Preparation method and application of microbial fertilizer for preventing and treating citrus diseases

Abstract

The invention relates to a preparation method and application of a microbial fertilizer for preventing and treating citrus diseases, and belongs to the technical field of fertilizer.The microbial fertilizer comprises beneficial microorganisms such as thiobacillus thiooxidans, nitrogen-fixing bacteria and saccharomycetes, humic acid, an AMS-SiO2 carrier, biochemical potassium fulvate and mineral elements. The preparation method comprises the following three key steps: firstly, combining beneficial bacteria with humic acid through a microbial flocculation technology to form a flocculation carrier; secondly, immobilizing paecilomyces lilacinus, bacillus and trichoderma by using the porous AMS-SiO2 carrier to form a complex microbial inoculant; the application of the fertilizer aims at citrus diseases such as citrus huanglongbing, canker and anthracnose, an annular furrow application, broadcast application prevention and chemical pesticide synergistic scheme is adopted, growth period management is combined, and by optimizing the carrier structure and flora compatibility, the fertilizer can be applied to citrus diseases such as citrus huanglongbing, canker and anthracnose. The method solves the problems of low survival rate of microorganisms in a soil environment, disease prevention time mismatch and the like, and obviously improves the disease prevention and control effect and the fruit quality.

Description

Technical Field

[0001] The present application relates to the field of fertilizer technology, and in particular to a method for preparing a microbial fertilizer for preventing and controlling citrus diseases and its application. Background Art

[0002] A preparation method and application of microbial fertilizer for citrus disease prevention and control: in-depth analysis based on background technology The citrus industry occupies an important position in the global agricultural economy. However, diseases that threaten its healthy growth, such as Huanglongbing, canker, and anthracnose, continue to pose severe challenges to yield and quality. For a long time, chemical control methods have been widely used because of their rapid effectiveness. However, problems such as pesticide residues, enhanced pathogen resistance, environmental pollution, and soil microecological imbalance caused by their excessive use have become increasingly prominent. As an important green alternative, microbial fertilizers have attracted much attention because they contain beneficial microorganisms that have the potential to antagonize pathogens, induce plant resistance, and activate nutrients. Looking at the microbial fertilizer products and related preparation methods currently on the market and recorded in technical literature for disease control, although they can show certain effects under specific experimental conditions, their stability, durability, and comprehensive disease prevention effects in actual field applications are often far below expectations. Technical shortcomings have led to numerous bottlenecks in application. Beneficial microorganisms are unable to establish effective and stable populations in the rhizosphere soil of citrus fruits. Existing product preparation technologies focus on increasing initial bacterial loads, paying insufficient attention to ensuring the survival, proliferation, and colonization of microbial agents in complex and variable soil environments, and in the face of the inevitable impact of chemical residues. Especially in the cultivation of high-value citrus varieties, the small amounts of necessary protective or therapeutic chemical fungicides to prevent key diseases are difficult to completely avoid. The disease prevention efficacy of many microbial products is significantly reduced or even eliminated once they come into contact with chemical residues or struggle to survive in soil microenvironments outside of ideal laboratory conditions. Existing preparation technologies often fail to establish an effective physical or biochemical buffering mechanism to provide an initial shelter for functional microbial communities, allowing them to slowly release into the environment after successful colonization. Furthermore, the occurrence and development of key citrus diseases have specific seasonal cycles and triggering conditions. Existing microbial fertilizer preparation methods often lack a sophisticated design for regulating nutrient release and microbial activity, resulting in a mismatch between peak microbial activity and the critical window for potential disease outbreaks. For example, before the rainy season arrives, many pathogens are active and diseases are prone to spreading. This is when the active support period of microbial fertilizers should reach its peak. However, the reality is that after many products are applied to the soil, the bacterial activity or nutrient stimulation of plant defenses may peak prematurely and disappear before the actual threat of a disease outbreak. Or, due to the lack of combined slow-release technology, the long-term protective effect cannot be maintained throughout the risk period. To address the challenges of complex diseases, the combination of multiple beneficial microorganisms has become a trend. However, existing preparation technologies generally provide a superficial understanding of the synergistic mechanisms between microbial communities and the scientific compatibility of bacterial strains. The microenvironments required by different strains may naturally conflict or be incompatible. Common simple physical mixing preparation methods not only fail to precisely maintain the active microbial niches of each bacterial strain, but may also lead to mutual antagonism or functional inhibition. The carrier selection and granulation processes of most products are relatively traditional, making them highly sensitive to soil moisture. They tend to disintegrate too quickly or become compacted by water absorption in moist soil, affecting the living environment of the functional microorganisms within and their migration to the rhizosphere.These physically unstable fertilizer granules are unable to effectively slow-release their beneficial active ingredients, nor can they form a physical barrier to create a protective microenvironment for microbial colonization. Furthermore, the microbial disease prevention potential and the delivery and release of essential plant nutrients are often separated, preventing the properties of fertilizer and pesticide from being truly integrated into a single solution.

[0003] In summary, there is an urgent need to develop a new method for preparing microbial fertilizers for the prevention and control of citrus diseases and its application on the market. Summary of the Invention

[0004] The present application provides a method for preparing a microbial fertilizer for preventing and controlling citrus diseases and its application, in order to solve the problems raised in the above-mentioned background technology.

[0005] To solve the above technical problems, the present application discloses a method for preparing a microbial fertilizer for preventing and controlling citrus diseases, comprising the following steps: a) Preparation of microbial flocculation carrier: Thiobacillus thiooxidans, nitrogen-fixing bacteria, and yeast were inoculated into a liquid culture medium containing glycerol and ammonium sulfate, cultured with shaking, humic acid was added and stirred, and CaCl2 solution was added and allowed to stand to form a flocculation carrier; b) Preparation of composite immobilized bacterial agents: AMS-SiO2 supports were synthesized by manipulating the pore structure using Pluronic P123 templates and polystyrene microspheres. After amino-thiol modification, Paecilomyces lilacinus, Bacillus licheniformis, Bacillus amyloliquefaciens, and Trichoderma were adsorbed and immobilized in a step-by-step manner. c) Granulation and maturation: Mix the nutrients and mineral agents, combine them with the microbial flocculation carrier through fluidized bed spraying, and dry and mature them into shape.

[0006] Furthermore, the nutrient agent includes biochemical potassium fulvic acid, molasses baking powder, seaweed powder and rare earth nitrate.

[0007] Furthermore, the mineralizer includes superphosphate, magnesium sulfate, borax and chelated zinc.

[0008] Furthermore, Thiobacillus thiooxidans, nitrogen-fixing bacteria and yeast were inoculated into a liquid culture medium containing 0.05 g / mL glycerol, 0.1 g / mL ammonium sulfate and pH 6.0 in a weight ratio of 15:10:8, and cultured with shaking at 30°C for 48 hours. 12 parts by weight of humic acid were added and stirred, and 0.1 mol / L CaCl2 solution was added and allowed to stand to form a flocculation carrier.

[0009] Furthermore, the synthesis of the AMS-SiO2 support includes dissolving 4.0 g of Pluronic P123 in 120 mL of 1.6 mol / L HCl, adding 0.8 g of polystyrene microspheres for ultrasonic dispersion, heating to 40°C, adding 9.2 mL of ethyl orthosilicate and 3.0 mL of styrene emulsion, and stirring at 600 rpm for 24 hours; the reaction solution is crystallized at 100°C for 48 hours, calcined at 550°C for 5 hours, and then the surface is modified by 3-aminopropyltriethoxysilane and 3-mercaptopropyltrimethoxysilane.

[0010] Furthermore, the adsorption ratio of the Paecilomyces lilacinus to the AMS-SiO2 carrier was 1:4 v / w, and the adsorption was allowed to stand at 28°C for 6 hours; the adsorption ratio of the Bacillus licheniformis and Bacillus amyloliquefaciens to the AMS-SiO2 carrier was 1:4, and the adsorption was allowed to stand at 28°C for 8 hours; the adsorption ratio of the Trichoderma to the AMS-SiO2 carrier was 1:2 v / w, and the adsorption was allowed to stand at 28°C for 6 hours.

[0011] Furthermore, the humic acid in step a) is Mengxi MX-HA90; the fluidized bed spraying conditions in step c) are an inlet air temperature of 30°C, an atomization pressure of 0.15 MPa, and an amount of microbial flocculation carrier of 45 parts by weight; the maturation conditions are 28°C and RH 60% for 48 hours, and the drying conditions are 40°C for 4 hours.

[0012] Furthermore, the nutrient agent in step c) is composed of biochemical potassium fulvic acid, molasses baking powder, seaweed powder and rare earth nitrate in a weight ratio of 8:7:5:5.

[0013] The present application also discloses the use of the microbial fertilizer obtained by the preparation method in preventing and controlling citrus Huanglongbing disease. Seven days before planting, 100 g / plant is applied in a circular ditch 20 cm deep and 15 cm wide, and then covered with soil and watered thoroughly with 15 L / plant.

[0014] The present application also discloses the use of the microbial fertilizer obtained by the preparation method in preventing and treating ulcer disease or anthrax, which is applied to the crown projection area at a rate of 50 g / m² 24 hours before the cumulative rainfall for three consecutive days is ≥30 mm.

[0015] The mechanism of action of the above-mentioned raw material components is as follows: In microbial fertilizers used for citrus disease control, the various raw material components work synergistically to form a comprehensive control system. Despite the complex and ever-changing soil environment and the persistent threat of disease, this method achieves an organic integration of microbial activity, nutrient supply, and disease suppression through carefully selected raw material combinations. Thiobacillus thiooxidans exerts a core antagonistic function, directly inhibiting the proliferation of pathogens such as Huanglongbing disease, while nitrogen-fixing bacteria provide nitrogen required by plants through nitrogen fixation, thereby enhancing citrus root vitality. Meanwhile, yeast participates in the organic matter fermentation process, promoting nutrient release and soil microecological balance. However, the survival of microorganisms in soil is often affected by chemical residues or competitive pressure. Therefore, humic acid is introduced as a key auxiliary. It not only stabilizes the microbial community through flocculation but also acts as a natural chelating agent to improve soil structure and create a living microenvironment for beneficial bacteria. The AMS-SiO2 carrier is the core innovation of this technology, with its multi-level pore structure providing physical shelter for microorganisms. Specifically, the carrier's internal pores adsorb and protect functional microorganisms from environmental shocks, ensuring they are not destroyed before colonizing the rhizosphere. This structure not only delays microbial release but also optimizes the matching of the bacterial population with the window of disease outbreak. Furthermore, amino and thiol modifications on the carrier surface enhance microbial adsorption, enabling efficient binding of Paecilomyces lilacinus, Bacillus licheniformis, Bacillus amyloliquefaciens, and Trichoderma. Paecilomyces lilacinus specifically targets vectors such as nematodes, while Bacillus and Trichoderma directly inhibit canker and anthrax pathogens by secreting antimicrobial substances. Despite the diverse needs of different strains, the hierarchical design of the carrier avoids interspecies conflict and instead promotes synergistic effects. Nutrients such as biochemical potassium fulvate and molasses baking powder not only provide an energy source for the microorganisms but also enhance disease resistance by stimulating the plant's own defense mechanisms. Seaweed extract, rich in bioactive substances, can induce the production of resistance proteins in citrus fruits, while rare earth nitrate acts as a trace element carrier, catalyzing enzymatic reactions and enhancing photosynthetic efficiency. Mineral elements such as superphosphate and magnesium sulfate ensure balanced plant nutrition and mitigate susceptibility to deficiencies. Borax and chelated zinc address the specific needs of citrus, indirectly reducing the risk of disease invasion by strengthening cell walls and enzymes. Therefore, all ingredients function not in isolation but form a chain reaction: microorganisms antagonize pathogens, nutrients strengthen the plant, and carrier mechanisms ensure long-lasting effects. Ultimately, this reduces reliance on chemical pesticides while achieving comprehensive disease prevention and control.

[0016] The mechanism of action of the above-mentioned preparation method is as follows: This preparation method transforms raw material components into a highly effective fertilizer through three key steps. Its mechanism of action is centered around optimizing microbial survival, ensuring disease prevention effectiveness, and integrating functionalities. First, during the preparation of the microbial flocculation carrier, while traditional methods can easily lead to bacterial inactivation, this technology stimulates bacterial activity through specific culture conditions. Thiobacillus thiooxidans, nitrogen-fixing bacteria, and yeast are cultured in a medium containing glycerol and ammonium sulfate to form a highly viable community. Subsequently, humic acid is added to induce flocculation through calcium ions, forming a stable carrier. This process not only protects the microorganisms from initial environmental stress but also, through the chelating properties of humic acid, lays the foundation for subsequent bacterial colonization. Second, the preparation of the composite immobilized bacterial agent is the core breakthrough of this method, resolving the issues of strain incompatibility and insufficient sustained release in existing technologies. The synthesis of the AMS-SiO2 carrier involves the manipulation of templates and microspheres to create a hierarchical pore structure. This structure provides a shelter for the microorganisms, ensuring their gradual release into the soil rather than a single exposure. The mechanism of action of the raw material components is as follows: In microbial fertilizers used for citrus disease prevention and control, the various raw material components work synergistically to form a comprehensive prevention and control system. Despite the complex and changeable soil environment and the persistent threat of diseases, this method achieves an organic integration of microbial activity, nutrient supply, and disease suppression through carefully selected raw material combinations. Thiobacillus thiooxidans plays a core antagonistic function, directly inhibiting the proliferation of pathogens such as Huanglongbing fungus, while nitrogen-fixing bacteria provide the nitrogen required by plants through nitrogen fixation, thereby enhancing the vitality of citrus roots. At the same time, yeast participates in the fermentation process of organic matter, promoting nutrient release and soil microecological balance. However, the survival of microorganisms in the soil is often affected by chemical residues or competitive pressure. Therefore, humic acid is introduced as a key auxiliary. It not only stabilizes the microbial community through flocculation, but also acts as a natural chelating agent to improve soil structure and create a living microenvironment for beneficial bacteria.

[0017] The AMS-SiO2 carrier is the core innovation of this technology, and its multi-level pore structure provides physical shelter for microorganisms. Specifically, the internal pores of the carrier can adsorb and protect functional microorganisms from environmental shocks, ensuring that they are not destroyed before they colonize in the rhizosphere. This structure not only delays the release of microorganisms, but also optimizes the matching of the bacterial flora with the window of disease outbreak. In addition, the amino and thiol modifications on the surface of the carrier enhance the adsorption capacity of microorganisms, allowing Paecilomyces lilacinus, Bacillus licheniformis, Bacillus amyloliquefaciens and Trichoderma to bind efficiently. Paecilomyces lilacinus specifically targets vectors such as nematodes, while Bacillus and Trichoderma directly inhibit canker and anthrax pathogens by secreting antibacterial substances. Although different strains have different needs, the hierarchical design of the carrier avoids conflicts between species and promotes synergistic effects.

[0018] Nutrient components such as biochemical potassium fulvic acid and molasses yeast not only provide an energy source for microorganisms but also enhance disease resistance by stimulating the plant's own defense mechanisms. Seaweed extract, rich in bioactive substances, can induce citrus to produce resistance proteins, while rare earth nitrates act as trace element carriers, catalyzing enzymatic reactions and enhancing photosynthetic efficiency. Mineral elements such as superphosphate and magnesium sulfate ensure balanced plant nutrition and prevent susceptibility to nutrient deficiencies. Borax and chelated zinc target the specific needs of citrus, indirectly reducing the risk of disease invasion by strengthening cell walls and enzyme function. Therefore, all ingredients do not exist in isolation, but rather form a chain reaction: microorganisms antagonize pathogens, nutrients strengthen the plant's constitution, and carrier mechanisms ensure long-term effects. Ultimately, this reduces reliance on chemical pesticides while achieving comprehensive disease prevention and control.

[0019] The mechanism of action of the preparation method is as follows: This preparation method transforms raw material components into highly effective fertilizers through three key processes. Its mechanism of action revolves around optimizing microbial survival, ensuring disease prevention effectiveness, and integrating functions. First, during the preparation of microbial flocculation carriers, although traditional methods can easily lead to bacterial inactivation, this technology stimulates strain activity through specific culture conditions. Thiobacillus thiooxidans, nitrogen-fixing bacteria, and yeast are cultured in a culture medium containing glycerol and ammonium sulfate to form a highly active community; humic acid is then added to induce flocculation through calcium ions to form a stable carrier. This process not only protects microorganisms from initial environmental stress, but also lays the foundation for subsequent bacterial colonization through the chelating properties of humic acid.

[0020] Secondly, the preparation of a composite immobilized bacterial agent is the core breakthrough of this method, resolving the existing issues of strain incompatibility and insufficient sustained release. The synthesis of the AMS-SiO2 carrier involves the manipulation of templates and microspheres to create a multi-level pore structure. This structure provides a shelter for the microorganisms, ensuring their gradual release into the soil rather than a single exposure.

[0021] After modification of the carrier with amino and thiol groups, the surface functional groups enhance the adsorption capacity of different microorganisms. A step-by-step adsorption strategy was employed for Paecilomyces lilacinus, Bacillus, and Trichoderma to avoid antagonism caused by mixing. For example, Paecilomyces lilacinus preferentially adsorbed and activated, followed by gradual binding to Bacillus and Trichoderma. This sequential adsorption not only maintained the independent microbial niches of each strain but also enhanced overall antibacterial efficacy through synergistic interactions. Although soil moisture and chemical residues often threaten microbial activity, the carrier's pore-buffering mechanism mitigates external shocks, enabling the microbial community to maintain peak activity during periods of high disease risk, such as before the rainy season. Finally, during the granulation and maturation stage, nutrients and minerals were integrated with the microbial carrier, achieving integrated functionality. The nutrients, including biochemical potassium fulvic acid, were uniformly coated onto the microbial carrier using fluidized bed spray technology to form a granular structure. This process ensured a slow release of nutrients while preventing thermal damage during the granulation process. The maturation process was carried out under controlled temperature and humidity, promoting the binding of microorganisms and nutrients, resulting in fertilizer granules with high physical stability. While traditional fertilizers tend to disintegrate in moist soil, the particle design of this method maintains structural integrity, ensuring a slow release of microorganisms and nutrients into the rhizosphere. Therefore, this entire preparation method not only addresses the issue of mismatched disease prevention timing but also transforms fertilizer into a long-lasting disease control tool through carrier protection, step-by-step immobilization, and slow-release technology.

[0022] Compared with the prior art, this application provides a method for preparing a microbial fertilizer for preventing and controlling citrus diseases and its application, which has the following beneficial effects: 1. This application uses the multi-level pore structure of the AMS-SiO2 carrier and optimized bacterial flora to not only reduce the incidence of Huanglongbing, but also ensure that the disease prevention time is precisely matched with the disease outbreak window. Spreading before the rainy season can maintain protection throughout the risk period. 2. This application integrates mineral agents and nutrients to not only increase the VC content of the fruit, with the soluble solids reaching 12.8°Brix, but also achieve a 15.3% increase in single fruit weight, thereby preventing and controlling diseases while promoting the healthy growth of citrus fruits; 3. This application uses a buffering mechanism for hole application to maintain a high canker disease inhibition rate when the concentration of chlorpyrifos is ≤ 0.10%, which not only reduces the amount of pesticide used but also alleviates the problem of soil microecological imbalance; 4. Despite the complex and changeable soil environment, this application uses step-by-step immobilization technology and flocculation carriers to form a stable population of beneficial microorganisms in the rhizosphere, thereby reducing the anthrax disease index to a level far lower than that of the control group, demonstrating its advantage in highly competitive soils; 5. Traditional methods require separate processing of fertilizers and pesticides, but this application integrates carrier slow-release, nutrient delivery and disease prevention into one. For example, through customized plans for the growth period, it not only improves operational convenience but also reduces labor costs. The comprehensive benefits far exceed those of a single technical solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the SEM image of the AMS-SiO2 carrier prepared in Example 1 of the present application. DETAILED DESCRIPTION

[0024] The preferred embodiments of the present application are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present application and are not used to limit the present application.

[0025] In addition, in this application, descriptions such as "first" and "second" are only used for descriptive purposes and do not specifically refer to the order or ranking, nor are they used to limit this application. They are only used to distinguish components or operations described with the same technical terms, and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one such feature. In addition, the technical solutions and technical features between the various embodiments can be combined with each other, but this must be based on the ability of ordinary technicians in this field to implement. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by this application.

[0026] Unless otherwise specified, the examples and comparative examples are parallel experiments with the same components, component contents, preparation steps, and preparation parameters. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are analytical reagents (AR) unless otherwise specified, which were purchased from commercial channels.

[0027] Thiobacillus thiooxidans was CGMCC 1.101 from the China General Microbial Culture Collection; nitrogen-fixing bacteria was ACCC 03101 from the China Agricultural Culture Collection; yeast was Saccharomyces cerevisiae SF-201 from Angel Yeast Co., Ltd.; humic acid was MX-HA90 from Inner Mongolia Mengxi Mining Co., Ltd.; Pluronic P123 was purchased from Sigma-Aldrich (USA), Mn = 5800 ± 500, EO 20 PO 70 EO 20Block copolymers; polystyrene microspheres were purchased from Merck (Germany), with a particle size of 1 μm; tetraethyl orthosilicate was purchased from Aladdin Reagent; 3-aminopropyltriethoxysilane was purchased from Nanjing Shuguang Chemical Group Co., Ltd., KH-550, with an amino group density ≥5.8 mmol / g; 3-mercaptopropyltrimethoxysilane was purchased from Gaide Chemical, KH-590, with a thiol group density ≥4.2 mmol / g; spores of Paecilomyces lilacinus were Bio-134513 from Beijing Biobowei Biotechnology Co., Ltd.; Bacillus licheniformis was CICC 10037 from the China Industrial Microorganism Culture Collection Center, and Bacillus amyloliquefaciens was GDMCC from the Guangdong Provincial Microorganism Culture Collection Center. 1.141; Trichoderma was TL-006 from Shandong Lvlong Biotechnology Co., Ltd.; biochemical potassium fulvate was purchased from BF-HA60 from Shandong Innovation Humic Acid Technology Co., Ltd.; molasses fermentation powder was purchased from NL-FM50 from Guangxi Nong Le Biotechnology Co., Ltd.; seaweed powder was purchased from OMAR-20 from Qingdao Haid Biological Group Co., Ltd.; rare earth nitrate was purchased from REO-N-46 from Baotou Rare Earth Research Institute; superphosphate was purchased from XY-12P from Hubei Xiangyun Group; magnesium sulfate was purchased from LM-MgSO4·7H2O from Sichuan Longmang Group; borax was purchased from XZ-B4O7 from Tibet Autonomous Region Mining Development Co., Ltd.; chelated zinc was purchased from DY-Zn-15 from Zhengzhou Diyi Ecological Agriculture Technology Co., Ltd. Example 1

[0028] This embodiment discloses a method for preparing a microbial fertilizer for preventing and controlling citrus diseases, comprising the following steps: Step 1: Thiobacillus thiooxidans, nitrogen-fixing bacteria, and yeast were inoculated into a liquid culture medium containing 0.05 g / mL glycerol, 0.1 g / mL ammonium sulfate, and pH 6.0 at a weight ratio of 15:10:8, and cultured at 30°C and 150 rpm for 48 hours. 12 parts by weight of humic acid were added, and the mixture was stirred at 40°C and 200 rpm for 30 minutes. 0.1 mol / L CaCl2 solution was added, and the mixture was allowed to stand at 25°C for 3 hours to prepare a microbial flocculation carrier.

[0029] Step 2: Add 4.0 g of Pluronic P123 (EO 20 PO 70 EO 20, Mn = 5800) was dissolved in 120 mL of 1.6 mol / L HCl solution, mechanically stirred at 250 rpm in a 35°C water bath for 45 minutes, 0.8 g of polystyrene microspheres was added, and ultrasonic dispersion was performed at 300 W for 12 minutes. The temperature was raised to 40°C, 9.2 mL of tetraethyl orthosilicate and 3.0 mL of styrene emulsion were added, and the temperature was maintained at 40°C ± 0.5°C. The reaction was stirred at 600 rpm for 24 hours. The reaction solution was transferred to a polytetrafluoroethylene autoclave, and the temperature was raised to 100°C at 1.5°C / min and maintained for 48 hours. After the crystallization was completed, it was cooled to 25°C at 0.8°C / min. The solid was filtered and washed three times with 200 mL of ultrapure water. The temperature was raised to 550°C at 2°C / min and maintained at this temperature for 5 hours. When the temperature was raised to 300°C under nitrogen, 2 g of the calcined support was dispersed in 50 mL of anhydrous ethanol and 1.2 mL 3-aminopropyltriethoxysilane, 1.05 mL 3-mercaptopropyltrimethoxysilane, ultrasonic treatment for 20 minutes to form a uniform suspension, the suspension was transferred to a round-bottom flask, installed with a condenser reflux device, stirred and refluxed at 300 rpm in a 78 ° C oil bath for 8 hours, cooled to room temperature, centrifuged at 8000 rpm for 10 minutes, washed with 50 mL of anhydrous ethanol and 50 mL of acetone three times each, vacuum dried at -0.09 MPa and 60 ° C for 12 hours, and sieved to obtain amino-mercaptosilica AMS-SiO2. 9 CFU / mL) was activated with 0.1 mol / L CaCl2, and Paecilomyces lilacinus was mixed with AMS-SiO2 at a ratio of 1:4 (v / w), and the mixture was allowed to stand at 28°C for 6 h. Bacillus licheniformis and Bacillus amyloliquefaciens were mixed with AMS-SiO2 at a ratio of 1:4 (v / w), and the mixture was allowed to stand at 28°C for 8 h. Trichoderma was mixed with AMS-SiO2 at a ratio of 1:2 (v / w), and the mixture was allowed to stand at 28°C for 6 h. The mixture was then centrifuged at 500 rpm for 15 min to obtain the composite immobilized bacterial agent.

[0030] Step 3: 8 parts of biochemical potassium humate, 7 parts of molasses baking powder, 5 parts of seaweed powder and 5 parts of rare earth nitrate are stirred and mixed by weight to prepare a nutrient agent, and 6 parts of superphosphate, 4 parts of magnesium sulfate, 3 parts of borax and 2 parts of chelated zinc are stirred and mixed to prepare a mineral agent. The mixture is stirred at 45 rpm at room temperature for 20 minutes, sprayed with 45 parts by weight of microbial flocculation carrier in a fluidized bed at an inlet air temperature of 30°C and an atomization pressure of 0.15 MPa, dried at 40°C for 4 hours, and matured at 28°C and RH 60% for 48 hours to obtain a microbial fertilizer for the prevention and control of citrus diseases.

[0031] Application Example 1 Targeted application techniques specifically address Huanglongbing (Huanglongbing) and canker / anthracnose. To control Huanglongbing vectors (psyllids / nematodes), dig a 20 cm deep and 15 cm wide circular trench seven days before planting. Apply 100 g of fertilizer granules evenly per plant, cover with soil, and water thoroughly at 15 L per plant. To prevent canker / anthracnose outbreaks, apply 50 g / m² of fertilizer to the canopy projection 24 hours before the local weather forecast indicates cumulative rainfall of 30 mm or more for three consecutive days.

[0032] The chemical pesticide synergistic plan specifically includes applying fertilizer in holes at a dose of 75 g per plant 12 hours before applying chlorpyrifos (concentration ≤0.10%±0.01%) to control leaf miners.

[0033] Specific management during the growth period includes digging a 15 cm deep hole 15 days before the spring shoots sprout and applying 150g / plant; during the young fruit stage, take 50g of fertilizer, add 1 L of pure water and shake for 30 minutes, filter the extract, dilute it to a concentration of 0.50% and spray it on the leaves; 20 days after the autumn shoots mature, apply 200g / plant in radial grooves 30 cm away from the main trunk.

[0034] The AMS-SiO2 in the microbial fertilizer for citrus disease control prepared in Example 1 was scanned with a scanning electron microscope, and the results were as follows: Figure 1 As shown in the figure, it can be seen that the carrier presents a multi-level pore structure. Example 2

[0035] This example discloses a microbial fertilizer for preventing and controlling citrus diseases. The mixture comprises: Paecilomyces lilacinus and AMS-SiO2 in a ratio of 1:3 (v / w), and the mixture is allowed to stand for adsorption at 28°C for 6 hours. The mixture comprises: Bacillus licheniformis and Bacillus amyloliquefaciens and AMS-SiO2 in a ratio of 1:5 (v / w), and the mixture is allowed to stand for adsorption at 28°C for 8 hours. The mixture comprises: Trichoderma spp. and AMS-SiO2 in a ratio of 1:3 (v / w), and the mixture is allowed to stand for adsorption at 28°C for 6 hours. The mixture is then centrifuged at 500 rpm for 15 minutes to obtain a composite immobilized bacterial agent. Other contents are consistent with those in Example 1. Example 3

[0036] This example discloses a microbial fertilizer for preventing and controlling citrus diseases. The mixture comprises: Paecilomyces lilacinus and AMS-SiO2 in a ratio of 1:4 (v / w), and the mixture is allowed to stand for adsorption at 28°C for 6 hours. The mixture comprises: Bacillus licheniformis and Bacillus amyloliquefaciens and AMS-SiO2 in a ratio of 1:6 (v / w), and the mixture is allowed to stand for adsorption at 28°C for 8 hours. The mixture comprises: Trichoderma spp. and AMS-SiO2 in a ratio of 1:4 (v / w), and the mixture is allowed to stand for adsorption at 28°C for 6 hours. The mixture is then centrifuged at 500 rpm for 15 minutes to obtain a composite immobilized bacterial agent. Other contents are consistent with those in Example 1.

[0037] Comparative Example 1 The difference from Example 1 is the lack of 0.8 g of polystyrene microspheres, and the other contents are the same.

[0038] Comparative Example 2 The difference from Example 1 is that 1.05 mL of MPTMS is missing, and the prepared product is not thiol-modified, and the other aspects are the same.

[0039] Comparative Example 3 The difference from Example 1 is that the mixed suspension of Paecilomyces lilacinus, Bacillus, and Trichoderma is adsorbed with AMS-SiO2 at a total bacterial liquid: carrier ratio of 1:4 (v / w), and the other ingredients are the same.

[0040] Performance Testing Performance tests were conducted on the embodiments and comparative examples. According to GB / T 17980.54-2004, the incidence rate was calculated in a high-incidence area of ​​Deqing, Guangdong (accumulative sample of 200 trees). Gonggan oranges (Citrus reticulata 'Gonggan'), 3-4 years old and 1.5-2.0 meters tall, were planted. They are a variety highly susceptible to Huanglongbing. In an experimental area in southern Jiangxi, the pathogen was artificially inoculated. Navel oranges (Citrus sinensis 'Newhall'), 3-4 years old and susceptible to canker, were planted. The lesion area was calculated after 15 days. A total of 200 trees were sampled. All healthy seedlings with no history of disease were selected. The planting density was 4 m×4 m. The soil pH was 5.5-6.5, and the organic matter content was ≥2.0%, which met the "Environmental Conditions for Green Food Citrus Production Areas" (NY / T 391-2013). Specific fertilization methods were strictly implemented in accordance with the disease-targeted application technology and growth period management in Application Example 1. The anthracnose disease index was tested by monitoring during the natural disease period (rainy season, June to August) and graded according to the "Specifications for Field Investigation of Crop Diseases" (GB / T 17980.54-2004): Grade 0 (disease-free), Grade 1 (lesions ≤ 5%), Grade 2 (6%-20%), Grade 3 (21%-50%), and Grade 4 (>50%). The vitamin C content in fruits was tested by randomly selecting 10 fruits for each treatment, peeling them, and squeezing out the juice for determination. The results are shown in Table 1.

[0041] Table 1 Test items Test conditions / methods Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Huanglongbing incidence Deqing, Guangdong, a high-incidence area (200 samples collected) 13.2% 11.7% 12.1% 28.5% 31.2% 25.8% Ulcer disease inhibition rate 15 days after artificial inoculation of pathogens (Gannan, Jiangxi) 70.1% 76.5% 73.8% 42.3% 38.7% 45.1% Anthrax Disease Index Natural onset period monitoring 15.7 12.3 13.9 32.6 35.1 28.9 Fruit VC content (mg / 100g) GB / T 6195-1986 42.5 41.8 42.0 36.2 35.6 37.1 Soluble solids (°Brix) NY / T 2637-2014 12.8 12.5 12.6 11.2 10.9 11.4 Single fruit weight increase (%) Weighing method of fruits during harvest +15.3 +14.7 +14.9 +8.2 +7.5 +9.1 As can be seen from Table 1, Example 1 uses the multi-level pore structure and step-by-step immobilization process of the AMS-SiO2 carrier, rare earth nitrate and seaweed powder synergistically activate the enzymatic reaction, and mineral elements enhance photosynthesis, achieving dual synergy of disease prevention and nutrient supply. Example 1 has the best overall performance; Comparative Example 1 lacks polystyrene microspheres, and the disordered carrier pores lead to uneven microbial adsorption and a decrease in disease inhibition rate; Comparative Example 2 lacks thiol-modified carrier protection, resulting in insufficient inhibition rate; Comparative Example 3 mixed adsorption, due to competition among bacterial species, the activity of Trichoderma is suppressed, and the anthrax index is increased.

[0042] Obviously, those skilled in the art may make various changes and modifications to this application without departing from the spirit and scope of this application. If these modifications and variations of this application fall within the scope of the claims of this application and their equivalents, this application is intended to include these modifications and variations.

Claims

1. A method for preparing a microbial fertilizer for preventing and controlling citrus diseases, characterized in that: The following steps are involved: a) Preparation of microbial flocculation carrier: Thiobacillus thiooxidans, nitrogen-fixing bacteria, and yeast were inoculated into a liquid culture medium containing glycerol and ammonium sulfate, cultured with shaking, humic acid was added and stirred, and CaCl2 solution was added and allowed to stand to form a flocculation carrier; b) Preparation of composite immobilized bacterial agents: AMS-SiO2 supports were synthesized by manipulating the pore structure using Pluronic P123 templates and polystyrene microspheres. After amino-thiol modification, Paecilomyces lilacinus, Bacillus licheniformis, Bacillus amyloliquefaciens, and Trichoderma were adsorbed and immobilized in a step-by-step manner. c) Granulation and maturation: Mix the nutrients and mineral agents, combine them with the microbial flocculation carrier through fluidized bed spraying, and dry and mature them into shape.

2. The method for preparing a microbial fertilizer for preventing and controlling citrus diseases according to claim 1, characterized in that: The nutrient agent comprises biochemical potassium fulvic acid, molasses fermentation powder, seaweed powder and rare earth nitrate.

3. The method for preparing a microbial fertilizer for preventing and controlling citrus diseases according to claim 1, characterized in that: The mineralizers include superphosphate, magnesium sulfate, borax and chelated zinc.

4. The method for preparing a microbial fertilizer for preventing and controlling citrus diseases according to claim 1, characterized in that: Thiobacillus thiooxidans, nitrogen-fixing bacteria and yeast were inoculated into a liquid culture medium containing 0.05 g / mL glycerol, 0.1 g / mL ammonium sulfate and pH 6.0 in a weight ratio of 15:10:8, and cultured with shaking at 30°C for 48 hours. 12 parts by weight of humic acid were added and stirred, and 0.1 mol / L CaCl2 solution was added and allowed to stand to form a flocculation carrier.

5. The method for preparing a microbial fertilizer for preventing and controlling citrus diseases according to claim 1, characterized in that: The synthesis of the AMS-SiO2 carrier includes dissolving 4.0 g of Pluronic P123 in 120 mL of 1.6 mol / L HCl, adding 0.8 g of polystyrene microspheres for ultrasonic dispersion, heating to 40°C, adding 9.2 mL of ethyl orthosilicate and 3.0 mL of styrene emulsion, and stirring at 600 rpm for 24 hours; the reaction solution is crystallized at 100°C for 48 hours, calcined at 550°C for 5 hours, and then the surface is modified with 3-aminopropyltriethoxysilane and 3-mercaptopropyltrimethoxysilane.

6. The method for preparing a microbial fertilizer for preventing and controlling citrus diseases according to claim 1, characterized in that: The adsorption ratio of the Paecilomyces lilacinus to the AMS-SiO2 carrier was 1:4 v / w, and the adsorption was allowed to stand at 28°C for 6 hours; the adsorption ratio of the Bacillus licheniformis and Bacillus amyloliquefaciens to the AMS-SiO2 carrier was 1:4, and the adsorption was allowed to stand at 28°C for 8 hours; the adsorption ratio of the Trichoderma to the AMS-SiO2 carrier was 1:2 v / w, and the adsorption was allowed to stand at 28°C for 6 hours.

7. The method for preparing a microbial fertilizer for preventing and controlling citrus diseases according to claim 1, characterized in that: The humic acid in step a) is Mengxi MX-HA90; the fluidized bed spraying conditions in step c) are an inlet air temperature of 30°C, an atomization pressure of 0.15 MPa, and a microbial flocculation carrier dosage of 45 parts by weight; the maturation conditions are 28°C and RH 60% for 48 hours, and the drying conditions are 40°C for 4 hours.

8. The method for preparing a microbial fertilizer for preventing and controlling citrus diseases according to claim 2, characterized in that: The nutrient agent in step c) is composed of biochemical potassium fulvic acid, molasses baking powder, seaweed powder and rare earth nitrate in a weight ratio of 8:7:5:

5.

9. Use of the microbial fertilizer obtained by the preparation method according to any one of claims 1 to 8 in preventing and treating citrus Huanglongbing, characterized in that: Seven days before transplanting, apply 100 g / plant in a circular ditch 20 cm deep and 15 cm wide, cover with soil and water thoroughly at 15 L / plant.

10. Use of the microbial fertilizer obtained by the preparation method according to any one of claims 1 to 8 in preventing and treating ulcer disease or anthrax, characterized in that: 24 hours before the cumulative rainfall for three consecutive days is ≥30 mm, apply 50 g / m² to the crown projection area.

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