mmol / L to 2.5 mmol / L.

5. The application according to claim 2, characterized in that, The concentration of spermidine in the biological agent is 1.6 mmol / L to 2.5 mmol / L.

6. The application according to claim 2, characterized in that, The biological agent is applied by smearing, spraying, or local penetration treatment.

7. The application according to claim 2, characterized in that, The biological agent is applied to the lesions and / or wounds on the branches and trunks of apple trees.

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