Microbial composition for preventing and treating citrus canker and application thereof

Abstract

The invention relates to the technical field of microbial compositions, in particular to a microbial composition for preventing and treating citrus canker and application of the microbial composition. According to the microbial composition provided by the invention, the pseudomonas Qingdao microbial agent, sodium lignin sulfonate, trehalose, glutathione, ascorbic acid, lecithin and trace elements (Zn, B and Mn) are taken as main raw materials, and the effects of efficiently inhibiting bacteria, remarkably inducing and improving the activity of key defensive enzymes of leaves, promoting synthesis and accumulation of disease-resistant substances and improving the disease resistance of the leaves are achieved through the matching effect of all the components. The diversity and richness of citrus phyllospheric bacterial communities are improved, and the purpose of constructing a stable community dominated by beneficial bacteria is achieved. In other words, the basic stress resistance of the plants can be enhanced, and meanwhile, the growth and metabolism activity of beneficial bacteria is also promoted, so that effective, eco-friendly and sustainable prevention and control of the citrus canker are achieved.

Description

Technical Field

[0001] This invention relates to the field of microbial composition technology, and more specifically, to a microbial composition for preventing and treating citrus canker and its application. Background Technology

[0002] Citrus canker, caused by Xanthomonas aureus, a citrus-pathogenic bacterium (Citrus canker). Xanthomonas citri subsp. citri Caused by [unclear], this is a devastating bacterial disease that seriously threatens the global citrus industry. The pathogen infects leaves, branches, and fruit, causing characteristic corky lesions. This not only reduces photosynthetic efficiency and weakens the tree, but also severely damages the appearance and quality of the fruit, rendering it commercially worthless and causing huge economic losses to the citrus industry.

[0003] The control of citrus canker mainly relies on chemical pesticides and agricultural measures, but both have significant drawbacks. Long-term, excessive use of Bordeaux mixture and copper hydroxide leads to copper accumulation in the soil, causing soil pollution, disrupting the soil's microecological balance, inhibiting the activity of beneficial microorganisms, and even becoming toxic to aquatic organisms. Furthermore, pathogens easily develop resistance to copper-based agents and antibiotics, leading to decreased efficacy and forcing increased dosages or changes in pesticides, creating a vicious cycle. Consumers are increasingly sensitive to chemical residues in agricultural products, and frequent use of chemical pesticides poses potential food safety risks, which is inconsistent with the development trend of green and organic agriculture. In addition, broad-spectrum chemical pesticides, while killing pathogens, also kill large amounts of beneficial microorganisms, disrupting the natural balance of the plant's microecological environment.

[0004] Biological control and plant health regulation represent a new direction for green pest control. Faced with the limitations of traditional control methods, developing environmentally friendly and sustainable green control strategies is imperative. Biological control, especially the use of beneficial microorganisms and their metabolites, and strategies that enhance disease resistance by activating the plant's own immune system (inducing systemic resistance, ISR), has become a research hotspot and a future development direction. Ideal biological control agents should not only effectively inhibit pathogens but also improve the overall health and resistance of plants.

[0005] Effective control of citrus canker urgently requires innovative breakthroughs. Developing a composite microbial composition integrating highly effective antagonistic microorganisms, immune-activating peptides, and synergistic trace elements is crucial. This composition can not only directly inhibit pathogens but also systematically enhance the intrinsic defense capabilities of citrus plants (activating defense enzymes and accumulating disease-resistant substances) and reshape a healthy foliar microbial community (increasing diversity, richness, and enrichment of beneficial bacteria). This multi-target synergistic approach of inhibiting pathogens, strengthening the plant itself, and optimizing the ecosystem offers a highly promising green, safe, and sustainable comprehensive solution to address the environmental pressures, drug resistance risks, and ecological damage caused by traditional chemical control methods. Summary of the Invention

[0006] The purpose of this invention is to address the problems existing in the prior art regarding the poor efficacy and high environmental risks of chemical fertilizers and pesticides in the control of citrus diseases, as well as the vicious cycle that easily leads to increased pathogen resistance. This invention provides a microbial composition and its application. The microbial composition provided by this invention uses *Pseudomonas tsinensis* (…). Pseudomonas qingdaonensis The main raw materials are inoculants, sodium lignosulfonate and trehalose, as well as glutathione, ascorbic acid, lecithin and trace elements. It has the characteristics of low cost and high efficacy, and can improve the disease resistance and production performance of citrus plants.

[0007] To achieve the above objectives, the first aspect of the present invention provides a microbial composition for preventing and treating citrus canker, the microbial composition comprising: component 1: glutathione, ascorbic acid, lecithin and trace elements; component 2: *Pseudomonas ts.* with accession number CCTCC NO: M 2023153. Pseudomonas qingdaonensis Ingredients: microbial agent, sodium lignosulfonate, and trehalose.

[0008] Furthermore, the content of *Pseudomonas tsinensis* in the microbial composition is 1 × 10⁻⁶. 12 -3×10 12 cfu / g.

[0009] Furthermore, in the composition, the weight ratio of glutathione, ascorbic acid, lecithin, trace elements, *Pseudomonas tsinensis* inoculum, sodium lignosulfonate, and trehalose is 0.8-1.2:8-12:12-18:3-6:2:1.2:6.8.

[0010] Furthermore, the sodium lignin sulfonate has a water content of 1-5% by weight, a lignin sulfonate content of 50-70% on a dry matter basis, a reducing sugar and carbohydrate content of 15-30%, and an inorganic salt content of 5-20%; and / or, the trace elements have a ZnSO4·H2O content of 30-45%, a Mn-sugar alcohol content of 40-45%, and a sodium octaborate tetrahydrate content of 10-30%.

[0011] The second aspect of the present invention provides the application of the microbial composition of the first aspect in improving the activity of plant leaf defense enzymes, and / or, increasing the production of disease-resistant substances in plant leaves, and / or, increasing the diversity and richness of phylloxera bacteria, and / or, in the prevention and control of citrus canker, wherein the pathogen of citrus canker is *Xanthomonas cereus* citrus pathogenic strain (…). Xanthomonas citri subsp. citri ).

[0012] A third aspect of the present invention provides a method for improving the activity of plant leaf defense enzymes, and / or increasing the production of disease-resistant substances in plant leaves, and / or increasing the diversity and richness of phyllodes bacteria, and / or preventing citrus canker, characterized in that the method comprises spraying the microbial composition described in the first aspect onto the surface of citrus leaves and fruits, wherein the amount of the microbial composition applied to the plant leaves is such that the amount of *Pseudomonas ts.* applied is 1 × 10⁻⁶. 14 -1×10 15 CFU / acre / application, component 1 is applied during the spring shoot stage and summer shoot stage respectively, and component 2 is applied 15-20 days after the application of component 1. The application frequency of the composition is more than once a year.

[0013] Through the above technical solution, the present invention can achieve at least the following beneficial effects: (1) The microbial composition provided by the present invention can produce abundant foliar organisms and a variety of functional enzymes, effectively improve the disease resistance of leaves, significantly increase the activity of defense enzymes such as superoxide dismutase (SOD), polyphenol oxidase (PPO), catalase (CAT) and phenylalanine ammonia-lyase, and promote the secretion of malondialdehyde, chlorophyll content, total phenol content and soluble protein, which promote plant growth and resist disease occurrence.

[0014] (2) The microbial composition provided by the present invention can form a phyllosphere bacterial community structure that is conducive to plant growth, and can significantly improve the diversity index such as the ACE index and Chao 1 index of plant phyllosphere bacteria, as well as the bacterial richness index such as the Simpson index and Shannon index, thereby forming a microecology that is conducive to plant growth and disease resistance.

[0015] (3) The microbial composition provided by the present invention adopts the method of spraying the agent at different growth stages of the plant, combining the dual approach of improving the plant's defense against diseases and inhibiting the growth and reproduction of pathogenic microorganisms, thereby significantly improving the plant's disease resistance and reducing the pathogenic microorganisms ( Xanthomonas citri subsp. citri The abundance of ) can reduce the incidence of diseases and has broad application prospects.

[0016] Biological Preservation This invention provides a strain of Pseudomonas tsinensis ( Pseudomonas qingdaonensis ), categorized and named as: Pseudomonas qingdaonensis YIM B08402 is deposited at the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, China, on February 20, 2023, with accession number CCTCC NO: M 2023153. The deposit information for this strain has been disclosed in a prior patent application with application number 202310455963.X. Detailed Implementation

[0017] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0018] The inventors of this invention discovered in their research that a microbial composition made by mixing glutathione, ascorbic acid, lecithin, and trace elements in a certain proportion, when sprayed on the leaves, can effectively increase the activity of foliar defense enzymes and enhance foliar disease resistance. Especially for citrus, spraying this microbial composition can promote the production of disease-resistant substances in citrus leaves, increase citrus yield, and has a good control effect on citrus canker. Spraying this microbial composition also significantly enhances the activity of the citrus plant's own defense enzymes, thus improving the plant's systemic resistance. Furthermore, it can significantly increase bacterial diversity indices such as the ACE index and Chao 1 index, as well as bacterial richness indices such as the Simpson index and Shannon index, forming a microecological environment conducive to plant growth and disease resistance, thereby reducing the incidence of diseases. Further research revealed that adding a Pseudomonas tsingiensis agent with preservation number CCTCCNO: M 2023153 to the microbial composition resulted in better control of citrus diseases and could further increase the yield of leaf defensive substances and enhance the activity of plant defensive enzymes.

[0019] Based on the above findings, a first aspect of the present invention provides a microbial composition comprising: Component 1: Glutathione, ascorbic acid, lecithin and trace elements; Component 2: *Pseudomonas tsingiensis* inoculum with preservation number CCTCC NO: M 2023153, sodium lignin sulfonate, and trehalose.

[0020] Preferably, the content of *Pseudomonas tsinensis* in the microbial composition is 1 × 10⁻⁶. 12 -3×10 12 cfu / g. Preferably 1.5 × 10⁻⁶ 12 -3×10 12 cfu / g. More preferably 2×10 12 -3×10 12 cfu / g.

[0021] According to a preferred embodiment of the present invention, the weight ratio of glutathione, ascorbic acid, lecithin, trace elements, *Pseudomonas tsinensis* inoculant, sodium lignosulfonate and trehalose in the composition is 0.8-1.2:8-12:12-18:3-6:2:1.2:6.8.

[0022] In this invention, there are no particular restrictions on the raw materials (such as glutathione, ascorbic acid, lecithin and trace elements) used in the above-mentioned microbial compositions. They can be any relevant products in the art that can be used to prepare microbial compositions, either commercially available products or products prepared by oneself according to the prior art.

[0023] The inventors discovered in their research that when specific raw materials are used, the obtained microbial composition exhibits better effects in enhancing the disease resistance and control of citrus fruits. Therefore, according to a preferred embodiment of the present invention, the sodium lignin sulfonate, on a dry matter basis, comprises 50-70% lignin sulfonate content, 15-30% reducing sugar and carbohydrate content, and 5-20% inorganic salt content. Preferably, the sodium lignin sulfonate contains 60-70% lignin sulfonate, 20-30% reducing sugar and carbohydrate, and 10-20% inorganic salt, on a dry matter basis. According to a preferred embodiment of the present invention, the trace elements, on a dry matter basis, contain 30-45% ZnSO4·H2O, 40-45% Mn-sugar alcohol, and 10-30% sodium octaborate tetrahydrate.

[0024] Preferably, the trace elements, on a dry matter basis, contain 35-45% ZnSO4·H2O, 42-45% Mn-sugar alcohol, and 15-30% sodium octaborate tetrahydrate.

[0025] In this invention, any *Pseudomonas tsingensis* inoculant that has the function of improving the disease resistance of citrus and controlling citrus diseases can be added to the above-mentioned microbial composition.

[0026] In this invention, there are no particular restrictions on the specific storage and use forms of the microbial composition. Since the raw materials used in this microbial composition are primarily solid, and the preparation method involves simply mixing the components in proportion and then dissolving them in water, the microbial composition can be stored or used directly in the form of the prepared solid dosage form.

[0027] A second aspect of the present invention provides the application of a microbial composition in the prevention and control of citrus diseases, and / or, in enhancing the production of disease-resistant substances in plant leaves, and / or, in increasing the activity of defense enzymes in citrus plant leaves, and / or, in improving the diversity and richness of foliar bacteria in citrus leaves.

[0028] According to a preferred embodiment of the present invention, the crop is citrus.

[0029] In this invention, the prevention and control of citrus diseases refers to preventing or reducing the occurrence of citrus diseases, or reducing the losses caused by diseases after they occur.

[0030] In this invention, improving the activity of defense enzymes in citrus plant leaves refers to increasing the activity of superoxide dismutase (SOD), polyphenol oxidase (PPO), catalase (CAT), and phenylalanine ammonia-lyase (PAL) in citrus leaves.

[0031] In this invention, enhancing the disease resistance substances in plant leaves refers to increasing the content of malondialdehyde, chlorophyll, total phenols, and soluble protein, thereby improving the plant's resistance to external stresses.

[0032] In this invention, improving the diversity and richness of phyllosphere bacteria in plant leaves means improving the Chao 1 richness index and ACE index, as well as the Shannon diversity index and Simpson diversity index.

[0033] Preferably, the disease is selected from citrus diseases. More preferably, it is citrus canker. More preferably, the pathogen causing the citrus canker is a bacterium of the genus Xanthomonas. Xanthomonas citri subsp. citri .

[0034] The specific characteristics of the microbial composition used in the method provided by this invention have been described above and will not be repeated here.

[0035] In this invention, there are no particular restrictions on the specific amount of the microbial composition used, as long as it can improve the disease resistance of citrus leaves / prevent citrus diseases.

[0036] According to some preferred embodiments of the present invention, the microbial composition is applied to citrus plants at a dosage of 300-500 g / mu / time. For example, it can be 300 g / mu / time, 320 g / mu / time, 350 g / mu / time, 400 g / mu / time, 420 g / mu / time, 500 g / mu / time, or any intermediate value between any two of the above values.

[0037] Preferably, the amount of the microbial composition used is such that the amount of *Pseudomonas tsao-sienna* applied to the plant leaves is 1 × 10⁻⁶. 14 -1×10 15 The composition is applied 1-3 times per year, with a concentration of CFU / acre / time.

[0038] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to further explain and illustrate the present invention, and are not intended to limit the present invention.

[0039] In the following examples, sodium lignosulfonate was purchased from Shanghai Yunzhe New Material Technology Co., Ltd., trehalose and glutathione were purchased from Guangdong Dingsheng Food Ingredients Co., Ltd., ZnSO4·H2O was purchased from Jinan Miaohong New Material Co., Ltd., Mn-sugar alcohol was purchased from Tianjin Zhonghe Shengtai Chemical Co., Ltd., sodium octaborate tetrahydrate was purchased from Zhengzhou Jiajie Chemical Products Co., Ltd., ascorbic acid was purchased from Anhui Yuansheng Biotechnology Co., Ltd., and lecithin was purchased from Henan Jiarun Biotechnology Co., Ltd.

[0040] Example 1: Preparation of Component 1 Weigh the raw materials according to the proportions in Table 2 and mix them evenly.

[0041] Table 1 Material Composition

[0042] Table 2 Microbial Composition Formulation

[0043] Example 2: Preparation of *Pseudomonas qingdaoense* inoculum This embodiment illustrates the preparation process of the *Pseudomonas qingnanensis* inoculant of the present invention. It includes the following steps: (1) The Qingdao Pseudomonas aeruginosa with preservation number CCTCC NO: M 2023153 was cultured in solid medium to obtain test tube culture; (2) Prepare liquid seed culture medium and inoculate test tube seeds in it for liquid culture to obtain liquid seeds; (3) Prepare a liquid fermentation medium and inoculate it with liquid seeds for fermentation.

[0044] In step (1), the *Pseudomonas tsinensis* is inoculated using a slant inoculation method, and the solid culture is carried out at a temperature of 25-30℃ for 40-50 hours. The solid culture medium includes: glucose 10-12 g / L, agar 15-20 g / L, beef extract 3-5 g / L, yeast extract 1-3 g / L, peptone 10-15 g / L, pH = 6.5-7.5.

[0045] In step (2), the test tubes are inoculated into liquid seed culture medium, and the liquid culture is carried out at a temperature of 25-30℃ and a rotation speed of 200-250r / min for 45-50h; the liquid seed culture medium includes: peptone 10-15g / L, beef extract 3-5g / L, sodium chloride 10-12g / L, pH=6.5-7.5.

[0046] In this step, the liquid seed culture medium is preferably sterilized at 120-125℃ for 20-30 minutes, and after cooling, 0.5-1.5 cm of the culture medium is inoculated into 100 mL of liquid seed culture medium. 2 The test tubes were cultured and the liquid seeds were obtained by culturing them at 28-30℃ on a shaker at 220-250 r / min for 45-50 h.

[0047] In step (3), the liquid seed is inoculated in the liquid fermentation medium at a volume ratio of 0.05-0.1:1 and treated for 45-50 hours at a temperature of 25-30℃ and a rotation speed of 200-250 r / min. The liquid fermentation medium includes: 20-23 g / L sucrose, 10-15 g / L peptone, 5-8 g / L yeast extract, 3-5 g / L potassium dihydrogen phosphate, 5-8 g / L ammonium sulfate, 2-4 g / L calcium carbonate, and pH = 6.5-7.5.

[0048] In this step, the liquid fermentation medium is preferably sterilized at 120-125℃ for 20-30 minutes, cooled, and then inoculated with liquid seed. It is also preferably cultured at 28-30℃ in a 50L fermenter with a rotation speed of 220-250r / min for 45-50 hours to obtain the fermentation broth.

[0049] The above preparation steps also include: after fermentation, centrifuging is performed using a high-speed centrifuge to obtain the concentrated fermentation broth of the strain, and sodium lignin sulfonate and trehalose are added sequentially according to the proportions in Table 2, followed by drying and pulverization to obtain a viable count of approximately 2 × 10⁻⁶. 12 CFU / g of Pseudomonas qingdaoensis solid inoculum.

[0050] Test Example 1 Select citrus orchards with uniform growth, dissolve the microbial composition from the above examples in water according to the dosages in Table 3, and spray it onto the citrus plants. Specifically, the microbial agent in component 1 is sprayed during the spring and summer shoot stages, and the composition in component 2 is sprayed 15-20 days after the application of component 1. The application frequency is once a year. Simultaneously, during the citrus harvest season, disease incidence is investigated in farmland where the microbial composition provided by this invention is applied (treatment group) and where it is not applied (control group). The control effect is calculated, and root zone and leaf samples are collected from citrus plants to determine the content of disease-resistant substances, leaf enzyme activity, and the diversity and richness of foliar bacteria.

[0051] Citrus canker disease survey grading standards (leaf): Grade 0, with no visible lesions on the leaves; Grade 1, with 1-3 scattered, typical cankerous spots on the leaves; Grade 2, with 4-10 lesions on the leaves; Grade 3: 11-25 lesions on the leaves, or lesions merging over a large area; Level 4: There are numerous lesions on the leaves (>25), or the lesions are almost completely merged and cover most of the leaf surface.

[0052] Citrus canker disease survey grading standards (fruit): Grade 0, no visible lesions; Grade 1: Fruit has 1-3 lesions, and the maximum diameter of a single lesion is <3mm; Grade 2: The fruit has 1-3 lesions, and the maximum diameter of a single lesion is ≥3mm; or the fruit has 4-6 lesions (regardless of size). Grade 3, with 7-10 lesions on the fruit (regardless of size); Grade 4: The fruit has more than 10 lesions (regardless of size), or the fruit falls off due to severe disease.

[0053] The following two formulas are used to calculate the prevention and control effect: Disease index = [(Σ(number of disease-grade plants × representative grade)) / (total number of plants × highest representative grade value)] × 100 Prevention and control efficacy (%) = [(Disease index of control group - Disease index of treatment group) / Disease index of control group] × 100% Methods for determining the activity of citrus plant leaf surface defense enzymes: Superoxide dismutase (SOD) (U·g) -1 Polyphenol oxidase (PPO) (U∙g) -1 Catalase (CAT) (nmol·min⁻¹·g) -1 ), phenylalanine ammonia-lyase (U·g) -1 All assays (FW) were performed using kits purchased from Suzhou Greens Biotechnology Co., Ltd., and the usage instructions were as per the product manual.

[0054] Methods for determining disease resistance substances on citrus plant leaves: Malondialdehyde (mmol / g) content was determined by the thiobarbituric acid reaction method, chlorophyll content (SPAD) was determined by a handheld chlorophyll meter, total phenol content (mg / g) was determined by the Folin-Ciocalteu method, and soluble protein (mg / g) was determined by the Coomassie Brilliant Blue G-250 staining method.

[0055] Phyllostachyological bacterial diversity assay: Plant cells were lysed by liquid nitrogen grinding, and bacterial DNA was extracted using the FastDNA® Spin Kit for Soil, with an A260 / A280 ratio of 1.8–2.0. Qualified DNA was sent to Meiji Biotechnology Co., Ltd. for high-throughput sequencing. Primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′) were used to amplify the V3–V4 regions of the 16S rRNA of phyllostachyological bacteria. The raw sequencing data underwent question sequence identification, inspection, and removal of chimeric sequences to obtain valid sequences from the samples. The sequence with the highest abundance in each OTU was used as the representative sequence for that OTU. Based on 97% sequence similarity, OTUs were merged and segmented to construct an OTU abundance matrix for each sample. Based on the OTU abundance matrix results, the number of OTUs in each sample was calculated; the Chao 1 richness index and ACE index were used to evaluate bacterial community diversity; and the Shannon diversity index and Simpson diversity index were used to evaluate bacterial community richness.

[0056] As can be seen from the data in Tables 3, 4, 5, and 6, the application of the microbial composition provided by this invention significantly enhanced the control effect of citrus canker compared to the control. It also significantly improved the activity of citrus leaf defense enzymes, the production of disease-resistant substances, and the diversity and abundance of foliar bacteria. Furthermore, the application of the microbial composition provided by this invention significantly reduced the incidence of soil-borne and fruit canker in citrus, indicating that the microbial composition provided by this invention can control citrus canker by improving plant systemic resistance and inhibiting the growth of pathogenic microorganisms.

[0057] Table 3. Test conditions and results of the experiment on the control effect of microbial compositions on citrus canker.

[0058] *The control group was a blank control group, and the pesticide was a commercially available chemical pesticide whose main component was cuprous oxide.

[0059] Table 4. Effects of microbial compositions on the activity of defensive enzymes in citrus leaves.

[0060] *The control group was a blank control group, and the pesticide was a commercially available chemical pesticide whose main component was cuprous oxide.

[0061] Table 5. Effects of microbial compositions on the production of disease resistance substances in citrus leaves.

[0062] *The control group was a blank control group, and the pesticide was a commercially available chemical pesticide whose main component was cuprous oxide.

[0063] Table 6. Effects of microbial compositions on bacterial diversity in citrus foliage.

[0064] *The control group was a blank control group, and the pesticide was a commercially available chemical pesticide whose main component was cuprous oxide.

[0065] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A microbial composition for preventing and treating citrus canker, characterized in that, The microbial composition comprises: component 1: glutathione, ascorbic acid, lecithin, and trace elements; component 2: *Pseudomonas tsao-siècle* with accession number CCTCC NO: M 2023153. Pseudomonas qingdaonensis Ingredients: microbial agent, sodium lignosulfonate, and trehalose.

2. The microbial composition for preventing and controlling citrus canker according to claim 1, characterized in that: The content of *Pseudomonas tsaoko* in the microbial composition is 1 × 10⁻⁶. 12 -3×10 12 cfu / g.

3. The microbial composition for preventing and controlling citrus canker according to claim 1, characterized in that: In the composition, the weight ratio of glutathione, ascorbic acid, lecithin, trace elements, Pseudomonas tsaokoyaki inoculum, sodium lignosulfonate and trehalose is 0.8-1.2:8-12:12-18:3-6:2:1.2:6.

8.

4. A microbial composition for preventing and controlling citrus canker according to any one of claims 1-3, characterized in that: The sodium lignosulfonate has a water content of 1-5% by weight (on dry matter), a lignosulfonate content of 50-70%, a reducing sugar and carbohydrate content of 15-30%, and an inorganic salt content of 5-20%; and / or, the trace elements have a ZnSO4·H2O content of 30-45%, a Mn-sugar alcohol content of 40-45%, and a sodium octaborate tetrahydrate content of 10-30%.

5. The use of the microbial composition according to any one of claims 1-3 in improving the activity of plant leaf defense enzymes, and / or, increasing the production of disease-resistant substances in plant leaves, and / or, increasing the diversity and richness of phylloxera bacteria, and / or, in the prevention and control of citrus canker, wherein the pathogen of citrus canker is *Xanthomonas cereus* citrus pathogenic strain (…). Xanthomonas citri subsp. citri ).

6. A method for increasing the activity of plant leaf defense enzymes, and / or increasing the production of disease-resistant substances in plant leaves, and / or increasing the diversity and abundance of phylloxera bacteria, and / or controlling citrus canker, characterized in that, The method includes spraying the microbial composition described in any one of 1-3 onto the surface of citrus leaves and fruits, wherein the amount of the microbial composition used is such that the amount of *Pseudomonas tsao-siennai* applied to the plant leaves is 1 × 10⁻⁶. 14 -1×10 15 CFU / acre / application, component 1 is applied during the spring shoot stage and summer shoot stage respectively, and component 2 is applied 15-20 days after the application of component 1. The application frequency of the composition is more than once a year.

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