Citrus huanglongbing biocontrol strain and application thereof

Abstract

The present application relates to the field of agricultural microorganisms and biological control of plant diseases, and discloses a strain of disease control bacteria of citrus Huanglongbing bacteria and its application, aiming at the defects of poor transmission of existing chemical agents and single function of conventional disease control bacteria, the present application provides a strain of Pseudomonas protegens, which has the functions of inhibiting bacterial diseases, decomposing inorganic phosphorus and organic phosphorus, and promoting growth by producing siderophores, the present application also discloses a composition containing the disease control strain, a fermentation liquid preparation method and its application in disease control and growth promotion, the strain competes and inhibits pathogenic bacteria by metabolizing siderophore complexed environmental iron, and transforms insoluble phosphorus to improve plant nutrient absorption, the present application takes into account the effects of antagonizing plant vascular diseases and repairing damaged root systems, the disease control composition provided has high survival rate and good dispersibility, and provides a stable and reliable technical solution for preventing and controlling citrus Huanglongbing and other diseases.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbiology and biological control technology of plant diseases, specifically to a biocontrol strain of Huanglongbing fungus for citrus and its application. Background Technology

[0002] Huanglongbing (HLB) is a devastating bacterial disease in the citrus industry. The pathogen mainly parasitizes the phloem tissue of citrus plants, hindering nutrient transport within the plant and leading to root decline, leaf yellowing, and plant death. Currently, conventional control of bacterial diseases such as HLB and canker in citrus mainly relies on antibiotics or copper-containing fungicides. However, after application, conventional chemical agents mostly adhere to the surface tissue of the plant and are difficult to effectively transport to the vascular bundles to reach the pathogens parasitizing the phloem, resulting in limited control efficacy. At the same time, long-term use of chemical agents alone can easily induce drug resistance in pathogens, and residual drugs can have a negative impact on the soil microecological environment.

[0003] To overcome the limitations of chemical control, biological control using microorganisms has become a common technique in this field. However, in actual field applications, most existing biocontrol strains suffer from unstable efficacy and limited functionality. When faced with the complex micro-ecological environment of nature, the colonization ability of conventional biocontrol bacteria is easily affected by environmental factors, making it difficult to maintain an effective population of viable bacteria. In addition, plants infected with citrus Huanglongbing (HLB) often suffer from severe absorption disorders and nutrient deficiencies due to vascular bundle damage. Most existing biocontrol bacteria focus only on the single antagonistic effect against pathogens and lack the ability to regulate the rhizosphere microenvironment. They cannot effectively degrade and transform insoluble minerals such as phosphorus and iron in the soil, making it difficult to meet the nutritional needs of infected plants to repair damaged roots and restore growth. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a biocontrol strain of Huanglongbing fungus for citrus and its application, which solves the problems of poor conductivity of existing chemical agents in plants, difficulty in reaching pathogenic bacteria in the phloem, and the inability of conventional single-function biocontrol bacteria to simultaneously inhibit pathogens and repair damaged plant roots.

[0005] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a biocontrol strain of *Huanglongbing* (citrus greening disease) fungus, employing the following technical solution: A biocontrol strain of Huanglongbing (HLB) of citrus, wherein the biocontrol strain is a Pseudomonas species that can inhibit plant bacterial pathogens and promote plant growth; the ability to promote plant growth includes the ability to produce siderophores, the ability to solubilize inorganic phosphorus, and the ability to solubilize organic phosphorus.

[0006] By employing the above technical solution, the selected biocontrol strain possesses both antagonistic and growth-promoting functions, thus achieving the technical effects of controlling vascular bacterial diseases and improving plant nutrient absorption. The mechanism of action of this biocontrol strain is as follows: Colonization and nutrient competition: This strain colonizes in the rhizosphere and phyllosphere of plants, secretes siderophores, and achieves the complexation of ferric ions in iron-deficient environments, blocking the source of iron nutrients required for the reproduction of pathogenic bacteria, and limiting the proliferation of Huanglongbing and canker bacteria in citrus.

[0007] Mineral element transformation: During colonization, this strain secretes low-molecular-weight organic acids and phosphatases, which degrade insoluble inorganic phosphate minerals and organic phosphorus compounds in the soil and substrate into water-soluble available phosphorus that can be absorbed and utilized by plant roots.

[0008] Promotes plant tissue repair: Increased available phosphorus and absorbable iron improve the nutrient supply status of plants infected by pathogens. Plants use nutrients to accumulate biomass, promote the development of the taproot and lateral root system, and compensate for tissue damage caused by pathogen infection through growth promotion.

[0009] Secondly, the present invention provides a biocontrol composition comprising the above-mentioned biocontrol strain of Huanglongbing fungus for citrus, using the following technical solution: A biocontrol composition comprising a biocontrol strain of citrus Huanglongbing (HLB) comprises the following components in weight percentages: a cell precipitate containing the biocontrol strain of citrus Huanglongbing: 15%-25%; a freeze-drying protectant: 3%-8%; a dispersant: 3%-8%; a wetting agent: 1%-5%; and the balance being a carrier.

[0010] By adopting the above technical solution, and by using live bacteria in combination with various excipients, a solid powder product with stable formulation, good dispersibility, and high survival rate is obtained. The freeze-drying protectant maintains the intracellular osmotic pressure during the dehydration stage; the dispersant and wetting agent reduce the surface tension of the solution, allowing the suspension to spread on the plant surface and in the rhizosphere soil, increasing the probability of live bacteria contacting the target pathogen.

[0011] Preferably, the freeze-drying protectant is trehalose, the dispersant is sodium lignosulfonate, the wetting agent is sodium dodecyl sulfate, and the carrier is diatomaceous earth; the biocontrol composition comprises the following components by mass percentage: 20% of the bacterial precipitate, 5% of the trehalose, 5% of the sodium lignosulfonate, 3% of the sodium dodecyl sulfate, and 67% of the diatomaceous earth; the effective viable count of the biocontrol composition is not less than 5.0 × 10^9 CFU / g.

[0012] By employing the above technical solutions, the hydroxyl groups on trehalose molecules form hydrogen bonds with the polar groups of proteins on the surface of bacterial cell membranes, replacing water molecules in a freeze-dried, dehydrated state to form an amorphous matrix, preventing ice crystals from piercing the cell membrane; sodium lignosulfonate provides steric hindrance and electrostatic repulsion, preventing bacterial particles from agglomerating when diluted with water for application; sodium dodecyl sulfate increases the wetting speed of solid formulations in water; diatomaceous earth, as an adsorption carrier, carries live bacteria and provides physical isolation, preventing the live bacteria from becoming inactive during storage.

[0013] Thirdly, the present invention provides a method for preparing a biocontrol fermentation broth containing the above-mentioned biocontrol strain of citrus Huanglongbing, using the following technical solution: A method for preparing a biocontrol fermentation broth containing a biocontrol strain of *Citrus citrus Huanglongbing* includes the following steps: (1) Inoculate a single colony of the biocontrol strain of citrus Huanglongbing into a liquid culture medium and culture it at a constant temperature of 28-32℃ and a rotation speed of 180rpm for 10-14 hours to obtain the seed liquid of the biocontrol strain; (2) The seed liquid of the biocontrol strain is inoculated into the fermentation medium at an inoculation amount of 1%-5% by volume, and fermented for 48-72 hours at a temperature of 28-32℃, a stirring speed of 200rpm and an aeration rate of 1.0vvm to obtain the biocontrol fermentation liquid.

[0014] By adopting the above technical solution, the viable cell count and metabolic activity of the fermentation product were increased due to the control of temperature, rotation speed, and aeration rate. A culture temperature of 28-32℃ is suitable for preventing the growth of Pseudomonas aeruginosa; a rotation speed of 200 rpm combined with an aeration rate of 1.0 vvm ensures the supply of dissolved oxygen in the fermentation system, meeting the cell division needs of aerobic bacteria during the logarithmic growth phase and reducing the impact of anaerobic metabolic byproducts on viable cells.

[0015] Preferably, in step (2), the composition of each liter of the fermentation medium is: 15 grams of sucrose, 10 grams of yeast extract, 5 grams of sodium chloride, and the remainder is water, and the pH value of the fermentation medium is controlled to be 7.0.

[0016] By employing the above technical solution, sucrose and yeast extract provide carbon and nitrogen sources. Sucrose provides carbon to support cell proliferation, while yeast extract provides amino acids and vitamins, promoting the synthesis of secondary metabolites such as siderophores and phospholytic enzymes. Sodium chloride maintains the osmotic pressure of the fermentation broth, and a pH of 7.0 provides a neutral buffer environment to prevent acidification of the culture medium from affecting the activity of the strain.

[0017] Fourthly, the present invention provides a method for preparing the above-mentioned biocontrol composition, which adopts the following technical solution: A method for preparing a biocontrol composition includes the following steps: S1. Take the biocontrol fermentation broth, centrifuge it at 8000 rpm for 15 minutes, discard the supernatant and collect the bottom bacterial precipitate; S2. Mix the bacterial precipitate with the freeze-drying protectant according to the ratio, and let it stand at room temperature for 20 minutes to allow the protectant to penetrate and protect the bacterial cells. S3. Then add the dispersant, the wetting agent and the carrier and continue to mechanically mix them evenly. Place the resulting mixture in a vacuum freeze dryer for freeze drying, and finally transfer it to an air jet mill to pulverize it so that its fineness passes through a 300-mesh standard sieve.

[0018] By adopting the above technical solution, the survival rate of wettable powders is improved due to the use of centrifugal collection and stepwise addition of excipients. The mechanism of this preparation process is as follows: Step 1: Live bacteria concentration and separation. 8000 rpm refrigerated centrifugation removes fermentation waste while minimizing the impact of mechanical shear heat on bacterial proteins.

[0019] Step 2: Protectant Infiltration. During mixing and settling at room temperature, trehalose permeates into the bacterial cells via its concentration gradient. A 20-minute settling time ensures the permeation process reaches equilibrium, avoiding insufficient protection due to too short a settling time or excessive energy expenditure by the bacteria due to too long a settling time.

[0020] Step 3: Mixing, Coating, and Dehydration. The addition of dispersants, wetting agents, and carriers creates physical isolation around the bacterial cells. Vacuum freeze-drying sublimates free water, preserving the dormant structure of the strain; air jet milling refines the product and improves the reconstitution rate.

[0021] Fifthly, the present invention provides an application of a biocontrol strain, biocontrol composition, or biocontrol fermentation broth of *Citrus citrus Huanglongbing*, employing the following technical solution: The above-mentioned biocontrol strains, biocontrol compositions, or biocontrol fermentation liquids of Huanglongbing fungus for citrus have been applied in the prevention and control of bacterial diseases in plants and the promotion of plant growth.

[0022] By adopting the above technical solutions, since Pseudomonas aeruginosa has a survival advantage in the plant rhizosphere and leaf environment, it is possible to obtain the ability to control a variety of systemic and localized plant bacterial diseases, thereby reducing the use of chemical agents and the problem of drug resistance.

[0023] Preferably, the bacterial plant diseases are selected from one or more of the following: citrus Huanglongbing, citrus canker, diseases caused by *Pseudomonas syringae*, and diseases caused by *Ralstonia solanacearum*. The specific implementation method of the application is as follows: the biocontrol composition or the biocontrol fermentation liquid is diluted 100-500 times with chlorine-free well water to obtain a biocontrol application solution; it is applied to the plants by simple root irrigation, simple foliar spraying, or trench irrigation combined with canopy spraying, with an application frequency of once every 10-30 days, for 3-4 consecutive treatments.

[0024] This invention provides a biocontrol strain of *Huanglongbing* (citrus greening disease) and its application. It has the following beneficial effects: 1. The defensive Pseudomonas strain used in this invention has the functions of antagonizing plant pathogenic bacteria and promoting plant growth. This strain inhibits the proliferation of citrus Huanglongbing bacteria by producing siderophores to compete for iron in the environment through metabolism. At the same time, it secretes organic acids and phosphatases to convert insoluble phosphorus into available phosphorus that can be absorbed by plants. This mechanism of combining disease prevention and growth promotion overcomes the defect of conventional chemical agents that are difficult to penetrate into the vascular bundles of plants to take effect. While controlling systemic diseases of the phloem, it also compensates for the plant tissue damage caused by pathogen infection.

[0025] 2. The biocontrol composition and preparation process of the present invention ensure high viable bacterial count and application performance of the product. During the preparation process, trehalose is used to penetrate the bacterial cells within a specific static time, replacing water molecules to maintain the osmotic pressure inside the cells and preventing physical damage to ice crystals during subsequent vacuum freeze-drying. Combined with the ratio of sodium lignosulfonate, sodium dodecyl sulfate and diatomaceous earth carrier, the final wettable powder maintains formulation stability during storage and has easy wetting and anti-agglomeration dispersion characteristics when diluted with water in the field.

[0026] 3. The application method of this invention improves the field control effect of biocontrol strains by combining root and leaf application. The live bacteria suspension directly adheres to the leaf surface to antagonize surface pathogens, while simultaneously colonizing the roots through rhizosphere irrigation, stimulating the upward transmission of acquired resistance signals along the vascular bundles, thereby inhibiting the deep-seated parasitic citrus Huanglongbing fungus. Combined with continuous treatment at specific intervals, it effectively replenishes the live bacteria population consumed in the field and maintains the antibacterial advantage in the plant microenvironment. Attached Figure Description

[0027] Figure 1 This is a flowchart of the method of the present invention. Detailed Implementation

[0028] The technical solutions in 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.

[0029] Please see Figure 1 This invention provides a biocontrol strain of Huanglongbing fungus for citrus and its application.

[0030] raw material: The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Unless otherwise specified, all pharmaceutical excipients, conventional culture medium components, and reagents are commercially available analytical grade or higher products. The biocontrol strains involved in this invention defend against *Pseudomonas*, and the test plant pathogens used for confrontation culture and disease control testing include *Pseudomonas syringae* DC3000, *Ralstonia solanacearum* XCC, and *Ralstonia solanacearum* GMI1000. The chemical control reagents involved in the comparative examples include copper hydroxide and streptomycin sulfate, both of which can be obtained through conventional commercial channels.

[0031] Preparation Example 1: This preparation example provides a biocontrol fermentation broth of *Pseudomonas aeruginosa*, comprising the following steps: a single colony of *Pseudomonas aeruginosa* is inoculated into a container containing LB liquid medium and cultured at a constant temperature of 30°C and a stirring speed of 180 rpm for 12 hours to obtain a *Pseudomonas aeruginosa* seed culture; the *Pseudomonas aeruginosa* seed culture is inoculated into a fermenter containing fermentation medium at an inoculation rate of 3% by volume, and fermented at a temperature of 30°C, a stirring speed of 200 rpm, and an aeration rate of 1.0 vvm for 60 hours to obtain a biocontrol fermentation broth of *Pseudomonas aeruginosa*. The effective viable count was determined by plate counting method to be 2.5 × 10^9 CFU / mL, wherein each liter of the fermentation medium contains 15 g of sucrose, 10 g of yeast extract, 5 g of sodium chloride, and the remainder is water, with a pH of 7.0.

[0032] Preparation Example 2: This preparation example provides a biocontrol fermentation broth of *Pseudomonas aeruginosa*, comprising the following steps: inoculating a single colony of *Pseudomonas aeruginosa* into a container containing LB liquid medium, and incubating it at a constant temperature of 28°C and a stirring speed of 180 rpm for 10 hours to obtain a *Pseudomonas aeruginosa* seed liquid; inoculating the *Pseudomonas aeruginosa* seed liquid into a fermenter containing fermentation medium at an inoculation amount of 1% by volume, and fermenting it at a temperature of 28°C, a stirring speed of 200 rpm, and an aeration rate of 1.0 vvm for 48 hours to obtain a biocontrol fermentation broth of *Pseudomonas aeruginosa*, the effective viable count of which was determined by plate counting method to be 1.1 × 10^9 CFU / mL, wherein the formulation of the fermentation medium is exactly the same as that in Preparation Example 1.

[0033] Preparation Example 3: This preparation example provides a biocontrol fermentation broth of *Pseudomonas aeruginosa*, comprising the following steps: inoculating a single colony of *Pseudomonas aeruginosa* into a container containing LB liquid medium, and incubating it at a constant temperature of 32°C and a stirring speed of 180 rpm for 14 hours to obtain a *Pseudomonas aeruginosa* seed liquid; inoculating the *Pseudomonas aeruginosa* seed liquid into a fermenter containing fermentation medium at an inoculation amount of 5% by volume, and fermenting it at a temperature of 32°C, a stirring speed of 200 rpm, and an aeration rate of 1.0 vvm for 72 hours to obtain a biocontrol fermentation broth of *Pseudomonas aeruginosa*, the effective viable count of which was determined by plate counting method to be 1.8 × 10^9 CFU / mL, wherein the formulation of the fermentation medium is exactly the same as that in Preparation Example 1.

[0034] Preparation Example 4: This preparation example provides a wettable powder for defense against Pseudomonas aeruginosa, comprising the following steps: Take the biocontrol fermentation broth of Pseudomonas aeruginosa obtained in Preparation Example 1, centrifuge at 8000 rpm for 15 minutes, discard the supernatant and collect the bottom bacterial precipitate; weigh 20% of the bacterial precipitate, 5% of trehalose, 5% of sodium lignosulfonate, 3% of sodium dodecyl sulfate and 67% of diatomaceous earth by mass percentage, first mix the bacterial precipitate and trehalose thoroughly and evenly, let stand at room temperature for 20 minutes to allow the trehalose to fully penetrate and protect the bacterial cells, then add sodium lignosulfonate, sodium dodecyl sulfate and diatomaceous earth and continue to mechanically mix evenly, place the resulting mixture in a vacuum freeze dryer for freeze drying to remove excess moisture, and finally transfer the dried material to an air jet mill for pulverization to make its fineness pass through a 300-mesh standard sieve, finally obtaining a wettable powder for defense against Pseudomonas aeruginosa with a viable count of not less than 5.0 × 10^9 CFU / g.

[0035] Example 1: This example provides a method for applying a biocontrol agent against Pseudomonas aeruginosa in the control of bacterial diseases and growth promotion of citrus, including the following steps: Take the biocontrol fermentation liquid against Pseudomonas aeruginosa prepared in Example 2, and dilute it by 100 times with chlorine-free well water to obtain a high-concentration biocontrol application solution; Select healthy potted citrus seedlings with basically uniform growth and citrus plants in the field suspected of being infected with citrus Huanglongbing in the early stage, and apply the high-concentration biocontrol application solution by simply irrigating the roots, so as to stimulate the systemic resistance of the plants through root absorption. The average amount of irrigated solution per plant is 500 ml, and the application frequency is once every 10 days, for a total of 3 consecutive treatments; During and after the treatment, the experimental area maintains conventional standard water and fertilizer management.

[0036] Example 2: This example provides a method for applying a biocontrol agent against Pseudomonas aeruginosa in the control of bacterial diseases and growth promotion of citrus, including the following steps: Take the biocontrol fermentation liquid against Pseudomonas aeruginosa prepared in Example 1, and dilute it 300 times with chlorine-free well water to obtain a medium-concentration biocontrol application solution; Select healthy potted citrus seedlings with similar growth as in Example 1 and citrus plants in the field suspected of being in the early stage of disease, and apply the medium-concentration biocontrol application solution by root irrigation combined with foliar spraying to synergistically control vascular bundle and leaf diseases. The average amount applied per plant is 300 ml, and at the same time, the leaves are evenly sprayed until both sides of the leaves are dripping with water. The application frequency is once every 20 days, and the treatment is repeated for 3 times; During and after the treatment, the experimental area is maintained with conventional standard water and fertilizer management.

[0037] Example 3: This example provides a method for applying a biocontrol agent against Pseudomonas aeruginosa in the control of bacterial diseases and growth promotion of citrus, including the following steps: Take the biocontrol fermentation liquid against Pseudomonas aeruginosa prepared in Example 3, and dilute it 500 times with chlorine-free well water to obtain a low-concentration biocontrol application solution; Select citrus plants suspected of being infected with citrus canker in the early stage in the field, and apply the low-concentration biocontrol application solution by simple foliar spraying, so that the live bacteria can directly contact the pathogens on the leaves. Spray each plant evenly on the leaves until both the upper and lower surfaces of the leaves are dripping with water. The application frequency is once every 30 days, and the treatment is repeated for 3 times; During and after the treatment, the experimental area is maintained with conventional standard water and fertilizer management.

[0038] Example 4: This example provides a method for applying a biocontrol agent against Pseudomonas aeruginosa in the control of bacterial diseases and growth promotion of citrus, including the following steps: Take the wettable powder against Pseudomonas aeruginosa prepared in Example 4, and dilute it by mixing 1 gram of powder with 500 ml of chlorine-free well water to obtain a solid preparation suspension; Select mature fruit-bearing trees in the field that have been clearly infected with citrus Huanglongbing or citrus canker, and dig a circular trench with a depth of 15 cm around the drip line at the root of the diseased tree. Apply the solid preparation suspension by combining trench irrigation with uniform spraying of the entire canopy. The total amount of medicine applied per tree is 5 liters, and the application frequency is once every 15 days, for a total of 4 treatments; After the treatment, backfill the circular trench and maintain conventional standard water and fertilizer management.

[0039] Comparative Example 1: Compared with Example 1, the difference is that no biocontrol agents were added, and only an equal amount of chlorine-free well water was used for irrigation as a blank control. All other aspects were the same.

[0040] Comparative Example 2: Compared with Example 1, the difference is that the biocontrol fermentation broth for defending against Pseudomonas aeruginosa was replaced with an aqueous solution of streptomycin sulfate at a concentration commonly recommended in agriculture; all other aspects are the same.

[0041] Comparative Example 3: Compared with Example 1, the difference is that the biocontrol fermentation broth of Pseudomonas aeruginosa was replaced with conventional commercially available fluorescent Pseudomonas aeruginosa fermentation broth with the same concentration of viable bacteria, and all other aspects are the same.

[0042] Comparative Example 4: Compared with Example 1, the difference is that the high-concentration biocontrol liquid was sterilized by high-pressure steam at 121 degrees Celsius for 20 minutes before use, that is, the inactivated fermentation liquid was used for application, and the rest were the same.

[0043] Comparative Example 5: Compared with Example 2, the difference is that the biocontrol fermentation broth of Pseudomonas aeruginosa was replaced with a fermentation broth of Bacillus amyloliquefaciens that only has antibacterial ability but not phosphorus solubilization and iron carrier production ability; all other aspects are the same.

[0044] Comparative Example 6: Compared with Example 4, the difference is that the wettable powder used to defend against Pseudomonas spp. did not contain trehalose as a freeze-drying protectant during the preparation process; otherwise, they were the same.

[0045] Test Example 1: Obtain *Pseudomonas syringae* DC3000, *Citrus canker* XCC, and *Ralstonia solanacearum* GMI1000 in the logarithmic growth phase, and adjust the bacterial concentration of each pathogen to 1×10^7 CFU / mL with sterile physiological saline. Spread 0.1 mL of each pathogen suspension evenly onto the surface of a nutrient agar plate. Use a sterile punch to make 6 mm holes in the center of the plate. Inject 50 μL of the *Pseudomonas* fermentation broth obtained in Example 1 into the holes. Incubate the plates at 28°C for 48 hours and observe the formation of clear inhibition zones.

[0046] The *Pseudomonas aeruginosa* fermentation broth obtained in Preparation Example 1 was inoculated into iron-deficient sucrose-asparagine liquid medium at a volume ratio of 1% and cultured at 30°C and 200 rpm for 48 hours with shaking. The fermentation broth was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected. 0.5 mL of the supernatant was mixed with 0.5 mL of CAS detection solution and reacted at room temperature in the dark for 1 hour. The color development of the reaction solution was observed, with uninoculated iron-deficient medium as a control. The *Pseudomonas aeruginosa* fermentation broth obtained in Preparation Example 1 was inoculated into NBRIP liquid medium with tricalcium phosphate as the phosphorus source at a volume ratio of 1% and cultured at 30°C and 200 rpm for 5 days with shaking. The fermentation broth was centrifuged at 10,000 rpm for 10 minutes to obtain the supernatant, and qualitative detection was performed using the molybdenum-antimony colorimetric method.

[0047] Test results showed that in confrontation cultures against *Pseudomonas syringae* DC3000, *Citrus canker* XCC, and *Ralstonia solanacearum* GMI1000, a clear, transparent inhibition zone was formed around the inoculation wells defending against *Pseudomonas*, demonstrating antagonistic activity and confirming that its antibacterial spectrum covers *Pseudomonas*, *Xanthomonas*, and *Rhizobium* genera. In colorimetric tests on iron-deficient medium and NBRIP culture systems, the reaction solution exhibited a typical positive colorimetric reaction, indicating that this strain can complex ferric ions in an iron-deficient environment and convert insoluble inorganic phosphorus into a soluble form. These qualitative data confirm the feasibility of applying this strain with both disease prevention and growth promotion functions.

[0048] Test Example 2: Potted citrus plants infected with citrus Huanglongbing (HLB) were selected and treated according to the protocols of Example 1 and Comparative Examples 1 to 4. Mature leaf samples were collected periodically before and after treatment, and total DNA was extracted from the midribs. qPCR amplification of HLB-forming bacteria was performed using specific primers, and the Ct values ​​before and after treatment were compared. A water control was also included. Disease-free potted citrus seedlings with uniform growth were selected and treated according to the protocols of Example 2 and Comparative Examples 1 and 5. After 60 days of treatment, the seedlings were removed, the substrate attached to the roots was washed off, and the development of the taproot was observed and root biomass was assessed.

[0049] Test results showed that in Example 1, after continuous application, the Ct value of pathogens detected by qPCR in the plant leaves increased compared to before treatment, indicating a decrease in the concentration of citrus Huanglongbing (HLB) in the plants. In Comparative Example 1, the Ct value of the blank control group showed a decreasing trend, indicating that the pathogens were in a state of natural proliferation. The Ct value changes in Comparative Example 2 (chemical agent treatment group) and Comparative Example 3 (conventional fluorescent Pseudomonas treatment group) were both inferior to those in Example 1. The Ct value change trend in Comparative Example 4 (inactivated group) was consistent with that of Comparative Example 1, indicating that the biocontrol effect against Pseudomonas depends on the metabolic activity of the live bacterial strain in the plant microenvironment.

[0050] In the growth-promoting test, the citrus seedlings in the treatment group of Example 2 showed better taproot length and overall fresh weight than the control group in Comparative Example 1. Comparative Example 5, using a single antibacterial biocontrol bacterium, showed a lower growth-promoting effect than Example 2. Combined with the results of phosphorus solubilization and siderophore production tests, the organic acids and phosphatases produced by *Pseudomonas* in the soil promote the release of insoluble nutrients, increasing the effective nutrient supply around the roots, allowing plants to accumulate biomass for root development. The comprehensive comparison of disease prevention and growth-promoting data verifies that *Pseudomonas* has a dual function of controlling vascular bacterial diseases and promoting root growth.

[0051] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A biocontrol strain of *Huanglongbing* fungus affecting citrus, characterized in that, The biocontrol strain was a defense against Pseudomonas protegens, named BCA. HLB1 It has the ability to inhibit plant bacterial pathogens and promote plant growth; the ability to promote plant growth includes the ability to produce iron carriers, the ability to solubilize inorganic phosphorus and the ability to solubilize organic phosphorus.

2. A biocontrol composition comprising the citrus Huanglongbing biocontrol strain of claim 1, characterized in that, It contains the following components by mass percentage: The bacterial precipitate containing the aforementioned biocontrol strains for citrus Huanglongbing is 15%-25%; Freeze-drying protectant: 3%-8%; Dispersant: 3%-8%; Wetting agent: 1%-5%; And the carrier of the remaining amount.

3. The biocontrol composition according to claim 2, characterized in that, The freeze-drying protectant is trehalose, the dispersant is sodium lignosulfonate, the wetting agent is sodium dodecyl sulfate, and the carrier is diatomaceous earth.

4. The biocontrol composition according to claim 3, characterized in that, It comprises the following components by weight percentage: 20% of the bacterial precipitate, 5% of the trehalose, 5% of the sodium lignosulfonate, 3% of the sodium dodecyl sulfate, and 67% of the diatomaceous earth; Preferably, the effective viable bacteria count of the biocontrol composition is not less than 5.0 × 10^9 CFU / g.

5. A method for preparing a biocontrol fermentation broth containing the citrus Huanglongbing biocontrol strain of claim 1, characterized in that, Includes the following steps: (1) Inoculate a single colony of the citrus Huanglongbing biocontrol strain into a liquid culture medium and culture it at a constant temperature of 28-32℃ and a rotation speed of 180 rpm for 10-14 hours to obtain the seed liquid of the biocontrol strain; (2) The seed liquid of the biocontrol strain is inoculated into the fermentation medium at an inoculation amount of 1%-5% by volume, and fermented for 48-72 hours at a temperature of 28-32℃, a stirring speed of 200rpm and an aeration rate of 1.0vvm to obtain the biocontrol fermentation liquid.

6. The preparation method according to claim 5, characterized in that, In step (2), each liter of the fermentation medium consists of: 15 grams of sucrose, 10 grams of yeast extract, 5 grams of sodium chloride, and the remainder is water, and the pH of the fermentation medium is controlled to be 7.

0.

7. A method for preparing the biocontrol composition according to any one of claims 2-4, characterized in that, Includes the following steps: S1. Take the biocontrol fermentation broth prepared according to claim 5 or 6, centrifuge it at 8000 rpm for 15 minutes, discard the supernatant and collect the bottom bacterial precipitate. S2. Mix the bacterial precipitate with the freeze-drying protectant according to the ratio, and let it stand at room temperature for 20 minutes to allow the protectant to penetrate and protect the bacterial cells. S3. Then add the dispersant, the wetting agent and the carrier and continue to mechanically mix them evenly. Place the resulting mixture in a vacuum freeze dryer for freeze drying, and finally transfer it to an air jet mill to pulverize it so that its fineness passes through a 300-mesh standard sieve.

8. The application of the citrus Huanglongbing biocontrol strain according to claim 1, the biocontrol composition according to any one of claims 2-4, or the biocontrol fermentation liquid prepared by the preparation method according to any one of claims 5-6 in the prevention and control of bacterial plant diseases and the promotion of plant growth.

9. The application according to claim 8, characterized in that, The plant bacterial diseases mentioned are selected from one or more of the following: citrus Huanglongbing, citrus canker, diseases caused by Pseudomonas syringae, and diseases caused by Ralstonia solanacearum.

10. The application according to claim 8, characterized in that, The specific implementation method of the application is as follows: the biocontrol composition or the biocontrol fermentation liquid is diluted 100-500 times with chlorine-free well water to obtain the biocontrol application liquid; Apply the solution to the plant by root irrigation, foliar spraying, or a combination of trench irrigation and canopy spraying. Apply the solution once every 10-30 days, for 3-4 consecutive treatments.

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