Application of a plant disease resistance activator with glycerol-3-phosphate as active ingredient to improve disease resistance of fruits and vegetables

Abstract

The application discloses application of a plant disease resistance activator with glycerol-3-phosphate as an active ingredient to improving disease resistance of fruits and vegetables, and also discloses application of glycerol-3-phosphate to disease prevention and treatment of fruits and vegetables, wherein the diseases of the fruits and vegetables include tomato gray mold, blueberry gray mold, blueberry anthracnose, pear ring rot or pear anthracnose. The application further provides a method for inducing disease resistance of fruits and vegetables, which comprises applying a solution of glycerol-3-phosphate or the plant disease resistance activator to the fruits and vegetables by spraying or infiltrating fruit and vegetable tissues. The application further provides application of glycerol-3-phosphate to improving the disease prevention and treatment effect of a biocontrol bacterium Staphylococcus equorum F1 on pear diseases, and a composite biocontrol bacterium agent composed of glycerol-3-phosphate and the Staphylococcus equorum F1. The glycerol-3-phosphate has the characteristics of long disease resistance duration, wide disease resistance spectrum and safety and environmental protection when applied as the disease resistance activator to the prevention and treatment of various diseases of the fruits and vegetables, and is a new strategy and feasible way for plant disease prevention and treatment.

Description

Technical Field

[0001] This invention relates to the field of plant disease control technology, and in particular to the application of a plant disease resistance activator with glycerol-3-phosphate as the active ingredient in improving the disease resistance of fruits and vegetables. Background Technology

[0002] my country has the world's largest planting area for vegetables and fruits, making them crucial pillars of the country's economic growth and farmers' income. However, various diseases severely restrict the healthy and sustainable development of my country's vegetable and fruit industries, resulting in a low overall export rate for Chinese fruits and vegetables. In particular, gray mold, anthracnose, and ring rot not only damage fruits and vegetables during their growing season but also during storage and transportation, and even under low-temperature conditions. These are the most serious diseases affecting fruit and vegetable production and storage; the incidence rate of these diseases directly translates to the loss rate, and these diseases can cause significant economic losses every year.

[0003] Currently, the control of diseases in vegetables and fruit trees mainly relies on chemical agents, ensuring that the crop surface is protected with pesticides before each rainfall. However, this inevitably leads to the large-scale and frequent use of chemical fungicides, posing a significant threat to food safety and easily resulting in the emergence of drug-resistant pathogens, damaging the ecological environment of vegetable and orchards. Therefore, many countries have already banned or prohibited a large number of highly toxic chemical fungicides for the control of fruit and vegetable diseases. Therefore, finding safe and efficient control products to replace chemical fungicides is an urgent problem to be solved, especially in the control of critical diseases in vegetable and fruit production.

[0004] Numerous studies have shown that controlling diseases by stimulating the plant's own defense system is an important approach to developing efficient, safe, and green pesticides. Plant activators are compounds that can induce disease resistance in plants. Currently, several commercially available plant activators are being used on crops such as tomatoes, grapes, and tobacco, achieving significant disease control effects.

[0005] However, the variety of disease resistance activators currently available on the market is limited, and effective disease resistance activators for fruits and vegetables are still lacking, requiring further development. Therefore, existing technologies need further improvement. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides an application of a plant disease resistance activator with glycerol-3-phosphate as the active ingredient in improving the disease resistance of fruits and vegetables, as well as a method for inducing disease resistance in fruits and vegetables. It also provides the application of glycerol-3-phosphate in controlling various fruit and vegetable diseases. Furthermore, glycerol-3-phosphate can be compounded with Staphylococcus equi as a compound biocontrol agent to further improve the control effect on various diseases of tomatoes, blueberries, pears, and other fruits and vegetables.

[0007] To address the above problems, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides an application of a plant disease resistance activator in improving the disease resistance of fruits and vegetables, wherein the active ingredient of the plant disease resistance activator is glycerol-3-phosphate.

[0009] The structure of the glycerol-3-phosphate is as follows:

[0010]

[0011] Currently, glycerol-3-phosphate is mainly used in the pharmaceutical industry, laboratories, and food and nutrition fields, such as as a vaccine adjuvant, as a nutritional additive, and as an intermediate in the preparation of other substances, and has a wide range of applications in the market.

[0012] However, during the research and development process, the inventors discovered that glycerol-3-phosphate also has the ability to activate the disease resistance of tomatoes, pears and blueberries to different diseases, that is, it has the biological activity of plant disease resistance activator. This activity gives glycerol-3-phosphate great application prospects in the biological control of fruit and vegetable diseases.

[0013] Experimental findings: Glyceryl-3-phosphate can enhance the activity of defense enzymes in tomato, pear and blueberry fruits, namely PAL, POD, SOD and PPO, and can also improve the expression of disease resistance genes, namely GLU and CHI; glyceryl-3-phosphate can also promote the accumulation of disease resistance substances in tomato, pear and blueberry fruits, namely lignin and total phenols.

[0014] Further experiments revealed that tomatoes, pears, and blueberries treated with glycerol-3-phosphate solution exhibited good resistance to various fruit and vegetable diseases, with this resistance being long-lasting and broad-spectrum. If stored, the effective period could reach more than four weeks. For example, tomatoes, blueberries, and pears treated with glycerol-3-phosphate showed significant control effects against gray mold, anthracnose, and ring rot, with control efficacy exceeding 65%, resulting in high economic benefits. Furthermore, this treatment is safe and environmentally friendly and does not lead to the emergence of drug-resistant pathogens.

[0015] Optionally, in the application, the fruits and vegetables include tomatoes, blueberries, and pears. In this embodiment of the invention, tomatoes, blueberries, and pears are used as representative fruits and vegetables for the experiment, but the invention is not limited to these three.

[0016] Based on the newly discovered biological activities 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.

[0017] Secondly, the present invention also provides the application of glycerol-3-phosphate in the prevention and control of fruit and vegetable diseases.

[0018] In this application, the fruit and vegetable diseases include: tomato gray mold, blueberry gray mold, blueberry anthracnose, pear ring rot, or pear anthracnose. Subsequent embodiments of this invention use the above-mentioned fruit and vegetable diseases as representatives in experiments, but the types of fruit and vegetable diseases are not limited to these.

[0019] Experiments have shown that, based on the activity of disease resistance activators, glycerol-3-phosphate can induce resistance in tomatoes, pears, and blueberries to the following fruit and vegetable diseases: tomato gray mold, pear anthracnose and ring rot, and blueberry gray mold and anthracnose. This demonstrates that glycerol-3-phosphate has a broad spectrum of applications in inducing disease resistance in fruits and vegetables. The pathogens of tomato gray mold, pear anthracnose and ring rot, and blueberry gray mold and anthracnose are, respectively, *Botrytis cinerea*, *Glomerella cingulata*, *Botryosphaeria dothidea*, *Botrytis cinerea*, and *Colletotrichum gloeosporioides*.

[0020] Experiments have shown that glycerol-3-phosphate can enhance the activity of defense enzymes in tomatoes, pears, and blueberries, increase the accumulation of disease-resistant substances, and induce the expression of disease-resistant genes.

[0021] Thirdly, the present invention also provides a method for inducing disease resistance in fruits and vegetables, wherein the method comprises applying the aforementioned glycerol-3-phosphate solution or the aforementioned plant disease resistance activator by spraying or soaking the fruit and vegetable tissues. The fruit and vegetable tissues include fruits or branches, etc.

[0022] Preferably, the fruit and vegetable tissue is fruit, and the harvested fruit and vegetables can be preserved by spraying or soaking the fruit to prevent the harvested fruit from being attacked by diseases, thereby achieving the preservation effect for more than 4 weeks.

[0023] Fourthly, this invention also provides the application of glycerol-3-phosphate in the preservation of fruits and vegetables after harvest. Based on the aforementioned disease prevention and control of fruits after harvest, glycerol-3-phosphate can be used as a fruit preservative to extend the storage time of fruits and vegetables, effectively avoiding losses caused by disease and rot during storage.

[0024] Optionally, in the method, the concentration of the glycerol-3-phosphate solution is 0.1 mmol·L⁻¹. -1 ~0.2mmol·L -1 .

[0025] Specifically, the fruits and vegetables include tomatoes, pears, or blueberries.

[0026] Optionally, the above method is as follows: 0.1 mmol·L -1 ~0.2mmol·L -1 Spray a glycerol-3-phosphate solution onto the surface of fruits and vegetables until water droplets run off; or, immerse the fruit in a solution with a concentration of 0.1 mmol·L⁻¹. -1 ~0.2mmol·L -1 A solution of glycerol-3-phosphate.

[0027] Preferably, the soaking time is at least 5 minutes. More optionally, the soaking time is controlled between 5 and 10 minutes.

[0028] 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.

[0029] Fifthly, this application also provides the application of glycerol-3-phosphate in improving the control effect of biocontrol bacteria Staphylococcus equorum F1 on pear diseases, wherein the accession number of Staphylococcus equorum F1 is CGMCC No. 21658.

[0030] Staphylococcus equorum F1 was disclosed in patent CN 114467975B, and detailed information about this strain can be found in that patent.

[0031] Optionally, the application method is to mix the bacterial suspension of Staphylococcus equorum F1 before use.

[0032] Optionally, the pear diseases are pear ring rot and pear anthracnose. Experiments have shown that the control efficacy of glycerol-3-phosphate and Staphylococcus equorum F1 against pear anthracnose is 65.85% and 43.90%, respectively, while the control efficacy of the combination of the two is as high as 86.59%; the control efficacy of glycerol-3-phosphate and Staphylococcus equorum F1 against pear ring rot is 66.67% and 47.44%, respectively, while the control efficacy of the combination of the two is as high as 88.46%; these all indicate that the combination of glycerol-3-phosphate and Staphylococcus equorum F1 has a synergistic effect and can significantly improve the control efficacy of Staphylococcus equorum F1 against a variety of diseases (such as pear ring rot and pear anthracnose).

[0033] In a sixth aspect, the present invention also provides a composite biocontrol agent comprising: the aforementioned Staphylococcus equorum F1 and glycerol-3-phosphate.

[0034] Seventhly, this application also provides the application of the aforementioned compound biocontrol agent in the prevention and control of plant diseases.

[0035] The present invention has the following beneficial effects:

[0036] 1. This invention discovers that glycerol-3-phosphate has the activity of stimulating plant disease resistance and can be used as an active ingredient of plant disease resistance activator to induce fruits and vegetables such as tomatoes, pears and blueberries to develop resistance to a variety of diseases. It can be developed into a plant disease resistance activator for the prevention and control of a variety of diseases in fruits and vegetables.

[0037] 2. Compared with traditional pesticides, the disease resistance activator provided by this invention has the following advantages:

[0038] (1) Glyceryl-3-phosphate itself has no bactericidal or bacteriostatic effect in vitro. It can only induce the plant's own immune system to resist the invasion of diseases under living conditions. The disease resistance induced by glyceryl-3-phosphate is a physiological metabolic response of the plant itself. The use of glyceryl-3-phosphate can also increase the activity of defense enzymes PAL, POD, SOD and PPO in tomatoes, pears and blueberries, and can induce a significant upregulation of disease resistance-related genes CLU and CHI. It also promotes the accumulation of disease resistance substances such as lignin and total phenols in the above fruits.

[0039] (2) The disease resistance induced in fruit trees has long-lasting and broad-spectrum characteristics. Tomatoes, pears and blueberries treated with glycerol-3-phosphate have significant control effects against tomato gray mold, pear anthracnose and ring rot, and blueberries gray mold and anthracnose. The control effect against tomato gray mold is 67.10%, the control effect against pear anthracnose and ring rot is 71.76% and 69.74% respectively, and the control effect against blueberry gray mold and anthracnose is 71.43% and 75.00% respectively, which has high economic benefits.

[0040] (3) The use of glycerol-3-phosphate can improve the control effect of biocontrol bacteria Staphylococcus equorum F1 on pear ring rot and pear anthracnose.

[0041] (4) It has the advantages of simple application method, low concentration, small dosage, environmental protection and safety; it also has the advantages of long resistance duration, broad disease resistance spectrum and no drug resistance. It is considered a new strategy and feasible approach for the prevention and control of plant diseases. Attached Figure Description

[0042] Figure 1 The control effects of different concentrations of glycerol-3-phosphate on pear ring rot;

[0043] Figure 2 Effects of glycerol-3-phosphate on mycelial growth and spore germination of *Gyromitra esculenta*, *Anthracnose causal agent*, and *Rhizoctonia solani*. A is colony diameter, B is spore germination rate.

[0044] Figure 3 The effect of glycerol-3-phosphate on the control of gray mold in blueberries;

[0045] Figure 4 The effect of glycerol-3-phosphate on the prevention and control of blueberry anthracnose;

[0046] Figure 5 Effects of glycerol-3-phosphate on the activity of defense enzymes in tomato, blueberry, and pear fruits;

[0047] Figure 6 The effect of glycerol-3-phosphate on the content of disease-resistant substances in tomato, blueberry and pear fruits;

[0048] Figure 7 The effect of glycerol-3-phosphate on the expression of disease resistance-related genes in tomato, blueberry, and pear fruits;

[0049] Figure 8 The effect of glycerol-3-phosphate mixed with Staphylococcus equi F1 on the prevention and control of pear ring rot. Detailed Implementation

[0050] 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.

[0051] Example 1: Control effect of different concentrations of glycerol-3-phosphate on pear ring rot

[0052] 1. Experimental Methods

[0053] (1) Prepare 0.05 mmol·L⁻¹ solutions respectively. -1 0.1 mmol·L -1 0.2 mmol·L -1 0.5 mmol·L -1 Pear ring rot pathogen was activated and cultured on PDA medium for 3 days using glycerol-3-phosphate aqueous solutions of various concentrations. Mycelial cakes with a diameter of 5 mm were then collected from the edge of the colony for later use.

[0054] (2) Select healthy pears of uniform size, wash them 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 wounds at the equator of the pears, each wound approximately 5.0 mm in diameter and 3.0 mm deep, with 3 wounds per fruit. Inoculate the wounds with the fungal cakes of the aforementioned ring rot pathogen. Observe and measure the diameter of the lesions 3-5 days after inoculation, and calculate the control effect.

[0055] Control effect = (diameter of lesions in control group - diameter of lesions in treatment group) / diameter of lesions in control group × 100%.

[0056] 2. Experimental Results and Analysis

[0057] For specific results on the preventive efficacy, please see Figure 1 As shown in the figure, the above-mentioned glycerol-3-phosphate solutions of different concentrations all have a certain control effect on pear ring rot, with concentrations of 0.1 mmol·L⁻¹. -1 0.2 mmol·L -1 and 0.5 mmol·L -1 The glycerol-3-phosphate solution showed more significant efficacy against pear ring rot, with efficacy rates of 65.79%, 69.74%, and 75.00%, respectively. This indicates that the glycerol-3-phosphate solution concentration is significantly higher at 0.1 mmol / L. -1 Subsequently, its efficacy against pear ring rot was only slightly improved. Therefore, to save costs, the optimal concentration of glycerol-3-phosphate solution is 0.1 mmol·L⁻¹. -1 ~0.2mmol·L -1 These two concentrations were subsequently used in the experiments.

[0058] Example 2: Glyceryl-3-phosphate has no effect on the growth and spore germination of various fruit and vegetable pathogens.

[0059] 2.1 Effects of glycerol-3-phosphate on the mycelial growth of *Gyromitra esculenta* (tomato gray mold), *Anthracnose causal agent* (blueberry anthracnose), and *Pyrus pyriformis* (pear ring rot).

[0060] (1) Experimental methods

[0061] ① Preparation of PDA medium supplemented with glycerol-3-phosphate: Weigh 200g of peeled potatoes, cut them into small pieces, boil them in water for 15-20 minutes, filter through four layers of gauze, add 20g of glucose and 15g of agar powder, and bring the volume to 1000mL. The pH should be natural. Add an aqueous solution of glycerol-3-phosphate to the above medium to make the final concentration 0.1 mmol·L⁻¹. -1 and 0.2 mmol·L -1 Sterilize at 121℃ with high-pressure steam for 20 minutes.

[0062] ② Activation of pathogens: Botrytis cinerea (tomato gray mold), Colletotrichum gloeosporioides (blueberry anthracnose), and Botryosphaeria dothidea (pear ring rot) were activated.

[0063] ③ Inoculate activated *Botrytis cinerea* (tomato gray mold), *Colletotrichum gloeosporioides* (blueberry anthracnose causal agent), and *Botryosphaeria dothidea* (pear ring rot causal agent) into the above-mentioned PDA medium, respectively. Incubate at a constant temperature of 25℃ in the dark for 3–5 days, and measure the colony diameter.

[0064] (2) Experimental Results and Analysis

[0065] Figure 2 Result 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.

[0066] 2.2 Effects of glycerol-3-phosphate on spore germination of *Gyromitra sesquiterpenes* (tomato gray mold), *Anthracnose spores* (blueberry anthracnose), and *Pyrus pyriformis* (pear ring rot).

[0067] (1) Experimental methods

[0068] Prepare conidial suspensions of *Gyromitra sesquiterpenes* (tomato gray mold), *Anthracnose causal agent* (blueberry anthracnose), and *Pyrus pyriformis* (pear ring rot) at a concentration of 10. 6 L -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.

[0069] (2) Experimental Results and Analysis

[0070] Figure 2 Result B showed: 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.

[0071] Example 3: Control effect of glycerol-3-phosphate on tomato gray mold and pear anthracnose

[0072] 1. Experimental Method:

[0073] Prepare 0.1 mmol·L -1 and 0.2 mmol·L -1 Glycerol-3-phosphate aqueous solution; tomato gray mold and pear anthracnose 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.

[0074] Select uniformly sized and healthy tomato and pear fruits, wash them thoroughly with running water, disinfect their surfaces with 75% alcohol, and then soak them in glycerol-3-phosphate aqueous solutions of different concentrations for 5-10 minutes, followed by air drying at room temperature. Make two puncture wounds on each tomato fruit at the equator, approximately 5.0 mm in diameter and 1.0 mm deep, and inoculate the wounds with the aforementioned tomato gray mold fungal cake. Make three puncture wounds on each pear fruit at the equator, approximately 5.0 mm in diameter and 3.0 mm deep, and inoculate the wounds with the aforementioned pear anthracnose fungal cake. Observe and measure the diameter of lesions 3-5 days after inoculation to calculate the control effect.

[0075] Control effect = (diameter of lesions in control group - diameter of lesions in treatment group) / diameter of lesions in control group × 100%.

[0076] 2. Experimental Results and Analysis

[0077] The results in Table 1 show that the concentration is 0.1 mmol·L⁻¹. -1 and 0.2 mmol·L -1 The glycerol-3-phosphate solutions at different concentrations showed control efficacy of 65.98% and 67.10% against tomato gray mold, and 67.42% and 71.76% against pear anthracnose, respectively. These results indicate that the glycerol-3-phosphate solutions at different concentrations have significant control effects against both tomato gray mold and pear anthracnose.

[0078] Table 1. Control effects of glycerol-3-phosphate on tomato gray mold and pear anthracnose.

[0079]

[0080] Example 4: Control effect of glycerol-3-phosphate on blueberry gray mold and blueberry anthracnose

[0081] 1. Experimental Method:

[0082] (1) Prepare a spore suspension of the blueberry gray mold fungus. Incubate the pathogen on a PDA at a constant temperature. After the hyphae have fully colonized the petri dish, continue incubation at a constant temperature for 7 days. Wash the petri dish with sterile water, then filter the conidia suspension through lens paper and adjust the concentration to 10. 6 pcs·mL -1 .

[0083] (2) Preparation of blueberry anthracnose spore suspension: The pathogen was cultured in the dark at a constant temperature on a PDA. When the mycelium grew to 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 anthracnose spore suspension was prepared and the concentration was adjusted to 10. 5 pcs·mL -1 .

[0084] (3) Prepare 0.1 mmol·L -1 and 0.2 mmol·L -1 Glycerol-3-phosphate aqueous solution: Select healthy blueberries of uniform size and spray them evenly with the above glycerol-3-phosphate aqueous solution until water droplets fall from the surface of the fruit. Use sterile water as a control.

[0085] (4) Two days after spraying glycerol-3-phosphate, inoculate with blueberry gray mold and blueberry anthracnose spore suspension and moisturize. Regularly observe and record the disease incidence of the fruit, count the fruit incidence rate, and calculate the control effect respectively.

[0086] Prevention and control effect = (incidence rate in control group - incidence rate in treatment group) / incidence rate in control group × 100%.

[0087] 2. Experimental Results and Analysis

[0088] (1) Experimental results showed that the incidence rate in the control group was 93.33%, while that in the control group was 0.1 mmol·L⁻¹. -1 and 0.2 mmol·L -1 The incidence rates of diseases treated with glycerol-3-phosphate were 31.11% and 26.67%, respectively, and their calculated control efficiencies were 66.67% and 71.43%, respectively. (See results below.) Figure 3 This result indicates that 0.1 mmol·L -1 and 0.2 mmol·L -1 Glyceryl-3-phosphate has a significant effect on the control of gray mold in blueberries.

[0089] (2) The results showed that the incidence rate in the control group was 84.44%, while that in the control group was 0.1 mmol·L⁻¹. -1 and 0.2 mmol·L -1 The incidence rates in the glycerol-3-phosphate treatment groups were 24.44% and 21.11%, respectively, and their control efficiencies against blueberry gray mold were calculated to be 71.05% and 75.00%, respectively. (See details...) Figure 4 The experimental results show that 0.1 mmol·L -1 and 0.2 mmol·L -1 Glyceryl-3-phosphate has a significant effect on the control of gray mold in blueberries.

[0090] Example 5: Mechanism of glycerol-3-phosphate inducing disease resistance in tomato, blueberry, and pear fruits

[0091] 1. Experimental Method:

[0092] The pear fruits were treated according to the method in Example 1. After surface disinfection, the fruits were placed in a solution of 0.2 mmol·L⁻¹. -1 Soak in an aqueous solution of glycerol-3-phosphate for 5–10 min; treat tomato fruits according to the method in Example 3, and after surface sterilization, immerse the fruits in 0.1 mmol·L⁻¹ water. -1 Soak in an aqueous solution of glycerol-3-phosphate for 5–10 min; treat blueberry fruits according to the method in Example 4, and after surface disinfection, spray with 0.2 mmol·L⁻¹. -1 Glycerol-3-phosphate aqueous solution; pear, tomato, and blueberry fruits treated with sterile water were used as controls. Samples were taken at 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 in each fruit.

[0093] Methods for determining the activity of various defensive enzymes and the content of disease-resistant substances in pear tissues: The reagent kits from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd. were used, and the methods were performed in accordance with the kit instructions.

[0094] Methods for detecting disease resistance-related genes: Quantitative PCR technology is used to detect gene expression levels.

[0095] 2. Experimental Results and Analysis

[0096] (1) From Figure 5 The results show that the 0.1 mmol·L -1 Or 0.2 mmol·L -1 Treatment of tomatoes, pears, and blueberries with glycerol-3-phosphate significantly increased the activities of phenylalanine ammonia-lyase (PAL), peroxidase (POD), superoxide dismutase (SOD), and polyphenol oxidase (PPO) in their tissues compared to the control, reaching 1.85–3.58 times the control level.

[0097] (2) From Figure 6 The results showed that, compared with the control, the accumulation of lignin and total phenols, disease-resistant substances, in the various fruit tissues was significantly increased after glycerol-3-phosphate treatment, ranging from 1.54 to 2.08 times that of the control.

[0098] (3) From Figure 7 The results showed that, compared with the control, the expression levels of the disease resistance genes β-1,3-glucanase (GLU) and chitinase (CHI) in various fruit tissues were significantly upregulated after glycerol-3-phosphate treatment, ranging from 2.54 to 6.54 times that of the control.

[0099] The above experimental results indicate that glycerol-3-phosphate can enhance the resistance of tomatoes, pears, and blueberries to gray mold, ring rot, and anthracnose by increasing the activity of defense enzymes in the fruits, increasing the accumulation of disease-resistant substances, and inducing the expression of disease-resistant genes.

[0100] Example 6: The combination of glycerol-3-phosphate and Staphylococcus equi F1 enhances the control efficacy against pear ring rot.

[0101] 1. Experimental Method:

[0102] (1) Prepare 0.2 mmol·L -1 A glycerol-3-phosphate aqueous solution was used to prepare a bacterial suspension of Staphylococcus equi F1 to a final concentration of 10. 6 cfu·L -1 Mix the two in equal volumes and set aside.

[0103] (2) Activate and culture the ring spot fungus and anthrax fungus on PDA medium for 3 days, and collect 5 mm diameter mycelial cakes from the edge of the colony for later use.

[0104] (3) Pear fruits were treated according to the method in Example 1. The experimental group fruits were soaked in the above-mentioned mixture of glycerol-3-phosphate and Staphylococcus equi F1 for 5-10 minutes and then air-dried at room temperature. Wounds were made at the equator of the pear, each wound approximately 5.0 mm in diameter and 3.0 mm deep, with 3 wounds per fruit. The above-mentioned fungal cakes of ring rot fungus and anthracnose fungus were inoculated at the wounds. Five days after inoculation, the diameter of the lesions was observed and measured, and the control effect was calculated.

[0105] In addition, multiple control groups were set up, each using 0.1 mmol·L⁻¹. -1 Glyceryl-3-phosphate, 10 6 cfu·L -1 Staphylococcus equi F1 or sterile water treatment, with other procedures the same as the experimental group.

[0106] 2. Experimental Results and Analysis

[0107] (1) The results in Table 2 show that all treatments except the sterile water treatment group have significant control effects on pear anthracnose. The control efficacy of the glycerol-3-phosphate treatment group and the Staphylococcus equi F1 treatment group against pear anthracnose is 65.85% and 43.90%, respectively, while the control efficacy of the experimental group using both is as high as 86.59%.

[0108] (2) Figure 8 The results showed that the control efficacy against pear ring rot was 66.67% and 47.44% for the glycerol-3-phosphate treatment group and the Staphylococcus equi F1 treatment group, respectively, while the control efficacy against pear ring rot was as high as 88.46% for the experimental group that used both treatments.

[0109] The above experimental results indicate that glycerol-3-phosphate can significantly improve the control effect of Staphylococcus equi F1 on pear anthracnose and ring spot disease.

[0110] Table 2. Control effect of glycerol-3-phosphate mixed with Staphylococcus equi F1 on pear anthracnose.

[0111]

[0112] 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. Glyceryl-3-phosphate is used to enhance the biocontrol of Staphylococcus equi ( Staphylococcus equorum The application of F1 in the control of pear diseases is characterized by, The Staphylococcus equi ( Staphylococcus equorum The accession number for F1 is CGMCC No. 21658; the application method is as follows: glycerol-3-phosphate is prepared to a concentration of 0.2 mmol·L⁻¹. -1 Staphylococcus equi F1 was prepared into a bacterial suspension using an aqueous solution, the final concentration of which was 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: Staphylococcus equi as described in claim 1 ( Staphylococcus equorum The final concentration of F1 was 10. 6 cfu·L -1 The bacterial suspension and glycerol-3-phosphate concentration were 0.2 mmol·L⁻¹. -1 An aqueous solution of both, mixed in equal volumes.

3. The application of the compound biocontrol agent as described in claim 2 in the prevention and control of plant diseases.

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