Lysozyme injection for inhibiting Candidatus Liberibacter asiaticum and use method and application thereof

Abstract

The invention relates to the field of plant disease control, in particular to a lysozyme injection for inhibiting Candidatus Liberibacter asiaticum as well as a use method and application of the lysozyme injection. Every 500 mL of the lysozyme injection contains more than or equal to 2 g of lysozyme, 1-2 g of potassium nitrate, 1-2 g of monopotassium phosphate, 0.5-1 g of chelated magnesium and the balance of water. The lysozyme injection can effectively inhibit citrus liberibacter asiaticum and related bacteria sinorhizobium fredii of the citrus liberibacter asiaticum. The invention not only provides a good basis for developing new drugs for preventing and treating citrus huanglongbing, but also can reduce the use of chemical pesticides and delay the pest resistance, and is beneficial to maintaining the balance of an ecological system and the sustainable development of agriculture.

Description

Technical Field

[0001] The present invention relates to the field of plant disease prevention and control, and in particular to a lysozyme injection for inhibiting citrus Huanglongbing fungus, and a use method and application thereof. Background Art

[0002] Citrus Huanglongbing (Huanglongbing) is currently the most devastating disease affecting citrus production, often referred to as the "cancer" of citrus. Newly infected trees typically die within one to two years, while older trees die or lose their fruiting capacity within three to five years. In severe cases, orchards can be destroyed. The disease is extremely widespread, occurring and spreading in over 50 countries and regions, including Asia, Africa, and South America. The causative agent of citrus Huanglongbing is a phloem-localized, Gram-negative bacterium, divided into three distinct species: the Asian species (Candidatus Liberibacter asiaticus), the American species (Candidatus Liberibacter americanus), and the African species (Candidatus Liberibacter africanus). However, the Asian species remains uncultivated. The Asian species is the primary cause of citrus Huanglongbing in China and is the most widespread worldwide. Its primary vector is the Asian citrus psyllid (Diaphorina citri Kuwayama), which is found in most citrus-producing regions worldwide. Currently, the main strategies for preventing and controlling citrus Huanglongbing include: using disease-free citrus seedlings for propagation, controlling its vector, the citrus psyllid, removing infected trees, breeding for Huanglongbing resistance / tolerance, thermal treatment for Huanglongbing, and chemical treatment. Propagating disease-free citrus seedlings requires a strictly isolated, virus-free nursery environment, regular pathogen testing, and specialized virus removal techniques such as stem-tip grafting, which are unaffordable for smallholder farmers. Controlling the vector, the citrus psyllid, can effectively curb the spread of citrus Huanglongbing, but it cannot fundamentally prevent it. While removing infected citrus plants can somewhat control the spread of citrus Huanglongbing, removing infected plants and replanting disease-free seedlings is costly and time-consuming for citrus production. The development of transgenic varieties resistant / tolerant to citrus Huanglongbing requires a lengthy safety assessment process, including whether these transgenic varieties could cause gene drift and lead to the emergence of superweeds, threatening the ecological environment, and whether they could affect the structure and population of soil rhizosphere microorganisms, thereby damaging the soil ecological environment. Citrus Huanglongbing is a systemic disease, and heat treatment is only effective for the above-ground parts of citrus, with less effect on the underground root system. Chemical treatment with antibiotics such as tetracycline and oxytetracycline only temporarily restores healthy growth to the plant, but the disease will recur after 1-3 years. Furthermore, antibiotics may carry the risk of residual contamination and drug resistance. Further research is needed to use antibiotics to treat citrus Huanglongbing.

[0003] Lysozymes are a class of enzymes widely found in animals, plants, and microorganisms. Their primary function is to destroy bacterial cell walls, leading to bacterial death. They can partially replace the function of antibiotics, making bacteria sensitive to lysozymes. Due to this property, egg white lysozyme (hereafter referred to as lysozyme) is used as a food additive, particularly in meat and dairy products, as a natural preservative. Most research on lysozymes has focused on medical applications. As an enzymatic antimicrobial, lysozymes participate in the metabolism of mucopolysaccharides and, when combined with oral and topical medications, can exert anti-inflammatory effects against microbial infections. However, in agriculture, the use of this enzyme for the prevention and treatment of citrus Huanglongbing has not been reported. Summary of the Invention

[0004] The purpose of the present invention is to provide a lysozyme injection for inhibiting citrus Huanglongbing fungus and a use method and application thereof.

[0005] To achieve the above object, the present invention adopts the following technical solutions: A lysozyme injection for inhibiting citrus Huanglongbing fungus, wherein each 500 mL of the lysozyme injection contains 2g or more of lysozyme, 1-2g of potassium nitrate, 1-2g of potassium dihydrogen phosphate, 0.5-1g of chelated magnesium, and the remainder is water; Furthermore, the lysozyme includes but is not limited to egg white lysozyme, animal lysozyme, plant lysozyme, microbial lysozyme, and bacterial phage lysozyme, and the chelated magnesium includes but is not limited to citrate chelated magnesium, EDTA chelated magnesium, amino acid chelated magnesium, and sugar alcohol chelated magnesium.

[0006] The preparation method of the lysozyme injection comprises the following steps: taking 2g or more of lysozyme, 1-2g of potassium nitrate, 1-2g of potassium dihydrogen phosphate and 0.5-1g of chelated magnesium, and preparing a mixed solution of 500ml with water to obtain the lysozyme injection.

[0007] Use of the above-mentioned lysozyme injection in preventing and treating citrus Huanglongbing; Furthermore, lysozyme injection was used on citrus trees using the hanging bottle infusion method; Furthermore, a hole is drilled in the main trunk of the citrus tree 15 to 20 cm above the ground, and the lysozyme injection solution is placed in a hanging bottle with an infusion tube and an infusion tube needle. The hanging bottle is then hung on a branch above the drill hole, and the infusion tube needle is inserted into the drill hole, so that the lysozyme injection solution is transported into the citrus tree through the drill hole.

[0008] The application of the above-mentioned lysozyme injection in inhibiting citrus Huanglongbing fungus.

[0009] The invention relates to an application of the above-mentioned lysozyme injection in inhibiting Sinorhizobium freundii.

[0010] The significant advantages of the present invention are: This study is the first to use lysozyme, a preservative used in the food industry, in research on the prevention and treatment of citrus Huanglongbing. The study found that lysozyme injection effectively inhibits the titer of the citrus Huanglongbing bacteria. This study not only provides a good foundation for the development of new drugs to inhibit citrus Huanglongbing, but also reduces the use of chemical pesticides, delays the development of pest resistance, and contributes to maintaining ecosystem balance and sustainable agricultural development. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 :Lysozyme injection was used in sweet orange trees infected with citrus Huanglongbing disease by hanging bottle infusion method.

[0012] Figure 2 : The changes in the content (titer) of citrus Huanglongbing disease in sweet orange trees infected with citrus Huanglongbing disease were analyzed by real-time fluorescence quantitative analysis before and after application of lysozyme solution or lysozyme injection.

[0013] Figure 3 :The results of the inhibition zone experiment of lysozyme injection on Sinorhizobium freundii.

[0014] Figure 4 : Statistical graph of the diameter of the inhibition zone of lysozyme injection against Sinorhizobium freundii. DETAILED DESCRIPTION

[0015] In order to make the contents of the present invention easier to understand, the technical solutions of the present invention are further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0016] In the following examples, the hen egg white lysozyme used was purchased from Beijing Solebow Technology Co., Ltd., with a product number of L8120, a CAS number of 12650-88-3, and an activity unit of 20,000 u / mg.

[0017] In the following examples, the chelated magnesium (C 10 H 12 N2O8MgNa2) was purchased from Shandong Yousuo Chemical Technology Co., Ltd., CAS No. 14402-88-1.

[0018] Example 1: Preparation of lysozyme injection: Dissolve 2 g of hen egg white lysozyme, 1 g of potassium nitrate, 1 g of potassium dihydrogen phosphate, and 1 g of chelated magnesium in pure water in sequence at room temperature to prepare 500 mL of a mixed solution to obtain lysozyme injection.

[0019] Preparation of lysozyme solution: Dissolve 2 g of hen egg white lysozyme in pure water at room temperature, and use pure water to prepare 500 mL of solution to obtain lysozyme solution.

[0020] Example 2: like Figure 1 As shown, lysozyme injection is administered to sweet orange trees using a hanging bottle infusion method: The old bark of a sweet orange tree infected with the Asian species of citrus Huanglongbing disease is scraped off with a spatula 15-20 cm above the ground. A 0.46 cm drill is then used to drill a hole at a 45-degree angle to the ground at the peeled area, ensuring that the hole reaches the phloem of the plant. The lysozyme injection is then placed in a hanging bottle with an infusion tube and a needle (the diameter of the drill hole matches the diameter of the needle). The hanging bottle is then hung on a branch above the drill hole. The needle is then inserted into the drill hole, allowing the lysozyme injection to be delivered through the drill hole into the citrus tree. A volume of 500 mL is infused, and the infusion rate is controlled to complete the infusion every 20 days. After the infusion is completed, the drill hole is sealed with beeswax. 20 days after the infusion, different leaves were taken from the sweet orange tree trunks, DNA from the main veins of the leaves were extracted, and the content of citrus Huanglongbing disease Asian species was analyzed by real-time fluorescence quantitative PCR, and the pathogen titer of the leaves of the susceptible plants was detected.

[0021] Using the hanging bottle infusion method to apply lysozyme solution or pure water to the sweet orange trees: The method is basically the same as the aforementioned method of using the hanging bottle infusion method to apply lysozyme injection to the sweet orange trees, except that the lysozyme injection is replaced by lysozyme solution or pure water.

[0022] Lysozyme injection was applied to sweet orange trees by spraying: lysozyme injection was used to spray the entire sweet orange tree infected with the Asian species of citrus Huanglongbing disease until the surface of all the leaves was completely wet; 20 days after spraying, different leaves on the sweet orange tree trunk were collected, DNA from the main veins of the leaves was extracted, and the content of the Asian species of citrus Huanglongbing disease was analyzed by real-time fluorescence quantitative PCR, and the pathogen titer of the leaves of the diseased plants was detected.

[0023] The results are as follows Figure 2 As shown: when lysozyme solution was applied to sweet orange trees using the hanging bottle infusion method, there was no significant change in the amount of bacteria on the leaves of the diseased plants before and after the infusion; when lysozyme injection was applied to sweet orange trees using the hanging bottle infusion method, the pathogen titer on the leaves of the diseased plants after injection was significantly lower than that before injection; when lysozyme injection was applied to sweet orange trees using the spraying method, the pathogen titer on the leaves of the diseased plants after spraying was significantly higher than that before spraying; when pure water was applied to sweet orange trees using the hanging bottle infusion method, the pathogen titer on the leaves of the diseased plants after injection was significantly higher than that before injection.

[0024] Example 3: Because the Asian strain of citrus Huanglongbing has not yet been isolated and purified, the antibacterial effect of lysozyme injection was tested using its closely related bacterium, Sinorhizobium freundii. This S. freundii strain was obtained from the China General Microbiological Culture Collection Center (CGMCC), strain number ACCC 15067.

[0025] The LB liquid medium was shaken to culture Sinorhizobium freundii until OD 600 To 0.6, a Sinorhizobium freundii culture solution was obtained. Using the pour plate method, the Sinorhizobium freundii culture solution was injected into LB solid medium cooled to approximately 50°C. The culture solution and medium were mixed thoroughly, poured into a Petri dish, and allowed to solidify horizontally at room temperature. After sterilization and drying, a filter paper piece was soaked in lysozyme injection solution for 10 seconds, then placed in the center of the test plate. The plate was incubated overnight at 28°C, and the size of the inhibition zone was measured. The diameter of the inhibition zone was measured using the cross-hatch method, taking the average value. The diameter was used to represent the size of the inhibition zone. Pure water, 50 μg / mL kanamycin solution, and lysozyme solution were used as controls.

[0026] The results are as follows Figure 3 Figure 4 As shown, lysozyme injection, lysozyme solution and kanamycin solution all had significant inhibitory effects on Sinorhizobium freundii.

[0027] The above description is only a preferred embodiment of the present invention. Any adjustments made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A lysozyme injection for inhibiting citrus Huanglongbing fungus, characterized by: Each 500 mL of the lysozyme injection contains lysozyme ≥2 g, potassium nitrate 1-2 g, potassium dihydrogen phosphate 1-2 g, chelated magnesium 0.5-1 g, and the remainder is water.

2. The lysozyme injection according to claim 1, characterized in that: The lysozyme includes but is not limited to egg white lysozyme, animal lysozyme, plant lysozyme, microbial lysozyme, and bacterial phage lysozyme. The chelated magnesium includes but is not limited to citrate chelated magnesium, EDTA chelated magnesium, amino acid chelated magnesium, and sugar alcohol chelated magnesium.

3. The method for preparing the lysozyme injection according to any one of claims 1 to 2, wherein: The following steps are involved: Take 2g or more of lysozyme, 1-2g of potassium nitrate, 1-2g of potassium dihydrogen phosphate and 0.5-1g of chelated magnesium, and prepare 500mL of a mixed solution with water to obtain lysozyme injection.

4. Use of the lysozyme injection according to any one of claims 1 to 2 in preventing and treating citrus Huanglongbing fungus.

5. The use according to claim 4, characterized in that: Lysozyme injection was administered to citrus trees using the drip infusion method.

6. The use according to claim 5, characterized in that: Drill a hole 15 to 20 cm above the ground in the main trunk of the citrus tree, put the lysozyme injection solution into a hanging bottle with an infusion tube and an infusion tube needle, then hang the hanging bottle on the branch above the drill hole, and then insert the infusion tube needle into the drill hole to allow the lysozyme injection solution to be transported into the citrus tree through the drill hole.

7. Use of the lysozyme injection according to any one of claims 1 to 2 in inhibiting citrus Huanglongbing fungus.

8. Use of the lysozyme injection according to any one of claims 1 to 2 in inhibiting Sinorhizobium freundii.

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