Application of glycerol-3-phosphate as active ingredient of plant disease resistance activator in improving disease resistance of apple tree

Abstract

The application discloses application of glycerol-3-phosphate in preparation of a plant disease resistance activator, and the glycerol-3-phosphate is also used as an active ingredient of the plant disease resistance activator to improve the disease resistance of apples. The glycerol-3-phosphate itself has no bactericidal or bacteriostatic effect in vitro, and can only induce the immune system of the plant to resist the invasion of diseases under the condition of living body. The disease resistance induced by the glycerol-3-phosphate belongs to the physiological metabolic response of the plant body itself, is environmentally friendly and safe, and does not produce drug resistance. The disease resistance of fruit trees induced by the glycerol-3-phosphate has the characteristics of long persistence and wide spectrum, and has a significant prevention effect on apple ring rot, rot disease, gray mold and anthracnose, and can be applied to the prevention and treatment of these diseases. In addition, the glycerol-3-phosphate can be compounded with Pseudomonas syringae B-1 to form a composite biocontrol agent, so that the prevention and treatment effect on apple diseases is significantly improved.

Description

Technical Field

[0001] This invention relates to the field of plant disease resistance technology, and more particularly to the application of glycerol-3-phosphate as an active ingredient in plant disease resistance activators to improve the disease resistance of apple trees. Background Technology

[0002] There are more than 100 diseases that harm apples. Depending on the affected parts, they can be divided into branch and trunk diseases, fruit diseases, leaf diseases, and root diseases.

[0003] Apple ring rot and apple anthracnose are two of the most serious rain-borne diseases affecting apples, distributed in all major apple-producing areas, especially in areas with heavy summer rainfall such as the Yellow River old course region and the Bohai Bay region. These diseases cause severe economic losses annually. In normal years, the incidence rate in bagged apple orchards is 10%-30%, reaching up to 80% in severely affected orchards, and can continue to cause damage during storage. The pathogens for both diseases originate from apple branches and trunks. Ring rot fungus can cause typical symptoms such as dry rot, galls, and cankers on branches and trunks, even leading to branch death or tree death. Anthracnose fungus mainly overwinters on 1-2 year old branches, but does not show obvious symptoms on the branches.

[0004] Apple tree canker is a typical branch and trunk disease, often referred to as apple tree cancer. It damages the main branches and trunk of the fruit tree, causing dead branches, dead trees, and even orchards, making it one of the most devastating diseases threatening the apple industry. Apple anthracnose leaf blight primarily affects leaves and fruit, causing premature leaf drop and severely reducing tree vigor and yield. It also damages fruit, producing small, circular brown spots, reducing the fruit's commercial value, and threatening the culling of some superior, susceptible varieties. Apple gray mold primarily affects fruit and can infect and spread during storage and transportation, even under low-temperature conditions, making it one of the most serious diseases affecting apples during storage.

[0005] Currently, the main method for controlling apple ring rot and anthracnose is bagging cultivation, which physically isolates the fruit from pathogens. However, with the increasing costs, environmental pollution, and labor shortages associated with bagging, bagless cultivation of apples has become an inevitable trend. However, the fundamental measure for controlling apple ring rot and anthracnose in bagless orchards is to eliminate the primary source of infection on the branches and trunks. Simultaneously, ensuring the fruit surface is protected with pesticides before each rainfall inevitably leads to the frequent and extensive use of chemical fungicides, posing a significant threat to food safety and easily resulting in the emergence of drug-resistant pathogens. Furthermore, in rainy years or regions, it is difficult to guarantee the effectiveness of pesticides, leading to a substantial increase in the incidence of these two fruit diseases and severe economic losses.

[0006] Currently, the control of apple tree rot and anthracnose leaf blight mainly relies on chemical agents. Ensuring that apple branches, fruit surfaces, or leaves are protected with pesticides before each rainfall inevitably leads to the extensive and frequent use of chemical fungicides, posing a significant threat to food safety and easily resulting in the emergence of drug-resistant pathogens, thus damaging the orchard's ecological environment. For the control of apple gray mold, the common practice is to soak the fruit in chemical agents, resulting in prominent problems such as excessive pesticide residues in the fruit.

[0007] Therefore, finding safe and effective control products to replace existing chemical fungicides is an urgent problem to be solved, especially in the prevention and control of important diseases in apple production.

[0008] Therefore, the existing technology needs further improvement. Summary of the Invention

[0009] To address the aforementioned problems, this invention provides the application of glycerol-3-phosphate in the preparation of plant disease resistance activators and a method for inducing disease resistance in apple trees. Based on the ability of glycerol-3-phosphate to induce improved resistance to various diseases in apples, this invention provides the application of glycerol-3-phosphate as an active ingredient in plant disease resistance activators to enhance apple disease resistance. Furthermore, it can be synergistically combined with *Pseudomonas syringae* B-1 as a compound biocontrol agent to improve the control effect against various apple diseases.

[0010] To achieve the above objectives, this application provides the following technical solution:

[0011] In the first aspect, this application also provides the application of glycerol-3-phosphate as an active ingredient of plant disease resistance activator in improving the disease resistance of apples.

[0012] The glycerol-3-phosphate of the present invention has the following structure:

[0013]

[0014] Extensive research has revealed that controlling diseases by stimulating the plant's own defense system is an important approach to developing efficient, safe, and green pesticides. These pesticides offer advantages such as long-lasting resistance, broad disease resistance spectrum, and no environmental pollution. Currently, various disease resistance activators have been applied to crops such as tomatoes, grapes, and tobacco. However, there are few disease resistance activators for fruit trees on the market, and further development is needed.

[0015] During the research and development process, the inventors discovered that glycerol-3-phosphate can activate the resistance of apple branches, fruits, and leaves to various diseases, and has the activity of a plant disease resistance activator, showing great application potential in the biological control of apple diseases.

[0016] Experiments have shown that glycerol-3-phosphate can enhance the activity of defense enzymes in apples, namely POD, SOD, GLU, PAL, and CHI; and glycerol-3-phosphate can increase the expression of disease-resistant genes, namely PR1, PR5, CLU, and CHI; and glycerol-3-phosphate can promote the accumulation of disease-resistant substances in apples, namely lignin and total phenols.

[0017] Furthermore, experiments have shown that apple trees treated with glycerol-3-phosphate solution develop resistance to a variety of apple diseases, and this resistance is characterized by long-lasting effectiveness and broad spectrum. Apple tissue treated with glycerol-3-phosphate has significant control effects on apple ring rot, anthracnose, canker, anthracnose leaf blight, and gray mold, with control efficacy exceeding 70%, resulting in high economic benefits.

[0018] Preferably, glycerol-3-phosphate induces resistance in apple trees to the following apple diseases: apple canker, apple anthracnose leaf blight, apple gray mold, apple ring rot, and apple anthracnose. It is evident that the disease resistance induced by glycerol-3-phosphate in apple trees has a broad spectrum and wide application range. The pathogens of apple canker, apple anthracnose leaf blight, apple gray mold, apple ring rot, and apple anthracnose are, in order, *Valsa mali*, *Glomerella cingulata*, *Botrytis cinerea*, *Botryosphaeria dothidea*, and *Colletotrichum gloeosporioides*.

[0019] Preferably, in the application, glycerol-3-phosphate is used to enhance the activity of defense enzymes in apples and increase the expression of disease-resistant genes.

[0020] Secondly, this application provides the application of glycerol-3-phosphate in the preparation of plant disease resistance activators.

[0021] Based on the aforementioned activity of glycerol-3-phosphate, it can be prepared into plant disease resistance activators in different formulations, such as glycerol-3-phosphate solutions. Furthermore, it can be combined with other adjuvants to prepare other formulations with better stability and easier storage and transportation.

[0022] Thirdly, this application provides the application of glycerol-3-phosphate in the prevention and control of apple tree canker, apple anthracnose leaf blight, apple gray mold, apple ring rot and apple anthracnose.

[0023] Experiments have shown that apples treated with glycerol-3-phosphate solution develop resistance to a variety of apple diseases, including apple canker, apple anthracnose leaf blight, apple gray mold, apple ring rot, and apple anthracnose. The control effect on these diseases is significant, and the resistance has long-lasting and broad-spectrum characteristics.

[0024] Preferably, the method is to apply the glycerol-3-phosphate solution directly by spraying or soaking the apple fruit.

[0025] More preferably, the concentration of the glycerol-3-phosphate solution is 0.1 mmol·L⁻¹. -1 ~0.2mmol·L -1 .

[0026] In other embodiments, glycerol-3-phosphate can also be used as an active ingredient and combined with other adjuvants to prepare different dosage forms of the drug for application, so as to improve its stability and duration of action.

[0027] Fifthly, this application also provides the application of glycerol-3-phosphate in improving the control effect of biocontrol fungus Pseudomonas syringae B-1 on apple diseases, wherein the accession number of Pseudomonas syringae B-1 is CCTCC NO:M2015813.

[0028] Pseudomonas syringae B-1 was disclosed in the patent with authorization announcement number CN111286477B. For details of this strain, please refer to the patent.

[0029] Optionally, the apple disease is apple ring rot or apple gray mold.

[0030] Preferably, the application method is as follows: the glycerol-3-phosphate solution and the bacterial suspension of Pseudomonas syringae B-1 are mixed before use.

[0031] Experiments have shown that the combination of glycerol-3-phosphate and Pseudomonas syringae B-1 has a synergistic effect, which can significantly improve the control efficacy of Pseudomonas syringae B-1 against a variety of diseases (such as apple gray mold and apple ring rot).

[0032] In a sixth aspect, this application also provides a compound biocontrol agent comprising: a bacterial suspension of Pseudomonas syringae B-1 and glycerol-3-phosphate.

[0033] Preferably, the concentration of the glycerol-3-phosphate is 0.1 mmol·L⁻¹. -1 ~0.5mmol·L-1 The concentration of the bacterial suspension is 10. 5 cfu·mL -1 ~10 9 cfu·mL -1 .

[0034] Seventhly, this application also provides a method for inducing disease resistance in apples, the method comprising: adding 0.1 mmol·L⁻¹ -1 ~0.2mmol·L -1 Spray a glycerol-3-phosphate solution onto the branches, leaves, or fruit until water droplets run off the surface; alternatively, soak the apple fruit in a solution with a concentration of 0.1 mmol·L⁻¹. -1 ~0.2mmol·L -1 A solution of glycerol-3-phosphate.

[0035] Preferably, the soaking time is at least 5 minutes. More preferably, the soaking time is controlled between 5 and 10 minutes.

[0036] The present invention has the following beneficial effects:

[0037] This invention discovers that glycerol-3-phosphate can act as a plant disease resistance activator, enhancing the disease resistance of apples. It can be developed into a plant disease resistance activator and applied to the control of various apple diseases. Compared with traditional pesticides, it has the following advantages:

[0038] (1) It has no bactericidal or bacteriostatic effect in isolation. It can only induce the plant's own immune system to resist the invasion of diseases under living conditions. The disease resistance induced by glycerol-3-phosphate is a physiological metabolic reaction of the plant itself, which is environmentally friendly and safe and does not produce drug resistance.

[0039] (2) The disease resistance induced in fruit trees has long-lasting and broad-spectrum characteristics. Apple branches and fruits treated with glycerol-3-phosphate have significant control effects on apple ring rot, canker, gray mold and anthracnose. The control effect on apple canker, a branch disease, is as high as 70.6%. The control effects on apple ring rot, anthracnose and gray mold, fruit diseases are as high as 83.3%, 71.6% and 71.6% respectively. The control effect on anthracnose leaf blight, a leaf disease, is as high as 85.6%. This shows that glycerol-3-phosphate has high economic benefits in the control of apple diseases.

[0040] (3) It has the advantages of simple usage, low concentration and small dosage.

[0041] (4) The use of glycerol-3-phosphate can also increase the activity of defense enzymes GLU, CHI, PAL, POD and SOD in apples, and can induce the significant upregulation of disease resistance-related genes PR1, PR5, CLU and CHI; it also promotes the accumulation of disease resistance substances such as lignin and total phenols in apples.

[0042] (5) The use of glycerol-3-phosphate can improve the control effect of biocontrol fungus Pseudomonas syringae B-1 on apple ring rot and apple gray mold. Attached Figure Description

[0043] Figure 1 The effects of different concentrations of glycerol-3-phosphate on mycelial growth and spore germination of *Aureobasidium pinnatifida* and *Aureobasidium anthracnose* are shown in Figure 1. A represents colony diameter, and B represents spore germination rate.

[0044] Figure 2 The effects of different concentrations of glycerol-3-phosphate on mycelial growth and spore germination of *Pseudomonas aeruginosa*, *Pseudomonas anthracnose*, and *Botrytis cinerea* were investigated. A represents colony diameter, and B represents spore germination rate.

[0045] Figure 3 The effect of glycerol-3-phosphate on the prevention and control of apple ring rot;

[0046] Figure 4 The effect of glycerol-3-phosphate on the prevention and control of apple anthracnose;

[0047] Figure 5 The effect of glycerol-3-phosphate on the control of gray mold in apples;

[0048] Figure 6 The effect of glycerol-3-phosphate on the control of apple diseases varies with the application interval;

[0049] Figure 7 The effect of glycerol-3-phosphate on the activity of defense enzymes in apple tissue;

[0050] Figure 8 The effect of glycerol-3-phosphate on the expression of disease resistance-related genes in apple tissues;

[0051] Figure 9 The effect of glycerol-3-phosphate on the activity of defense enzymes in apple tissue;

[0052] Figure 10 The effect of glycerol-3-phosphate on the content of disease-resistant substances in apple tissue;

[0053] Figure 11 The effect of glycerol-3-phosphate on the expression of disease resistance-related genes in apple tissues;

[0054] Figure 12 A mixture of glycerol-3-phosphate and Pseudomonas syringae B-1 is used for the control of gray mold in apples;

[0055] Figure 13 A mixture of glycerol-3-phosphate and Pseudomonas syringae B-1 is used to control apple ring rot. Detailed Implementation

[0056] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0057] Example 1: Control effect of different concentrations of glycerol-3-phosphate on gray mold in apples

[0058] 1. Experimental Methods

[0059] Configure 0.05, 0.1, 0.2, and 0.5 mmol·L⁻¹ -1 Multiple concentrations of glycerol-3-phosphate aqueous solution were used to activate and culture the botrytis cinerea on PDA medium for 3 days. Mycelial cakes with a diameter of 5 mm were then collected from the edge of the colony for later use.

[0060] Select healthy Fuji apples of uniform size, wash them thoroughly with running water, disinfect their surface with 75% alcohol, and then soak them in glycerol-3-phosphate aqueous solutions of different concentrations for 5-10 minutes. Allow them to air dry at room temperature. Make three puncture wounds on each apple at the equator, each approximately 5.0 mm in diameter and 3.0 mm deep. Inoculate the puncture sites with the aforementioned gray mold fungal cake. Five days after inoculation, observe and measure the diameter of the lesions to calculate the control effect.

[0061] Control effect = (diameter of lesions in control group - diameter of lesions in treatment group) / diameter of lesions in control group × 100%.

[0062] 2. Experimental Results and Analysis

[0063] The specific control effects are shown in Table 1. As can be seen from the table, the above-mentioned glycerol-3-phosphate solutions at different concentrations (0.1 mmol·L⁻¹) all showed significant control effects against apple gray mold. -1 0.2 mmol·L -1 and 0.5 mmol·L -1 The control efficacy of glycerol-3-phosphate solution against apple gray mold was 71.59%, 68.18%, and 70.45%, respectively. This shows that the control efficacy against apple gray mold is essentially maximized when the concentration of glycerol-3-phosphate solution reaches 0.1 mmol / L. Therefore, for cost-effectiveness, a concentration of 0.1 mmol / L is preferable for the use of glycerol-3-phosphate solution. -1 ~0.2mmol·L -1These two concentrations will be the primary focus of subsequent experiments.

[0064] Table 1. Control efficacy of glycerol-3-phosphate against gray mold in apples.

[0065]

[0066] Note: Different letters after the data in the same column indicate significant differences in the data.

[0067] Example 2: Glyceryl-3-phosphate has no effect on various apple disease pathogens.

[0068] 2.1 Effects of glycerol-3-phosphate on the mycelial growth of *Aureobasidium perfringens* and *Aureobasidium anthracnose*

[0069] (1) Experimental method:

[0070] Prepare PDA medium by adding an aqueous solution of glycerol-3-phosphate to a final concentration of 0.1 mmol·L⁻¹. -1 and 0.2 mmol·L -1 Activated *Botryosphaeria dothidea*, the pathogen causing apple ring rot, and *Colletotrichum gloeosporioides*, the pathogen causing anthracnose, were inoculated into the aforementioned PDA medium, respectively. Both bacteria are common pathogens in this field. The cultures were incubated in the dark at 25°C for 3–5 days, and the colony diameter was measured.

[0071] The PDA culture medium used above has the following components and formula: 200g of peeled potatoes are weighed, cut into small pieces, boiled in water for 15-20 minutes, filtered through four layers of gauze, and then 20g of glucose and 15g of agar powder are added to make up to 1000mL. The pH value is natural, and the medium is sterilized by autoclaving at 121℃ for 20 minutes.

[0072] (2) Experimental Results and Analysis

[0073] Figure 1 The results from A showed that 0.1 mmol·L -1 and 0.2 mmol·L -1 Glyceryl-3-phosphate had no significant effect on the colony diameter of *Aureobasidium perfringens* and *Aureobasidium anthracnose*, indicating that this concentration of the compound did not inhibit the mycelial growth of the pathogens.

[0074] 2.2 Effects of glycerol-3-phosphate on spore germination of *Aureobasidium perfringens* and *Aureobasidium anthracnose*

[0075] (1) Experimental method:

[0076] Prepare spore suspensions of *Pseudomonas aeruginosa* and *Pseudomonas aeruginosa* at a concentration of 10. 5pcs·mL -1 Glycerol-3-phosphate aqueous solution was added to the above spore suspensions to achieve a final concentration of 0.1 mmol·L⁻¹. -1 and 0.2 mmol·L -1 Place them at 25℃ and observe the spore germination for 12 to 24 hours.

[0077] (2) Experimental Results and Analysis

[0078] from Figure 1 The result of B shows that 0.1 mmol·L -1 and 0.2 mmol·L -1 Glyceryl-3-phosphate had no significant effect on the germination of spores of *Aureobasidium perfringens* and *Aureobasidium anthracnose*, indicating that this concentration of the compound did not inhibit the germination of pathogenic spores.

[0079] 2.3 Effects of glycerol-3-phosphate on the mycelial growth of apple tree rot fungus, anthracnose leaf blight fungus, and gray mold fungus

[0080] (1) Experimental method:

[0081] Prepare PDA medium by adding an aqueous solution of glycerol-3-phosphate to a final concentration of 0.1 mmol·L⁻¹. -1 and 0.2 mmol·L -1 Activated *Valsa mali* (causing apple tree rot), *Glomerella cingulata* (causing anthracnose leaf blight), and *Botrytis cinerea* (causing gray mold) were inoculated into the aforementioned PDA medium, respectively. All of these fungi are common pathogens in this field. The cultures were incubated in the dark at 25°C for 3–5 days, and the colony diameter was measured.

[0082] The PDA culture medium used above has the following components and formula: 200g of peeled potatoes are weighed, cut into small pieces, boiled in water for 15-20 minutes, filtered through four layers of gauze, and then 20g of glucose and 15g of agar powder are added to make up to 1000mL. The pH value is natural, and the medium is sterilized by autoclaving at 121℃ for 20 minutes.

[0083] (2) Experimental Results and Analysis

[0084] Figure 2 The results from A showed that 0.1 mmol·L -1 and 0.2 mmol·L -1 Glyceryl-3-phosphate had no significant effect on the colony diameter of the above-mentioned pathogens, indicating that this concentration of compound did not inhibit the mycelial growth of the pathogens.

[0085] 2.4 Effects of glycerol-3-phosphate on spore germination of apple tree rot fungus, anthracnose leaf blight fungus, and gray mold fungus

[0086] (1) Experimental method:

[0087] Prepare conidial suspensions of *Pseudomonas aeruginosa*, *Pseudomonas anthracnose*, and *Botrytis cinerea* at a concentration of 10. 6 pcs·mL -1 Glycerol-3-phosphate aqueous solution was added to the above spore suspensions to achieve a final concentration of 0.1 mmol·L⁻¹. -1 and 0.2 mmol·L -1 Place them at 25℃ and observe the spore germination for 12 to 24 hours.

[0088] (2) Experimental Results and Analysis

[0089] Figure 2 The results for B showed that 0.1 mmol·L -1 and 0.2 mmol·L -1 Glyceryl-3-phosphate had no significant effect on the germination of the above-mentioned pathogenic fungi spores, indicating that this concentration of compound did not inhibit the germination of pathogenic fungi spores.

[0090] Example 3: Control effect of glycerol-3-phosphate on apple ring rot, apple anthracnose, apple gray mold, and apple tree rot pathogens.

[0091] 1. Experimental Method:

[0092] Prepare 0.1 mmol·L -1 and 0.2 mmol·L -1 Glyceryl-3-phosphate aqueous solution, apple ring rot fungus, apple anthracnose fungus, apple gray mold, and apple tree rot fungus were activated and cultured on PDA medium for 3 days, and mycelial cakes with a diameter of 5 mm were collected from the edge of the colony for later use.

[0093] (1) Select healthy Fuji apples of uniform size, wash them thoroughly with running water, disinfect their surface with 75% alcohol, soak them in the above-mentioned aqueous solution for 5-10 minutes, and air dry them at room temperature. Make punctures at the equator of the apples, with each wound approximately 5.0 mm in diameter and 3.0 mm deep, and make 3 wounds per fruit. Inoculate the puncture sites with fungal cakes of apple ring rot fungus, anthracnose fungus, or gray mold fungus. Observe and measure the diameter of the lesions 5 days after inoculation, and calculate the control effect.

[0094] Control effect = (diameter of lesions in control group - diameter of lesions in treatment group) / diameter of lesions in control group × 100%.

[0095] (2) Select healthy 1-2 year old apple branches with uniform growth, and burn them with a soldering iron. The wounds should be about 3 mm long and 1 mm deep. Inoculate each wound with 20 μL of the above-mentioned glycerol-3-phosphate aqueous solution. After drying at room temperature, inoculate with the above-mentioned activated apple tree rot fungus. Use the treatment of inoculating with sterile water and then inoculating with apple tree rot fungus as a control. Observe the disease status of the branches regularly. Measure the length of the lesions 5 days after inoculation and calculate the control effect.

[0096] Control effect = (Length of lesions in control group - Length of lesions in treatment group) / Length of lesions in control group × 100%.

[0097] 2. Experimental Results and Analysis

[0098] (1) From Figure 3 and Figure 4 The results showed that the above-mentioned glycerol-3-phosphate solutions at different concentrations had significant control effects on apple ring rot and apple anthracnose, with a concentration of 0.1 mmol·L⁻¹. -1 and 0.2 mmol·L -1 The glycerol-3-phosphate solution showed control efficacy of 83.3% and 80.7% against apple ring rot, and 70.5% and 71.6% against apple anthracnose, respectively.

[0099] (2) From Figure 5 The results showed that the concentration was 0.1 mmol·L⁻¹. -1 and 0.2 mmol·L -1 The glycerol-3-phosphate solution showed control efficacy of 71.6% and 68.2% against gray mold in apples, respectively, indicating that it has a significant control effect on gray mold in apples.

[0100] (3) The results in Table 2 show that two different concentrations of glycerol-3-phosphate solution have significant control effects on apple tree canker, with a concentration of 0.1 mmol·L⁻¹. -1 and 0.2 mmol·L -1 The efficacy of the glycerol-3-phosphate solution was 50.6% and 70.6%, respectively, as shown in Table 2.

[0101] Table 2. Control efficacy of glycerol-3-phosphate against putrefactive bacteria.

[0102]

[0103] Example 4: Control effect of glycerol-3-phosphate on apple anthracnose leaf blight

[0104] 1. Experimental method: Prepare 0.1 mmol·L⁻¹ solution. -1 and 0.2 mmol·L -1A spore suspension of *Anthracnose leaf blight* fungus was prepared using an aqueous solution of glycerol-3-phosphate. The pathogen was cultured in the dark at a constant temperature on a PDA. When the mycelium reached 2 / 3 of the plate, the aerial mycelium was scraped off with an inoculation loop. Orange-yellow conidial horns were observed to appear after 2-3 days. The conidial suspension was then prepared and the concentration was adjusted to 10. 5 pcs·mL -1 .

[0105] Select one- to two-year-old apple branches with fully expanded leaves of uniform growth and spray them evenly with the above-mentioned glycerol-3-phosphate aqueous solution until water droplets fall from the leaves. Use sterile water as a control. Three days after spraying, inoculate with a suspension of apple anthracnose leaf blight spores. After planting in the field, keep the soil moist with plastic bags for 24 hours. Regularly observe and record the disease incidence on the leaves. Count the number of leaf lesions three days after inoculation and calculate the control effect.

[0106] Control effect = (Number of lesions in control group - Number of lesions in treatment group) / Number of lesions in control group × 100%.

[0107] 2. Experimental Results and Analysis

[0108] As shown in Table 3, 0.1 mmol·L -1 and 0.2 mmol·L -1 Glyceryl-3-phosphate showed control efficacy of 85.2% and 85.6% against apple anthracnose leaf blight, respectively.

[0109] Table 3. Control efficacy of glycerol-3-phosphate against apple anthracnose leaf blight

[0110]

[0111] Example 5: Changes in the control effect of glycerol-3-phosphate with application interval

[0112] 1. Experimental Methods

[0113] After treating the apple fruit according to the method in Example 1, creating wounds, 0.1 mmol·L⁻¹ was applied. -1 Glyceryl-3-phosphate aqueous solution was sprayed evenly on the fruit and allowed to air dry at room temperature. At 6h, 12h, 24h, 48h, 72h and 96h after spraying, apple anthracnose fungal cakes were inoculated at the wound site to determine the change in the disease control effect of glyceryl-3-phosphate with the time interval of its application.

[0114] 2. Experimental Results and Analysis

[0115] The results are as follows Figure 6 As shown, inoculation with pathogens at different time intervals all showed significant control effects. When the interval was greater than 72 hours, the control effect on apple anthracnose was the most significant, with a control efficacy of over 70%.

[0116] Example 6: Mechanism of disease resistance induced by glycerol-3-phosphate in different parts of apple

[0117] 6.1 Mechanism of Glycerol-3-phosphate-induced disease resistance in apple fruit

[0118] (1) Experimental method:

[0119] Apple fruits were treated according to the method in Example 1. After surface sterilization, the fruits were then subjected to 0.1 mmol·L⁻¹ water. -1 Fruits were soaked in an aqueous solution of glycerol-3-phosphate for 5–10 min, with sterile water treatment serving as a control. Samples were taken at 0, 6, 12, 24, 48, 72, and 96 h after treatment to determine the changes in the activity of defense enzymes and disease resistance-related genes in the apple fruits.

[0120] The activity of various enzymes was detected using kits from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., following the instructions in the kit's manual. The expression levels of disease resistance-related genes were detected using quantitative PCR.

[0121] (2) Experimental Results and Analysis

[0122] from Figure 7 and Figure 8 The results show that:

[0123] (1) The 0.1 mmol·L -1 After treatment of apple fruits with glycerol-3-phosphate, the activities of peroxidase (POD), superoxide dismutase (SOD), β-1,3-glucanase (GLU), and chitinase (CHI) in the tissues were significantly increased compared with the control.

[0124] (2) The expression levels of the disease course-related protein (PRs) genes PR1 and PR5 were significantly upregulated, indicating that glycerol-3-phosphate can enhance the resistance of apple fruits to ring rot and anthracnose by increasing the activity of defense enzymes in apple fruits and inducing the expression of disease resistance-related genes.

[0125] 6.2 Mechanism of disease resistance induced by glycerol-3-phosphate in apples

[0126] (1) Experimental methods

[0127] After treating the apple branches according to the method described in the previous embodiments and disinfecting the surface, spray with 0.2 mmol·L⁻¹. -1 Glycerol-3-phosphate aqueous solution; apple leaves were treated according to the method in the example, and after surface disinfection, 0.1 mmol·L⁻¹ was sprayed. -1 Glycerol-3-phosphate aqueous solution; apple fruits were treated according to the method in the examples, and after surface sterilization, the fruits were placed in 0.1 mmol·L⁻¹ water. -1Apple tissue was soaked in an aqueous solution of glycerol-3-phosphate for 5–10 min, with sterile water-treated apple tissue serving as a control. Samples were taken at 12, 24, and 48 h after treatment to determine the changes in the activity of defense enzymes, the content of disease-resistant substances, and the trends of disease-resistant genes within the apple tissue.

[0128] Methods for determining the activity of various defensive enzymes and the content of disease-resistant substances in apple tissue: The reagent kits from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd. were used, and the methods were performed in accordance with the kit instructions.

[0129] Methods for detecting disease resistance-related genes: The expression levels of each gene were detected using existing conventional quantitative PCR technology.

[0130] (2) Experimental Results and Analysis

[0131] from Figure 9 , Figure 10 and Figure 11 The results showed that: ① 0.1 mmol·L -1 Or 0.2 mmol·L -1 After treatment of apple tissues with glycerol-3-phosphate, the activities of phenylalanine ammonia-lyase (PAL) and peroxidase (POD) in the tissues were significantly increased compared with the control. At the same time, the contents of disease-resistant substances lignin and total phenols were significantly increased, and the expression levels of disease-resistant genes β-1,3-glucanase (GLU) and chitinase (CHI) were significantly upregulated. This indicates that glycerol-3-phosphate can enhance the resistance of apples to canker, anthracnose leaf blight and gray mold by increasing the activity of defense enzymes in apple fruits, promoting the accumulation of disease-resistant substances, and inducing the expression of disease-resistant genes.

[0132] Example 7: The combination of glycerol-3-phosphate and Pseudomonas syringae B-1 improves the control efficacy against apple diseases.

[0133] 7.1 Control of gray mold in apples

[0134] (1) Experimental methods

[0135] Prepare 0.2 mmol·L -1 A bacterial suspension of *Pseudomonas syringae* B-1 was prepared using an aqueous solution of glycerol-3-phosphate to achieve a final concentration of 10. 6 cfu·mL -1 Mix the two in equal volumes and set aside.

[0136] The gray mold pathogen was activated and cultured on PDA medium for 3 days. Mycelial cakes with a diameter of 5 mm were collected from the edge of the colonies for later use. Apple fruits were treated according to the method in Example 1, soaked in the above-mentioned mixture of glycerol-3-phosphate and *Pseudomonas syringae* B-1 for 5–10 min, and air-dried at room temperature. Three wounds were made on each fruit at the equator, each approximately 5.0 mm in diameter and 3.0 mm deep. The wounds were then inoculated with the above-mentioned gray mold mycelial cakes. A 0.2 mmol·L⁻¹ solution was used. -1 Glyceryl-3-phosphate, 10 6 cfu·mL -1 *Pseudomonas syringae* B-1 and sterile water treatment were used as controls. Five days after inoculation, the diameter of the lesions was observed and measured, and the control effect was calculated.

[0137] Control effect = (diameter of lesions in control group - diameter of lesions in treatment group) / diameter of lesions in control group × 100%.

[0138] (2) Experimental Results and Analysis

[0139] All the above treatments showed significant control effects against apple gray mold. The control efficiencies of glycerol-3-phosphate solution and *Pseudomonas syringae* against apple gray mold were 70% and 71.35%, respectively, while the combined treatment achieved a control efficacy as high as 87.5%. (See results below.) Figure 12 .

[0140] 7.2 Prevention and control of apple ring rot

[0141] (1) Experimental methods

[0142] Preparation concentration of 10 5 cfu·L -1 A bacterial suspension of the biocontrol bacterium *Pseudomonas syringae* B-1 was mixed with a glycerol-3-phosphate solution to achieve a final concentration of 0.1 mmol·L⁻¹. -1 .

[0143] Healthy apple fruits were selected, and after surface disinfection, wounds were made. Apple ring rot fungus was inoculated with the fungal cake according to the method in Example 1 above. Glycerol-3-phosphate treatment alone and biocontrol B-1 treatment alone were used as controls. The control effect was calculated according to the formula in Example 1.

[0144] (2) Experimental Results and Analysis

[0145] The results are as follows Figure 13As shown, glycerol-3-phosphate significantly enhances the control effect of *Pseudomonas syringae* B-1. The control effects of glycerol-3-phosphate and *Pseudomonas syringae* B-1 alone on apple ring rot were 74.2% and 82.3%, respectively. The control effect of the combined treatment of glycerol-3-phosphate and *Pseudomonas syringae* B-1 on apple ring rot reached 94.4%. Therefore, glycerol-3-phosphate has a significant synergistic effect on *Pseudomonas syringae* B-1, greatly improving its control effect on apple ring rot.

[0146] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. The application of glycerol-3-phosphate in improving the control efficacy of biocontrol fungus *Pseudomonas syringae* B-1 against apple diseases, characterized in that... The preservation number of *Pseudomonas syringae* B-1 is CCTCC NO: M2015813; the application method is as follows: glycerol-3-phosphate is prepared to a concentration of 0.2 mmol·L⁻¹. -1 A bacterial suspension of *Pseudomonas syringae* B-1 was prepared using an aqueous solution of 10 ppm. 5 cfu·L -1 -10 6 cfu·L -1 The bacterial suspension is mixed with an equal volume of glycerol-3-phosphate aqueous solution before use.

2. A compound biocontrol agent, characterized in that, include: The bacterial suspension of *Pseudomonas syringae* B-1 and glycerol-3-phosphate as described in claim 1, wherein the preservation number of *Pseudomonas syringae* B-1 is CCTCC NO: M2015813; the final concentration of *Pseudomonas syringae* B-1 is 10. 5 cfu·L -1 -10 6 cfu·L -1 The concentration of the glycerol-3-phosphate is 0.2 mmol·L⁻¹. -1 An aqueous solution of both, mixed in equal volumes.

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