Application of bacillus subtilis L1-21 strain in preparation of biological agent for preventing and treating citrus huanglongbing

Abstract

The invention discloses application of a bacillus subtilis L1-21 strain in preparation of a biological agent for preventing and treating citrus huanglongbing, and belongs to the technical field of agricultural microorganisms. The strain of the bacillus subtilis L1-21 is classified and named as bacillus subtilis and is preserved in the China General Microbiological Culture Collection Center, and the address of the preservation unit is No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing; the preservation date is May 04, 2018; the preservation number of the strain is CGMCC No. 15726. The invention provides a biological agent taking bacillus subtilis L1-21 as a core component and a systematic application method of the biological agent. The prevention and treatment effect of the biological agent reaches 90% or above, yellow leaves of diseased trees turn green, new roots regenerate, normal fruits are born, the biological agent has the advantages of being efficient, environmentally friendly and low in cost, the loss of orange farmers can be remarkably reduced, and the quality and yield of oranges can be improved.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological control, in particular to application of bacillus subtilis L1-21 strain in preparation of biological agents for preventing and treating citrus Huanglongbing. BACKGROUND

[0002] Citrus Huanglongbing (HLB) is a worldwide destructive disease, which is an immune deficiency disease of citrus. Citrus planting areas in nearly 50 countries and regions in the world are infected with the disease. On September 15, 2020, Huanglongbing was listed in the directory of class I crop diseases and pests by the Ministry of Agriculture and Rural Affairs. It is mainly transmitted by citrus psylla and grafting, and can infect many Rutaceae plants such as citrus, fortune orange and bunge orange. Chemical pesticides cannot penetrate the phloem and directly kill Huanglongbing. Once Huanglongbing occurs, it has a great influence on the quality and yield of fruits, and causes serious economic losses.

[0003] Huanglongbing is transmitted by citrus psylla and scion (disease seedling). Huanglongbing prevention and control is extremely difficult, and after more than 100 years, an ideal control method has not been found. The most commonly used method is the so-called “three-axle” technology of “killing psylla, planting healthy seedlings, and cutting diseased trees”, but it can only delay the disease progression and cannot really stop the spread of the disease, resulting in cutting trees and planting new trees in the orchard, and the phenomenon of ancestor and grandson plants in the same garden. Citrus has been produced at high cost. The diseased trees generally lose the normal fruiting ability in about 3 years.

[0004] Citrus Huanglongbing is a gram-negative bacterium belonging to the phylum Proteobacteria, the class Alphaproteobacteria, the order Rhizobiales, the family Rhixobiaceae, and the genus Liberibacter. The shape of the pathogen is mainly round or oval. The pathogen is a phloem-specific parasitic bacterium, and three species of Huanglongbing have been found so far: Asian Liberibacter (CLas), African Liberibacter (CLaf) and American Liberibacter (CLam). The disease caused by the pathogen CLas triggers the plant's own immune response. The Asian subspecies is the most widely distributed and poses the greatest threat to the world's citrus industry. The Asian subspecies is mainly distributed in China. The methods for detecting the pathogen mainly include ordinary PCR and real-time fluorescent quantitative PCR. Candidatus

[0005] In the main citrus producing areas in China, the disease generally occurs in late May and is most serious in August and September. In spring and summer, it rains a lot, and in autumn, it is dry, so the disease is more serious. The disease is more serious in low-lying orchards with poor drainage, closed tree crowns, and insufficient fertilization. Young trees are more resistant than old trees, and 4-8 year-old trees are more susceptible. The occurrence of citrus psylla is more serious, and the occurrence of Huanglongbing is also more serious. Huanglongbing damages the roots, leaves, flowers and fruits of citrus, and the distribution of Huanglongbing pathogen in citrus is also uneven.

[0006] ​The typical symptom of Huanglongbing disease is similar to that of deficiency symptom, showing mottling and yellowing. At the early stage of the disease, some new shoot leaves are yellow, and the yellow shoots first appear at the top of the tree canopy and then spread gradually. After 1-2 years, the whole plant is affected. The branches die from the top down, and the wood of the diseased branches becomes orange rose in color, and finally the whole plant dies. The diseased trees have sparse canopies, many dead branches, small plants, few fruits, unevenly colored fruits, and poor quality.

[0007] Root rot is a direct symptom of Huanglongbing disease, and the severity is symmetrical to the aboveground branches. At the early stage of the disease, the roots are not rotted, and the leaves are yellow and shed. The fine roots and branch roots begin to rot, and then spread to the lateral roots and main roots, and the cortex is broken and separated from the xylem until death.

[0008] Because of the lack of effective chemical control agents and disease-resistant varieties, the prevention and control of Huanglongbing disease is still a worldwide problem. The most commonly used "three board axe" technology in production can only delay the disease progression, but cannot really stop the spread of the disease, resulting in cutting trees and replanting in the orchard, and the phenomenon of grandparent and grandchild plants in the same garden. Citrus has been produced at high cost. The diseased trees generally lose the normal fruiting ability in about 3 years. Therefore, it is urgent to find and feasible control measures to reduce production cost and improve prevention and control effect, and to establish the confidence of citrus growers.

[0009] There are a large number of microorganisms (endophytes) in the plant body, and they form a stable balance. From the economic point of view, plant endophytes can be divided into "beneficial", "harmful" and "neutral" (the functional group is currently unknown). These microorganisms play the role of second genome in the plant body, helping the plant to improve disease resistance, insect resistance, drought resistance, waterlogging resistance and other stress resistance. However, due to the large amount of chemical pesticides used by people, especially broad-spectrum fungicides (up to 18-22 times per year), the number of plant endophytes is reduced, the function of the second genome is lowered, and the auxiliary role of the endophyte allies is reduced, so that the plant grows in adversity as a lone soldier, with low resistance and easy to be attacked by diseases and pests. SUMMARY

[0010] In order to solve the problems of the prior art, the purpose of the present application is to provide an application of Bacillus subtilis L1-21 strain in preparing biological preparation for preventing and treating Huanglongbing disease of citrus.

[0011] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: In a first aspect, the present application provides an application of Bacillus subtilis L1-21 strain in preparing biological preparation for preventing and treating Huanglongbing disease of citrus.

[0012] In a second aspect, the present application provides a biological preparation of Bacillus subtilis L1-21 strain for application.

[0013] In a third aspect, the application provides a preparation method of the biological agent of the Bacillus subtilis L1-21 strain.

[0014] In a first aspect, the application provides an application of the Bacillus subtilis L1-21 strain in preparing a biological agent for preventing and treating citrus Huanglongbing disease. Bacillus subtilis The Bacillus subtilis L1-21 strain is classified as Bacillus subtilis and preserved in the China General Microbiological Culture Collection Center, located at No. 1, Yihuang West Road, Beijing, China, on May 4, 2018, with a preservation number of CGMCC No. 15726.

[0015] Further, the method for using the biological agent comprises the following steps: (1) Digging: before autumn, a ring ditch and a side ditch are opened within a crown drip line of a diseased citrus tree, with a ditch depth of 20 cm. After rotten lateral roots are removed, the biological agent is applied at 20-50 L / plant, wherein 30-40 L is applied to a plant with a crown diameter of ≥2 m, and 10-20 L is applied to a plant with a crown diameter of <2 m; (2) Drip irrigation: 30 days after the first application, the biological agent is diluted by 100 times, and is applied to the roots through drip irrigation, once every 15 days for 2 times, with an application amount of 20-50 L / plant; (3) Spraying: before the spring shoots of citrus sprout, the biological agent is diluted by 100 times and is sprayed on the back of leaves, once in the morning and once in the evening, once every 20 days for 3 times, with an application amount of 20-50 L / plant.

[0016] Further, in step (1), the ditch opening position is adjusted according to the tree vigor: the ditch is opened close to the main stem when the tree vigor is weak, and the ditch is opened away from the main stem but within the drip line when the tree vigor is strong.

[0017] Further, in step (2), the drip irrigation diluent is injected into the ditched area through a fertilizer gun, and the wetness in the ditch is used as a criterion; in step (3), the spraying time is cloudy, early morning or evening, and the wetness of the back of leaves to near water dripping is used as a criterion.

[0018] In a second aspect, the application provides a biological agent of the Bacillus subtilis L1-21 strain for use, which is composed of the following components in mass percentage: 3%-5% of the Bacillus subtilis L1-21 strain CGMCC No. 15726 bacterial liquid, the effective viable bacterial count of the bacterial liquid being ≥1.0×10 10 cfu / mL; 0.4%-1% of a water-soluble fertilizer, the water-soluble fertilizer being selected from one or a combination of potassium fulvate, fish protein or amino acid, and the components being equal in amount when combined; 0.1%-0.3% of a cosolvent, the cosolvent being selected from one or a combination of glycerol and emulsifiable concentrate, and the components being equal in amount when combined; and the balance being water, with a pH value of 6.2-7.2.

[0019] The third aspect of the application provides a preparation method of a biological preparation of Bacillus subtilis L1-21 strain, comprising the following steps: (1) Primary seed culture of the strain with the preservation number of CGMCC No. 15726: inoculate various bacteria strains stored at low temperature on a primary seed culture medium, and culture at a culture temperature of 37 DEG C and a rotation speed of 120 rpm / min for 36 h in a shaking bed to obtain a primary seed liquid, wherein the primary seed culture medium is LB liquid culture medium with a pH of 7.0-7.2; (2) Secondary seed culture: inoculate the primary seed liquid into a secondary seed culture medium in a seed tank at an inoculation amount of 10%, and culture at 30 DEG C, a rotation speed of 100 rpm / min, and a ventilation amount of 0.72-1.6 m 3 / h, with the tank pressure kept at 0.05-0.08 MPa for 36-48 h to obtain a secondary seed liquid, wherein the secondary seed culture medium is LB liquid culture medium with a pH of 7.0-7.2; (3) Fermentation culture: inoculate the secondary seed liquid into a fermentation culture medium in a fermentation tank at an inoculation amount of 10%, and culture at 30 DEG C, a rotation speed of 100 rpm / min, and a ventilation amount of 0.72-1.6 m 3 / h, with the tank pressure kept at 0.05-0.08 MPa for 48-72 h, and the Landy culture medium is selected, so that the effective viable cell count in each fermentation liquid after culture is >=1x10 10 cfu / mL, the fermentation liquid is packaged in a plastic bucket and stored in a ventilated and dry place; (4) Pour 3-5% of the fermentation liquid of the Bacillus subtilis L1-21 strain obtained in step (3) into a mixer and provide a culture medium, control the temperature at 28-30 DEG C, maintain stirring and oxygenation, after the secondary maturation is completed, add water-soluble fertilizer 0.4-1% and a solubilizer 0.1-0.3%, continue to maintain the ventilation and stirring conditions, the pH value is 7.2, pour into a storage tank after 72 h, and then package into products to prepare the biological preparation of the Bacillus subtilis L1-21 strain.

[0020] Beneficial effects: The prevention and treatment effect of the application reaches more than 90%, the yellow leaves of diseased trees turn green, new roots regenerate and normal fruits are formed, and the application has the advantages of high efficiency, environmental protection and low cost, can significantly reduce the loss of citrus farmers, and improve the quality and yield of citrus.

[0021] Compared with the prior art, the application has the following advantages: (1) The preparation takes probiotics as the core and does not contain chemical pesticide components, has the characteristics of non-toxicity, non-pollution and non-residue, reduces the damage to the soil and the ecological system, and meets the needs of green agriculture and sustainable development.

[0022] (2) Compared with the traditional "three-ax method" (frequent tree cutting, seedling replacement, and pesticide application), the application reduces the dependence on chemical agents and manual labor through the combination and scientific application of bacterial agents, has a simple preparation process (Example 1), low use cost, can maintain the health of orchards for a long time, and reduces the economic burden of orange farmers.

[0023] (3) Root repair and growth: the rotten roots are removed through the "one digging" step to stimulate the regeneration of active roots Figure 2 ), in combination with bacterial agents and organic fertilizer, to promote the development of new roots and enhance the nutrient absorption capacity. Immune activation: the plant immune system is activated after the endogenous colonization of the bacterial agent, the disease resistance is improved, and the pathogen infection is reduced (verified in the specification).

[0024] (4) Fruit quality improvement: the diseased trees produce normal fruits Figure 3 after recovery, the fruits are uniformly colored and have improved sugar content, and the yield is increased by 20%~30% compared with the traditional method (Example 2).

[0025] (5) The "one digging, two dripping, and three spraying" three-step method forms a comprehensive prevention and control system: digging: root repair and direct penetration of bacterial agents to solve the problem of root rot; drip irrigation: drip irrigation to strengthen the colonization of probiotics and nutrient supply in the roots; spraying: leaf spraying to activate the immune barrier of the leaves, which can promote the enrichment of beneficial fungi. The three steps synergistically significantly improve the sustainability and stability of the prevention and control effect, and avoid the limitations of single means. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0027] Figure 1 It is a schematic diagram of the diseased tree leaves of the application before spraying on April 25, 2025. 80% of the leaves are yellow, and the tree vigor is weak.

[0028] Figure 2 It is a schematic diagram of the diseased tree leaves of the application 40 days after spraying on June 4, 2025. All the yellow leaves turn green, and the growth is vigorous.

[0029] Figure 3 It is a schematic diagram of the diseased tree leaves of the application 70 days after spraying on July 6, 2025. All the yellow leaves turn green, and the growth is vigorous.

[0030] Figure 4The schematic diagram of the opening ring ditch of the present application. For strong trees, the ring ditch is opened slightly far from the trunk, but must be within the drip line. Remove the rotten lateral roots, cut or shear part of the active roots, and apply 100 times diluted liquid of the biocontrol agent and organic fertilizer.

[0031] Figure 5 The schematic diagram of the opening side ditch of the present application. For weak trees, the side ditch is opened close to the trunk. Remove the rotten lateral roots, cut or shear part of the active roots, and apply 100 times diluted liquid of the biocontrol agent and organic fertilizer.

[0032] Figure 6 The schematic diagram of the new root growth of the present application. After 40 days of application of the endophyte and organic fertilizer, new roots grow from the broken surface of the active roots.

[0033] Figure 7 The symptom diagram before spraying of the present application. The tree is weak and small, and the leaves are mottled and yellow.

[0034] Figure 8 The symptom diagram of the present application for the growth of normal fruits. The tree is vigorous, the fruits are full, the color is uniform, the skin is thin and smooth, and the taste is sweet. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0036] In the present application, the term "and / or" describes the association relationship of the associated objects, which means that there can be three kinds of relationships, for example, A and / or B can mean that A exists alone, A and B exist together, and B exists alone. Wherein A and B can be singular or plural. The character " / " generally represents an "or" relationship between the associated objects before and after it.

[0037] In the present application, "one or more" means one or more, and "multiple" means two or more. "One or more of the following" or similar expressions means any combination of these items, including any combination of single item or multiple items. For example, "one or more of a, b, or c", or "one or more of a, b, and c", can mean a, b, c, a~b (i.e. a and b), a~c, b~c, or a~b~c, wherein a, b, and c can be single or multiple.

[0038] It should be understood that the size of the sequence number of the above processes in various embodiments of the present application does not mean the order of execution, and part or all of the steps can be executed in parallel or in sequence, and the execution order of the processes should be determined according to their functions and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0039] The first aspect of the embodiments of the present application provides an application of Bacillus subtilis L1-21 strain in preparing a biological preparation for preventing and treating citrus Huanglongbing disease. The classification name of the Bacillus subtilis L1-21 strain is Bacillus subtilis Bacillus subtilis , which is preserved in the China General Microbiological Culture Collection Center, located at No. 1, Xibaixili, Chaoyang District, Beijing, China, with a preservation date of May 4, 2018, and a preservation number of CGMCC No. 15726.

[0040] In some embodiments, the method for using the biological preparation comprises the following steps: (1) Digging: Before autumn, open ring ditches and side ditches within the water dripping line of the crown of diseased citrus trees, with a ditch depth of 20 cm. After removing the rotten lateral roots, 20-50 L of the biological preparation is applied per plant, wherein 30-40 L is applied to plants with a crown diameter of ≥2 m, and 10-20 L is applied to plants with a crown diameter of <2 m; (2) Drip irrigation: 30 days after the first application, the biological preparation is diluted 100 times, and is applied to the roots through drip irrigation, once every 15 days for 2 times, with an application amount of 20-50 L per plant; (3) Spraying: Before the spring shoots of citrus sprout, the biological preparation is diluted 100 times and sprayed on the back of the leaves, once in the morning and once in the evening, once every 20 days for 3 times, with an application amount of 20-50 L per plant.

[0041] In some embodiments, in step (1), the ditch opening position is adjusted according to the tree vigor: when the tree vigor is weak, the ditch is opened close to the trunk, and when the tree vigor is strong, the ditch is opened away from the trunk but within the water dripping line.

[0042] In some embodiments, in step (2), the drip irrigation dilution liquid is injected into the ditched area through a fertilizer gun, and the wetness in the ditch is used as the criterion; in step (3), the spraying time is cloudy, early morning or evening, and the spraying amount is used as the criterion that the back of the leaves is wet to near water dripping.

[0043] The second aspect of the embodiments of the present application provides a biological preparation of Bacillus subtilis L1-21 strain for application, which is composed of the following components in mass percentage: 3%-5% of Bacillus subtilis L1-21 strain CGMCC No. 15726 bacterial liquid, the effective viable bacterial count of the bacterial liquid is ≥1.0×10 100.4%~1%, the water-soluble fertilizer is selected from one or more combinations of potassium fulvic acid, fish protein or amino acid, and when combined, the components are equal in amount; a dissolution aid 0.1%~0.3%, the dissolution aid is selected from one or a combination of glycerol and emulsified oil, and when combined, the components are equal in amount; the balance is water, and the pH value is 6.2~7.2.

[0044] The third aspect of the embodiment of the present application provides a preparation method of a biological preparation of the Bacillus subtilis L1-21 strain, comprising the following steps: (1) primary seed culture of the strain with the preservation number of CGMCC No. 15726: inoculate various strains preserved at low temperature on a primary seed culture medium, and shake culture at a culture temperature of 37 DEG C and a rotation speed of 120 rpm / min for 36 h to obtain a primary seed liquid, the primary seed culture medium is LB liquid medium with a pH value of 7.0~7.2; (2) secondary seed culture: inoculate the primary seed liquid into a secondary seed culture medium in a seed tank at an inoculation amount of 10%, and culture at 30 DEG C, a rotation speed of 100 rpm / min, and a ventilation amount of 0.72~1.6 m 3 / h, with the tank pressure kept at 0.05~0.08 MPa for 36~48 h to obtain a secondary seed liquid; the secondary seed culture medium is LB liquid medium with a pH value of 7.0~7.2; (3) fermentation culture: inoculate the secondary seed liquid into a fermentation culture medium in a fermentation tank at an inoculation amount of 10%, and culture at 30 DEG C, a rotation speed of 100 rpm / min, and a ventilation amount of 0.72~1.6 m 3 / h, with the tank pressure kept at 0.05~0.08 MPa for 48~72 h, and the Landy medium is selected, when the effective viable bacterial count in each fermentation liquid after culture is >=1x10 10 cfu / mL, the fermentation liquid is packaged in a plastic barrel and stored in a ventilated and dry place; (4) pour 3~5% of the fermentation liquid of the Bacillus subtilis L1-21 strain obtained in step (3) into a mixer and provide a culture medium, control the temperature at 28~30 DEG C, maintain stirring and oxygenation, after the secondary maturation is completed, further add water-soluble fertilizer 0.4~1% and dissolution aid 0.1~0.3%, continue to maintain the ventilation and stirring conditions, the pH value is 7.2, after 72 h, pour into a storage tank, and then package into products to prepare the biological preparation of the Bacillus subtilis L1-21 strain. Example 1

[0045] The application of the Bacillus subtilis L1-21 strain in the preparation of a biological preparation for preventing and treating citrus Huanglongbing, the classification and naming of the Bacillus subtilis L1-21 strain is Bacillus subtilis Bacillus subtilis, and is preserved in China General Microbiological Culture Collection Center, Beijing, China, with the address of No. 1, Beichen West Road, Beijing, China; the preservation date is May 4, 2018; and the preservation number is CGMCC No. 15726.

[0046] The method for using the biological agent of the present application comprises the following steps: (1) Digging: before autumn, open ring ditch and side ditch in the drip line of the diseased citrus tree crown, the ditch is 20 cm deep, and after the rotten lateral roots are dug out, the biological agent is applied at 20-50 L / plant, wherein 30-40 L is applied to the plant with a crown diameter of greater than or equal to 2 m, and 10-20 L is applied to the plant with a crown diameter of less than 2 m; the ditch position is adjusted according to the tree vigor: the ditch is opened close to the main stem when the tree vigor is weak, and the ditch is opened away from the main stem but in the drip line when the tree vigor is strong.

[0047] (2) Drip irrigation: 30 days after the first application, the biological agent is diluted by 100 times, and is applied to the root through drip irrigation, once every 15 days, for 2 times, and the application amount is 20-50 L / plant; the drip irrigation diluent is injected into the ditch area through the fertilizer gun, and the wetness in the ditch is used as the criterion; in step (3), the spraying time is cloudy, early morning or evening, and the spraying amount is used as the criterion that the back of the leaf is wet to nearly water dripping.

[0048] (3) Spraying: before the spring shoots of citrus germinate, the biological agent is diluted by 100 times and sprayed on the back of the leaf, once in the morning and once in the evening, once every 20 days, for 3 times, and the application amount is 20-50 L / plant. Example 2

[0049] The biological agent of the Bacillus subtilis L1-21 strain for application of the present application is composed of the following components in mass percentage: Bacillus subtilis L1-21 strain CGMCC No. 15726 bacterial liquid 4%, the effective viable bacterial count of the bacterial liquid is greater than or equal to 1.0 x 10 10 cfu / mL; water-soluble fertilizer 0.6%, the water-soluble fertilizer is amino acid, and the dissolution aid is 0.1%-0.3%, the dissolution aid is selected from two combinations of glycerol and emulsified oil, and each component is equal in combination; the balance is water, and the pH value is 6.6. Example 3

[0050] The difference between Example 3 and Example 2 is that the biological agent of the Bacillus subtilis L1-21 strain for application of the present application is composed of the following components in mass percentage: Bacillus subtilis L1-21 strain CGMCC No. 15726 bacterial liquid 3%, the effective viable bacterial count of the bacterial liquid is greater than or equal to 1.0 x 10 10 cfu / mL; water-soluble fertilizer 1%, the water-soluble fertilizer is potassium fulvic acid; dissolution aid 0.3%, the dissolution aid is selected from glycerol; the balance is water, and the pH value is 6.2. Example 4 Example 4 differs from Example 2 in that:

[0051] The biological preparation of the Bacillus subtilis L1-21 strain for application of the present application is composed of the following components in mass percentage: Bacillus subtilis L1-21 strain CGMCC No. 15726 bacterial solution 5%, the effective viable bacterial count of the bacterial solution ≥1.0×10 10 cfu / mL; water-soluble fertilizer 0.4%, the water-soluble fertilizer is potassium fulvic acid and fish protein, and when combined, the components are equal in amount; cosolvent 0.1%, the cosolvent is selected from emulsifiable concentrate, and the balance is water, with a pH value of 7.2. Example 5 Example 5 differs from Example 2 in that:

[0052] The biological preparation of the Bacillus subtilis L1-21 strain for application of the present application is composed of the following components in mass percentage: Bacillus subtilis L1-21 strain CGMCC No. 15726 bacterial solution 5%, the effective viable bacterial count of the bacterial solution ≥1.0×10 10 cfu / mL; the water-soluble fertilizer is a combination of fish protein and two kinds of amino acids, and when combined, the fish protein and amino acids are equal in amount; the balance is water, with a pH value of 6.5. Example 6

[0053] The preparation method of the biological preparation of the Bacillus subtilis L1-21 strain of the present application comprises the following steps: (1) Primary seed culture of the strain with preservation number CGMCC No. 15726: inoculate various bacterial strains preserved at low temperature on a primary seed culture medium, and under the conditions of a culture temperature of 37℃ and a rotation speed of 120 rpm / min, shake the bed for 36 h to obtain a primary seed solution, the primary seed culture medium is LB liquid medium, with a pH value of 7.0; (2) Secondary seed culture: inoculate the primary seed solution into a secondary seed culture medium in a seed tank at an inoculation amount of 10%, and under the conditions of a culture temperature of 30℃, a rotation speed of 100 rpm / min, a ventilation amount of 0.72 m 3 / h, and a tank pressure kept at 0.08 MPa, culture for 38 h to obtain a secondary seed solution; the secondary seed culture medium is LB liquid medium, with a pH value of 7.1; (3) Fermentation culture: inoculate the secondary seed solution into a fermentation culture medium in a fermentation tank at an inoculation amount of 10%, and under the conditions of a culture temperature of 30℃, a rotation speed of 100 rpm / min, a ventilation amount of 0.72 m 3 / h, and a tank pressure kept at 0.08 MPa, culture for 48 h to obtain a fermentation solution, and the Landy culture medium is selected, and the effective viable bacterial count in each fermentation solution after culture is ≥1×10 10When the viable cell count is 1 x 10 (4) 5% of the fermentation broth of the Bacillus subtilis L1-21 strain obtained in step (3) is poured into a mixer and a culture medium is provided, the temperature is controlled at 30°C, stirring and aeration are maintained, after secondary maturation is completed, 1% water-soluble fertilizer and 0.3% solubilizer are added, the aeration and stirring conditions are continuously maintained, the pH value is 7.2, after 72 h, it is poured into a storage tank, then packaged into products, to obtain the biological preparation of the Bacillus subtilis L1-21 strain. Example 7

[0054] Example 7 is different from Example 6 in that the preparation method of the biological preparation of the Bacillus subtilis L1-21 strain of the application comprises the following steps: In step (1), the strain with the preservation number of CGMCC No. 15726 is subjected to primary seed culture: various strains preserved at low temperature are inoculated on a primary seed culture medium, under the conditions of a culture temperature of 37°C and a rotation speed of 120 rpm / min, the seed culture is carried out on a shaking table for 36 h, to obtain a primary seed liquid, the primary seed culture medium is LB liquid medium, and the pH value is 7.2; In step (2), secondary seed culture: the primary seed liquid is transferred to a secondary seed culture medium in a seed tank at an inoculation amount of 10%, under the conditions of a culture temperature of 30°C, a rotation speed of 100 rpm / min, and a ventilation amount of 1.6 m 3 / h, the tank pressure is kept at 0.05 MPa, and the culture is carried out for 48 h, to obtain a secondary seed liquid; the secondary seed culture medium is LB liquid medium, and the pH value is 7.0; In step (3), fermentation culture: the secondary seed liquid is inoculated into a fermentation culture medium in a fermentation tank at an inoculation amount of 10%, under the conditions of a culture temperature of 30°C, a rotation speed of 100 rpm / min, and a ventilation amount of 1.2 m 3 / h, the tank pressure is kept at 0.05 MPa, and the culture is carried out for 72 h, Landy culture medium is selected, and the viable cell count in each fermentation liquid after culture is ≧1 x 10 10 cfu / mL, the fermentation liquid is packaged in a plastic bucket and stored in a ventilated and dry place; In step (4), 5% of the fermentation broth of the Bacillus subtilis L1-21 strain obtained in step (3) is poured into a mixer and a culture medium is provided, the temperature is controlled at 30°C, stirring and aeration are maintained, after secondary maturation is completed, 1% water-soluble fertilizer and 0.3% solubilizer are added, the aeration and stirring conditions are continuously maintained, the pH value is 7.2, after 72 h, it is poured into a storage tank, then packaged into products, to obtain the biological preparation of the Bacillus subtilis L1-21 strain. Example 8

[0055] The difference between Example 8 and Example 6 is that the preparation method of the biological agent of Bacillus subtilis strain L1-21 of the present invention includes the following steps: In step (1), the strain with accession number CGMCC No. 15726 was subjected to primary seed culture: various strains preserved at low temperature were inoculated onto primary seed culture medium and cultured on a shaker at a temperature of 37℃ and a rotation speed of 120 rpm / min for 36 h to obtain primary seed liquid. The primary seed culture medium was LB liquid medium with pH 7.1. In step (2), secondary seed culture: at an inoculum volume of 10%, the primary seed culture is transferred to the secondary seed culture medium in the seed tank, and culture is carried out at 30°C, 100 rpm / min, and an aeration rate of 1.2 m³ / min. 3 The culture was carried out at a pressure of 0.06 MPa for 36 hours to obtain a secondary seed culture. The secondary seed culture medium was LB liquid medium with a pH of 7.2. In step (3), fermentation culture: The secondary seed culture was inoculated into the fermentation medium in the fermenter at a 10% inoculum rate. The fermentation was carried out at 30°C, with a rotation speed of 100 rpm / min and an aeration rate of 1.6 m³ / min. 3 The fermentation broth was incubated at a pressure of 0.06 MPa for 62 hours using Landy medium. The viable cell count in each fermentation broth after incubation was ≥1 × 10⁻⁶. 10 When the concentration of CFU / mL is 1000 mg / mL, the fermentation broth should be packaged in a plastic bucket and stored in a well-ventilated and dry place. In step (4), 4% of the fermentation broth of Bacillus subtilis strain L1-21 obtained in step (3) is poured into a mixer and culture medium is provided. The temperature is controlled at 28°C, and stirring and oxygenation are maintained. After the second ripening is completed, 0.4% water-soluble fertilizer and 0.2% co-solvent are added, and the aeration and stirring conditions are maintained. The pH value is 7.2. After 72 hours, it is poured into a storage tank and packaged into a product to obtain the biological agent of Bacillus subtilis strain L1-21. Example 9

[0056] This invention relates to a bacterium isolated from healthy early-maturing Satsuma mandarin orange seedlings of the 'Xingjin' variety in Panxi Town, Huaning County, Yunnan Province, which exhibits good inhibitory effects against citrus Huanglongbing (HLB). Quantitative real-time PCR analysis showed an inhibition rate exceeding 80%. Morphological and molecular identification confirmed it to be Bacillus subtilis. Bacillus subtilis L1-21, through in vitro inoculation with bacterial culture, demonstrated its excellent endogenetic activity. Bacillus subtilis ( Bacillus subtilis The gene sequences of the identification sites of L1-21 are 16S (PP886200.1), rpoB (PP898322.1), and gyrB (PP898321.1).

[0057] Compared to the traditional "three-pronged approach" of removing diseased trees, which costs farmers 200 yuan per tree, applying beneficial endophytic bacteria can restore the growth of Huanglongbing trees and help them produce normal fruit, increasing citrus yield. On the other hand, it also improves the quality of citrus, increasing the relative sugar content of the fruit by 5%, thereby increasing farmers' income.

[0058] This invention, through the application of Bacillus subtilis L1-21 inoculant combined with a systematic approach (one dig, two drops, three sprays), achieves a control effect of over 90% against citrus Huanglongbing (verified in Example 2), significantly reversing diseased tree symptoms (yellowing leaves turning green, new shoot growth), and restoring plant health. Figure 1 , Figure 3 ). Preparation method of microbial inoculants

[0059] The above-mentioned strains are cultured and fermented according to their biological characteristics.

[0060] (1) Primary seed culture Various strains of bacteria preserved at low temperature were inoculated onto the corresponding liquid culture medium and cultured in a shaker at a temperature of 37℃ and a rotation speed of 120 rpm / min for 36 h to obtain the primary seed culture. The seed culture medium was LB liquid medium with a pH of 7.0~7.2.

[0061] (2) Secondary seed culture At an inoculum volume of 10%, the primary seed culture was transferred to the secondary seed culture medium in the seed tank. The medium was incubated at 30°C, with a rotation speed of 100 rpm / min and an aeration rate of 0.72–1.6 m³ / min. 3 The culture was carried out at a pressure maintained at 0.05~0.08MPa for 36~48h to obtain secondary seed culture. The seed culture medium was LB liquid medium with a pH of 7.0~7.2.

[0062] (3) Fermentation culture At an inoculum volume of 10%, the secondary seed culture was inoculated into the fermentation medium in the fermenter. The fermentation was carried out at 30°C, with a rotation speed of 100 rpm / min and an aeration rate of 0.72–1.6 m³ / min. 3 The fermentation broth was incubated at a pressure maintained at 0.05–0.08 MPa for 48–72 hours using Landy medium. The effective viable cell count in each fermentation broth after incubation was ≥1 × 10⁻⁶. 10 When the concentration of cfu / mL is 1000 mg / mL, the fermentation broth should be packaged in a plastic container and stored in a well-ventilated and dry place.

[0063] (4) Preparation of biological agents Bacillus subtilis ( Bacillus subtilis L1-21 CGMCC No.15726 fermentation broth (effective viable count ≥ 1×10⁻⁶) 10The second seed liquid is poured into the mixer and provided with the culture medium, the temperature is controlled at 28-30 DEG C, the stirring and aeration are maintained, after the secondary maturation is completed, the water-soluble fertilizer 0.4-1% and the dissolving agent 0.1-0.3% are added, the aeration and stirring are continuously maintained, the pH value is 7.2, after 72 hours, the product is poured into the storage tank and packaged.

[0064] (5) Culture medium The components of the first seed liquid include: 10 g / l of proteose peptone, 5 g / l of yeast powder and 10 g / l of sodium chloride. Example 10 The prevention and treatment effect of the method of the application on citrus Huanglongbing

[0065] Test site: In Menling Town, Huichang County, Ganzhou City, Jiangxi Province, a test demonstration was carried out, and the actual application area reached 1000 mu.

[0066] Method: On April 25, 2025, shallow trenches were dug and organic fertilizer was applied. 20-40 L of bio-organic fertilizer was poured in. The bio-organic fertilizer included 100 g of bacterial powder (10 10 cfu / g), 0.5 g of protein, 3 L of water, and fermentation for 24 hours. Before use, 4 kg of protein water-soluble fertilizer was added, and diluted with 1000 L of water. Each tree with a tree crown diameter of more than 2 m was applied with 40 L of the above water-soluble solution, and each tree with a tree crown diameter of less than 2 m was applied with 20 L of the above water-soluble solution.

[0067] On the evening of April 25, 2025, 10 7 cfu / mL of L1-21 bacterial liquid was sprayed on the back of the leaves until the beginning of dripping.

[0068] On June 4, 2025, and July 6, 2025, 10 7 cfu / mL of L1-21 bacterial liquid was sprayed once. Root drenching, trench application, leaf spraying, and pruning were carried out. The prevention effect was particularly obvious, and the trees became green and healthy (see the following Figure 1 to Figure 3 Effect diagram of the yellowing of the leaves of the diseased trees turning green. Figure 1 Schematic diagram of the leaves of the diseased trees before spraying on April 25, 2025. 80% of the leaves were yellow, and the tree vigor was weak. Figure 2 Schematic diagram of the leaves of the diseased trees 40 days after spraying on June 4, 2025. All the yellowing leaves turned green, and the growth was vigorous. Figure 3 Schematic diagram of the leaves of the diseased trees 70 days after spraying on July 6, 2025. All the yellowing leaves turned green, and the growth was vigorous. Example 11 Process for preventing and treating citrus Huanglongbing

[0069] Preparation: 50 mL of biocontrol bacteria solution and 1 kg of potassium fulvate or rotten rapeseed cake are added into 1 L of water for standby.

[0070] Implementation steps: Dig: The length of the ditch is determined according to the tree vigor and disease condition. For the trees with good vigor and not extremely serious disease, two sides or ring ditch can be dug; for the trees with weak vigor, side ditch is opened. The ditch is about 20 cm deep. The width of the ditch from the base of the tree is determined by digging to the active roots. Part of the active roots are cut or pruned. That is, for the plants with weak vigor, the ring ditch is opened near the trunk, otherwise, for the plants with strong vigor, the ring ditch is opened slightly far from the trunk, but it must be within the drip line. The 100-fold dilution liquid of the above-mentioned biocontrol agent, endophyte and organic fertilizer are applied. After 40 days, new roots grow at the broken surface of the active roots. The process is shown in Figure 4 to Figure 6 . Figure 4 It is a schematic diagram of the ring ditch of the present application. For the plants with strong vigor, the ring ditch is opened slightly far from the trunk, but it must be within the drip line. The rotten lateral roots are dug out, and part of the active roots are cut or pruned. The 100-fold dilution liquid of the above-mentioned biocontrol agent and organic fertilizer are applied. Figure 5 It is a schematic diagram of the side ditch of the present application. For the plants with weak vigor, the side ditch is opened near the trunk. The rotten lateral roots are dug out, and part of the active roots are cut or pruned. The 100-fold dilution liquid of the above-mentioned biocontrol agent and organic fertilizer are applied. Figure 6 It is a schematic diagram of the new root growth of the present application. After 40 days of application of endophyte and organic fertilizer, new roots grow at the broken surface of the active roots.

[0071] Drip irrigation: drip irrigation is carried out in the dug fertilization ditch, or injection is carried out in the dug fertilization ditch by using a fertilization gun. The amount of water is also determined by the moisture in the ditch. After the above-mentioned biological agent is diluted by 300-500 times, root drip irrigation is carried out once every 15 days, and continuous drip irrigation is carried out for 2 times. 50 L per plant is applied.

[0072] Spray: on cloudy or light rain days, or before 8 o'clock in the morning or after sunset in the evening, the biological agent is diluted by 200 times, and then directly sprayed on the back of the leaf. The amount of water is determined by the wetting of the back of the leaf to near water dripping. Spray once every 10-15 days in the early stage, and spray for 3 times. Thereafter, spray once every 20-30 days. The bacteria solution cannot dry out within 1 hour after spraying. Before harvesting, the diseased trees bear normal fruits, as shown in Figure 7 and Figure 8 . Figure 7 It is a symptom diagram before spraying of the present application. The tree vigor is weak, and the leaf is mottled and yellow. Figure 8 It is a symptom diagram of the normal fruiting of the present application. The growth is vigorous, the fruit is full, the color is uniform, the skin is thin and smooth, and the taste is sweet.

[0073] The above shows and describes the basic principles, main features and advantages of the present application. Those skilled in the art should understand that the present application is not limited to the above-mentioned embodiments, and the above-mentioned embodiments and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and the scope of protection of the present application is defined by the appended claims, the specification and their equivalents.

Claims

1. The application of a Bacillus subtilis strain L1-21 in the preparation of a biological agent for controlling citrus Huanglongbing (HLB), characterized in that: The Bacillus subtilis strain L1-21 is classified as Bacillus subtilis. Bacillus subtilis It is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; deposit date: May 4, 2018; Accession number: CGMCC No. 15726.

2. The application according to claim 1, characterized in that: The method of using the biological agent includes the following steps: (1) Digging: Before autumn, dig a ring trench and side trenches inside the drip line of the canopy of diseased citrus trees. The trenches are 20 cm deep. After digging out the rotten side roots, apply 20-50 L of biological agent per tree. For trees with a canopy diameter ≥ 2 m, apply 30-40 L, and for trees with a canopy diameter < 2 m, apply 10-20 L. (2) Drip irrigation: 30 days after the first application, dilute the biological agent 100 times and apply it to the roots by drip irrigation once every 15 days for two consecutive times. The application rate is 20~50 L / plant. (3) Spraying: Before the spring shoots of citrus sprout, dilute the biological agent 100 times and spray it on the back of the leaves once in the morning and once in the evening, once every 20 days for 3 consecutive times. The application rate is 20~50 L / plant.

3. The application according to claim 1, characterized in that: In step (1), the trenching location is adjusted according to the tree vigor: when the tree vigor is weak, the trench is dug close to the trunk; when the tree vigor is strong, the trench is far from the trunk but within the drip line.

4. The application according to claim 1, characterized in that: In step (2), the drip irrigation diluent is injected into the trench area through the fertilizer gun until the trench is moistened; in step (3), the spraying time is cloudy, early morning or evening, and the spraying amount is until the underside of the leaves is moistened to the point that water droplets slide down.

5. A biological agent of Bacillus subtilis strain L1-21 for use in the application described in claim 1, characterized in that: The biological agent comprises the following components by weight percentage: 3%~5% of Bacillus subtilis L1-21 strain CGMCC No. 15726 bacterial solution, wherein the effective viable count of the bacterial solution is ≥1.0×10⁻⁶. 10 cfu / mL; water-soluble fertilizer 0.4%~1%, wherein the water-soluble fertilizer is selected from one or more combinations of potassium humate, fish protein or amino acids, and each component is in equal amounts when combined; cosolvent 0.1%~0.3%, wherein the cosolvent is selected from one or two combinations of glycerol and emulsifiable concentrate, and each component is in equal amounts when combined; the balance is water, and the pH value is 6.2~7.

2.

6. A method for preparing a biological agent of Bacillus subtilis strain L1-21 as described in claim 5, characterized in that... Includes the following steps: (1) The strain with accession number CGMCC No. 15726 was subjected to primary seed culture: various strains preserved at low temperature were inoculated on primary seed culture medium and cultured on a shaker at a temperature of 37℃ and a rotation speed of 120 rpm / min for 36 h to obtain primary seed liquid. The primary seed culture medium was LB liquid medium with pH 7.0~7.

2. (2) Secondary seed culture: At an inoculum volume of 10%, the primary seed culture was transferred to the secondary seed culture medium in the seed tank. The culture was kept at 30°C, with a rotation speed of 100 rpm / min and an aeration rate of 0.72~1.6 m³ / min. 3 The culture was carried out at a pressure of 0.05~0.08MPa for 36~48h to obtain a secondary seed culture; the secondary seed culture medium was LB liquid medium with a pH of 7.0~7.

2. (3) Fermentation culture: Inoculate the secondary seed culture into the fermentation medium in the fermenter at an inoculation rate of 10%, and maintain the culture at 30℃, a rotation speed of 100 rpm / min, and an aeration rate of 0.72~1.6 m³ / min. 3 The fermentation broth was incubated at a pressure maintained at 0.05–0.08 MPa for 48–72 hours using Landy medium. The effective viable cell count in each fermentation broth after incubation was ≥1 × 10⁻⁶. 10 When the concentration of CFU / mL is 1000 mg / mL, the fermentation broth should be packaged in a plastic bucket and stored in a well-ventilated and dry place. (4) Pour 3-5% of the fermentation broth of Bacillus subtilis strain L1-21 obtained in step (3) into a mixer and provide culture medium. Control the temperature at 28-30℃, maintain stirring and oxygenation. After the second ripening is completed, add 0.4-1% water-soluble fertilizer and 0.1-0.3% co-solvent. Continue to maintain aeration and stirring conditions. The pH value is 7.

2. After 72 hours, pour into a storage tank and package into a product to obtain the biological agent of Bacillus subtilis strain L1-21.

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