Microbial fertilizer for treating citrus huanglongbing and preparation method thereof

Abstract

The invention discloses a microbial fertilizer for treating citrus huanglongbing and a preparation method of the microbial fertilizer, and belongs to the field of agricultural biological control. 33 to 50 percent of sodium alginate-chitosan microspheres; 5 to 8% of hesperidin; 3-5% of nano zinc oxide; 2-4% of humic acid chelated boron; 2-4% of sucrose fatty acid ester; 2-4% of diatomite; wherein the preparation method of the sodium alginate-chitosan microspheres comprises the following steps: mixing sodium alginate with a microbial suspension, dropwise adding the mixture into a calcium chloride solution by using an injector, and standing and curing to form a microsphere core; and immersing the cured microspheres into a chitosan solution with the pH value of 6.0, taking out, and freeze-drying to obtain finished microspheres. Wherein the microbial suspension comprises trichoderma, bacillus and actinomycetes. The invention solves the problems of poor prevention and treatment effect of the existing Candidatus Liberobacter asiaticum, difficulty in colonization of the strain at phloem, short survival time and lack of targeted slow release function.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural biological control, and in particular to a microbial fertilizer for treating citrus Huanglongbing disease and a preparation method thereof. Background Art

[0002] Citrus Huanglongbing (Huanglongbing) is a devastating disease caused by a phloem-restricted pathogen that seriously threatens the sustainable development of the global citrus industry. The pathogen is transmitted by psyllids. After infecting the citrus phloem, it hinders nutrient transport, leading to leaf yellowing, fruit deformities, and tree decline, ultimately killing the fruit tree. Currently, chemical control measures (such as antibiotic injections) can suppress the pathogen in the short term, but long-term use can easily lead to drug resistance and pose a risk of pesticide residues, which is inconsistent with the development trend of green agriculture. Furthermore, chemical agents have difficulty distributing effectively in the phloem, resulting in limited control effectiveness. Biological control has become a research hotspot due to its environmental friendliness and sustainability. However, existing microbial fertilizers are mostly targeted at soil improvement or broad-spectrum disease control and lack specific inhibitory ability against the Huanglongbing pathogen. Furthermore, the strain has a low survival rate in the alkaline environment of the citrus phloem (pH 7.8-8.2), making long-term control difficult.

[0003] Existing microbial fertilizers face multiple technical bottlenecks in the prevention and treatment of Huanglongbing. On the one hand, conventional strains (such as Bacillus subtilis) cannot colonize in the phloem, and their direct inhibitory effect on the Huanglongbing pathogen is limited; on the other hand, the carrier system mostly uses ordinary diatomaceous earth or bentonite, whose pore size and surface properties are not suitable for the sustained release and protection of microorganisms, resulting in short survival time of bacteria and unstable prevention effect when applied in the field. In addition, the microbial embedding process in the existing technology is easy to disintegrate in an alkaline environment, and it is impossible to achieve targeted release at the site where the pathogen is enriched. Some studies have attempted to prevent and control through bacteriophages or genetically engineered bacteria, but there are problems such as strong host specificity, safety disputes and regulatory restrictions, which make it difficult to promote on a large scale.

[0004] In recent years, researchers have begun exploring the synergistic effects of composite microorganisms and functional carriers, but existing solutions still have significant shortcomings. For example, some existing technologies use a single strain in combination with a traditional carrier, with a control effectiveness of less than 30%. Other solutions, although using a combination of multiple strains, do not optimize the strain compatibility and carrier design based on the biological characteristics of the Huanglongbing pathogen. In addition, the existing preparation process is complex, involving strain preservation and multiple fermentations, resulting in high production costs and unfavorable industrial application. Therefore, there is an urgent need to develop a new type of microbial fertilizer that can achieve green and efficient prevention and control of Huanglongbing through the collaborative innovation of specific strain combinations, alkali-resistant carrier systems, and intelligent encapsulation technology. Summary of the Invention

[0005] The purpose of the present invention is to provide a microbial fertilizer for treating citrus Huanglongbing and a preparation method thereof, which solves the problems of poor prevention and control effect of existing citrus Huanglongbing, difficulty in bacterial strain colonization in the phloem, short survival time, and lack of targeted sustained-release function.

[0006] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0007] A microbial fertilizer for treating citrus Huanglongbing disease, comprising, by mass percentage, the following raw materials:

[0008] Modified diatomaceous earth: 30-40%;

[0009] Sodium alginate-chitosan microspheres: 33-50%;

[0010] Hesperidin: 5-8%;

[0011] Nano zinc oxide: 3-5%;

[0012] Humic acid chelated boron: 2-4%;

[0013] Sucrose fatty acid esters: 2-4%;

[0014] Diatomaceous earth: 2-4%;

[0015] The sodium alginate-chitosan microsphere preparation steps include: mixing sodium alginate with a microbial suspension, dripping it into a calcium chloride solution with a syringe, and letting it stand to solidify to form a microsphere core; immersing the solidified microspheres in a chitosan solution with a pH of 6.0, taking them out and freeze-drying them to obtain finished microspheres; wherein the microbial suspension includes Trichoderma, Bacillus and Actinomycetes.

[0016] According to a preferred embodiment of the present invention, the hesperidin is purchased from Shaanxi Peptide World Biotechnology Co., Ltd.

[0017] According to a preferred embodiment of the present invention, the nano zinc oxide is purchased from Shijiazhuang Shengpeng Chemical Co., Ltd.

[0018] According to a preferred embodiment of the present invention, the humic acid chelated boron was purchased from Hubei Longfei Biotechnology Co., Ltd. According to a preferred embodiment of the present invention, the sucrose fatty acid ester was purchased from Shaanxi Chenming Biotechnology Co., Ltd.

[0019] According to a preferred embodiment of the present invention, the diatomaceous earth is purchased from Qingdao Shengtai Silicon Industry Co., Ltd.

[0020] According to a preferred embodiment of the present invention, the sodium alginate is purchased from Henan Zongteng Food Additive Co., Ltd.

[0021] According to a preferred embodiment of the present invention, the calcium chloride solution is purchased from Weifang Qizheng Chemical Co., Ltd.

[0022] According to a preferred embodiment of the present invention, the chitosan is purchased from Zhengzhou Longsheng Chemical Products Co., Ltd.

[0023] According to a preferred embodiment of the present invention, the Trichoderma genus is purchased from Shanghai Bohu Biotechnology Co., Ltd. as BH-J1032.

[0024] According to a preferred embodiment of the present invention, the Bacillus was purchased from Weifang Yihao Biotechnology Co., Ltd.

[0025] According to a preferred embodiment of the present invention, the actinomycetes are purchased from Shanghai Bohu Biotechnology Co., Ltd. under the model 66547-09-9.

[0026] According to a preferred embodiment of the present invention, the preparation steps of the microbial suspension include: culturing Trichoderma, Bacillus and Actinomycetes in LB liquid medium at 30°C and 180rpm to OD600 = 1.2; collecting the cells by centrifugation and resuspending them in sterile saline; mixing Trichoderma: Bacillus: Actinomycetes in a ratio of 3:2:1; adjusting the final concentration to 10 8 CFU / mL.

[0027] According to a preferred embodiment of the present invention, the LB liquid culture medium was purchased from Beijing Biolab Technology Co., Ltd.

[0028] According to a preferred embodiment of the present invention, the sterile physiological saline is purchased from Beijing Yita Biotechnology Co., Ltd.

[0029] In this invention, Trichoderma directly destroys the pathogen's cell wall structure by secreting chitinase; Bacillus produces antimicrobial peptides that inhibit protein synthesis in the Huanglongbing pathogen; and Actinomycetes interfere with pathogen signal transduction through secondary metabolites. In a 3:2:1 ratio, the three bacteria form a synergistic antibacterial network, with the enzymatic hydrolysis of Trichoderma creating a pathway for the antimicrobial substances of the other two bacteria.

[0030] The Trichoderma genus in this invention specifically hydrolyzes the β-1,4-glycosidic bonds in the cell wall of the Huanglongbing pathogen by secreting cell wall-degrading enzymes such as β-1,3-glucanase and chitinase. Its mechanism of action includes: oxidative cleavage of pathogen exopolysaccharides, secretion of peptaibol antimicrobial peptides that disrupt the integrity of the pathogen's cell membrane; and competitive occupation of the citrus phloem niche. Bacillus sporogenes synthesize lipopeptide antibiotics via a non-ribosomal pathway, disrupting the cell membrane bilayer structure; inhibiting pathogen cyst formation; disrupting cell membrane potential equilibrium; and simultaneously secreting competitive iron ion capture to inhibit pathogen growth. Actinomycetes produce polyketide and aminoglycoside secondary metabolites that inhibit the DNA dependency of CLas; and disrupt peptidoglycan biosynthesis by blocking the initiation of protein synthesis by RNA polymerase.

[0031] According to a preferred embodiment of the present invention, the mass volume ratio concentration of the calcium chloride solution is 3%; the mass volume ratio concentration of the chitosan solution is 1%; and the particle size of the finished microspheres is 100-300 μm.

[0032] According to a preferred embodiment of the present invention, the preparation steps of the modified diatomaceous earth include: contacting natural diatomaceous earth with 2MHCl solution to obtain a mixture; centrifuging the mixture and washing it with deionized water to pH = 7.0; drying it at 105°C for 12 hours, crushing it to D50 = 5μm to obtain impurity-removed diatomaceous earth; mixing the impurity-removed diatomaceous earth with Na2CO3, and calcining it at 550°C in a muffle furnace for 2 hours; washing it with 0.5M HNO3 solution to remove residual alkali; obtaining pore-expanded diatomaceous earth; dissolving 3-aminopropyltriethoxysilane in an ethanol / water mixture at a volume ratio of 2%, adding the pore-expanded diatomaceous earth, wherein the solid-liquid ratio is 1:10; refluxing at 70°C for 4 hours, washing with ethanol 3 times, and vacuum drying at 80°C to obtain primary modified diatomaceous earth; soaking the primary modified diatomaceous earth with citrus phloem extract for 2 hours; and obtaining modified diatomaceous earth after vacuum drying at 50°C.

[0033] According to a preferred embodiment of the present invention, the natural diatomite is purchased from Shengzhou Huali Diatomite Products Co., Ltd.

[0034] According to a preferred embodiment of the present invention, the HCl solution is purchased from Guangdong Xinchengyuan Technology Co., Ltd.

[0035] According to a preferred embodiment of the present invention, the Na2CO3 is purchased from Wuhan Chujiang Haoyu Chemical Technology Development Co., Ltd.

[0036] According to a preferred embodiment of the present invention, the muffle furnace is purchased from Nanjing Kejie Testing Technology Development Co., Ltd.

[0037] According to a preferred embodiment of the present invention, the HNO3 solution is purchased from Guangdong Wengjiang Chemical Reagent Co., Ltd.

[0038] According to a preferred embodiment of the present invention, the 3-aminopropyltriethoxysilane is purchased from Shanghai Hansi Chemical Co., Ltd.

[0039] According to a preferred embodiment of the present invention, the ethanol is purchased from Shandong Xuchen Chemical Technology Co., Ltd.

[0040] According to a preferred embodiment of the present invention, the citrus phloem extract was purchased from Jining Fangyu Chemical Co., Ltd.

[0041] In the present invention, the diatomite treated with 2M HCl produces a mesoporous structure (10-20nm), which is then calcined at 550℃ and reacted with Na2CO3 to form through-holes. The specific surface area is increased from the original 20m 2 / g increased to 65-80m 2 / g. 3-Aminopropyltriethoxysilane (APTES) modification converts surface silanol (Si-OH) groups into amino groups (-NH2), reversing the zeta potential from -25mV to +12mV. This charge reversal property enables it to immobilize microorganisms through electrostatic adsorption (microbial cell surfaces are usually negatively charged, with an isoelectric point of pH 3-4) and hydrogen bonding. Flavonoids such as hesperidin and naringenin in citrus phloem extracts adsorb on the diatomaceous earth surface through π-π stacking, forming a biomolecular corona. This modification produces a triple effect: (1) enhancing affinity with citrus vascular cells (through plant pattern recognition receptors PRRs); (2) providing quorum sensing signal molecule analogs to induce microbial chemotaxis; and (3) slowly releasing active ingredients in an alkaline environment (pH 8.0-8.2).

[0042] The Ca in the 3% calcium chloride solution of the present invention is 2+ It forms an "egg-box" structure with the carboxyl group on the G unit of sodium alginate. 2+ Coordinated with 8 oxygen atoms. The protonation degree of chitosan is about 60% at pH = 6.0. 3+ With alginate-COO - The double crosslinking results in a microsphere swelling rate of less than 10% in simulated gastric fluid (pH = 1.2), but up to 300% in a phloem environment (pH = 8.0). The multi-level pore structure formed by freeze-drying produces a capillary effect. The release of microorganisms exhibits three phases: (1) rapid release (surface-adsorbed bacteria, approximately 25%) within 0-24 hours; (2) linear release (bacteria within the pores, approximately 50%) within 24-72 hours; and (3) sustained release (core-embedded bacteria, approximately 25%) after 72 hours.

[0043] In this method, modified diatomaceous earth serves as a primary carrier, forming a "bacteria reservoir" in the rhizosphere, which is transported upward by plant transpiration. Sodium alginate-chitosan microspheres serve as secondary carriers for targeted release in the phloem, forming a particle size gradient delivery system. The rigid structure of the diatomaceous earth provides mechanical protection for the microspheres, preventing soil compression. The pH-responsive nature of the microspheres compensates for the limited release of diatomaceous earth in alkaline environments. When the ambient pH exceeds 7.8, chitosan deprotonates, causing the PIC to dissociate, while the coordination bonds of calcium alginate reorganize, achieving intelligent release in pathogen-rich areas.

[0044] The modified diatomite in the present invention is modified by amino surface modification to enhance electrostatic adsorption with microbial cell membranes, and its 10-20nm pore size structure provides an ideal habitat for bacteria. Sodium alginate-chitosan microspheres are modified by Ca 2+Cross-linking forms an "egg-box" structure, which slowly dissolves and releases bacteria in an alkaline environment (pH = 8.0-8.2), while chitosan coating increases the adhesion and retention time of the microspheres in the phloem.

[0045] According to a preferred embodiment of the present invention, the contact conditions of natural diatomaceous earth and HCl solution are: stirring at 60°C for 2 hours; the solid-liquid ratio of natural diatomaceous earth and HCl solution is 1:5; the impurity-removed diatomaceous earth and Na2CO3 are mixed in a mass ratio of 1:0.3; the pore size of the expanded diatomaceous earth is 10-20 nm.

[0046] In this invention, hesperidin acts as a plant defense activator, enhancing citrus systemic resistance by upregulating PR protein gene expression; nano-zinc oxide (20-30nm) enhances its ability to penetrate pathogen cell membranes through surface effects; humic acid-chelated boron promotes vascular repair and improves nutrient transport; and sucrose fatty acid esters increase leaf permeability by reducing surface tension.

[0047] The present invention also provides a method for preparing the microbial fertilizer for treating citrus Huanglongbing disease, comprising the following steps:

[0048] S1, removing moisture from the modified diatomaceous earth and passing the mixture through a 200-mesh sieve, then mixing the modified diatomaceous earth, nano zinc oxide, and humic acid chelated boron to obtain a mixture;

[0049] S2, hesperidin was dissolved in 5% ethanol and then sprayed into the mixture, and then sodium alginate-chitosan microspheres were mixed with the mixture;

[0050] S3, finally adding sucrose fatty acid ester and diatomaceous earth to prepare microbial fertilizer.

[0051] According to a preferred embodiment of the present invention, in step S1, the modified diatomaceous earth is dried at 80° C. for 2 hours to remove moisture.

[0052] According to a preferred embodiment of the present invention, in step S2, after adding the sodium alginate-chitosan microspheres, the temperature is controlled to be ≤35°C.

[0053] According to a preferred embodiment of the present invention, diatomaceous earth is added to make the moisture content of the microbial fertilizer reach 5-8%.

[0054] According to a preferred embodiment of the present invention, the method for preparing the microbial fertilizer for treating citrus Huanglongbing disease further comprises: ultrasonically dispersing the nano zinc oxide with a 0.1% sodium citrate solution for 10 minutes before mixing.

[0055] The beneficial effects of the present invention are:

[0056] In terms of maintaining microbial activity, the synergistic effect of sodium alginate-chitosan microsphere encapsulation technology and a modified diatomaceous earth carrier system significantly improves the survival rate and colonization ability of functional microorganisms in the alkaline environment of citrus phloem. The composite microsphere structure not only provides a physical protective barrier for microorganisms, but its pH-responsive properties also enable targeted release of antibacterial ingredients in areas where pathogens are concentrated, ensuring that the antibacterial ingredients continue to exert their effects in key areas.

[0057] In terms of disease control, the specially formulated combination of Trichoderma, Bacillus, and Actinomycetes produces a synergistic antibacterial effect, effectively inhibiting the growth and reproduction of the Huanglongbing pathogen. Combined with plant immune activators like hesperidin, this enhances the systemic resistance of citrus trees while promoting the functional recovery of damaged vascular bundles, achieving a comprehensive control effect.

[0058] In terms of optimizing application performance, the addition of functional components such as nano-zinc oxide and humic acid-chelated boron improves the fertilizer's transport and distribution within citrus plants. The unique preparation process ensures excellent product stability, making it unaffected by fluctuations in ambient temperature and humidity during field application, significantly extending its effective duration. DETAILED DESCRIPTION

[0059] The following specific implementation methods are only used to further illustrate the present application and should not be understood as limiting the scope of protection of the present application. Technicians in this field may make some non-essential improvements and adjustments to the present application based on the above application content.

[0060] 1. Implementation

[0061] Example 1

[0062] 1. Preparation of Microbial Suspension

[0063] (1) Trichoderma culture: Take the Trichoderma strain from the -80℃ glycerol tube, streak it on a PDA plate, and culture it in the dark at 28℃ for 5 days. Spore suspension: Elute the spores with sterile water containing 0.05% Tween-80, and adjust the blood cell count to 1×10 7 Spores / mL; Fermentation culture: inoculate LB liquid medium (100 mL / 250 mL Erlenmeyer flask) at 5% inoculum, shake at 30°C and 180 rpm for 48 h; Detection: sample and measure OD600 every 12 h, terminate the culture when OD600 = 1.20 ± 0.05 (usually 45-50 h); Bacteria collection: centrifuge at 8000 rpm for 10 min (rotor radius 8 cm, RCF = 7168 × g), discard the supernatant and resuspend: wash twice with pre-cooled sterile saline (0.85% NaCl), and finally resuspend to 3.0 × 10 9 CFU / mL (verified by plate spreading method).

[0064] (2) Bacillus culture: culture Bacillus to OD600 = 1.20 according to the above conditions; heat treatment: kill the vegetative body (retain the spores) in a 70℃ water bath for 10 min; adjust the concentration to 2.0×10 9 spores / mL (microscope counting).

[0065] (3) Actinomycete culture: Gao's medium No. 1 (liquid volume 50 mL / 250 mL bottle), static culture at 30°C for 7 days, mycelium treatment: ultrasonic disruption (power 300 W, working 2 s / interval 3 s, total time 5 min); filtration: use 5 μm filter membrane to remove unbroken mycelium, adjust to 1.0×10 9 CFU / mL.

[0066] (4) Mixing: Mix aseptically: 150 mL of Trichoderma suspension, 100 mL of spore suspension, and 50 mL of actinomycete suspension by vortexing (3000 rpm, 2 min); final concentration: 1.08 × 10 8 CFU / mL (allowable error ±5%).

[0067] 2. Preparation of Sodium Alginate-Chitosan Microspheres

[0068] (1) Preparation of sodium alginate solution: Accurately weigh 10.00 g of sodium alginate, add 500 mL of deionized water, stir magnetically (500 rpm, 50°C water bath) for 2 h, sterilize under high pressure (121°C, 15 min), and then cool to 25±1°C.

[0069] (2) Bacterial liquid mixing: 300 mL of microbial suspension was mixed with 500 mL of sodium alginate solution and magnetically stirred (200 rpm, 10 min) to avoid bubble formation; viscosity was measured using a Brookfield DV2T viscometer, 350 ± 20 cP (spindle #3, 60 rpm) at 25 °C.

[0070] (3) Droplet formation into pellets: Apparatus: peristaltic pump (BT100-2J) connected to a 22G flat-tip needle (inner diameter 0.41 mm); parameters: flow rate 5.0 mL / min, needle distance 10 cm from the liquid surface, CaCl2 solution depth 15 cm; crosslinking: 3% CaCl2 solution (sterilized and cooled to 4°C in advance), magnetic stirring (100 rpm); curing time: 30.0 ± 0.5 min (timer control).

[0071] (4) Chitosan coating: Chitosan solution: 1.00 g chitosan (85% deacetylation) was dissolved in 100 mL 1% acetic acid, and the pH was adjusted to 6.00 ± 0.05 with NaOH. Coating conditions: microsphere to solution volume ratio 1:5, constant temperature shaker (25°C, 100 rpm) for 10 min. Washing: Rinse three times with sterile deionized water (100 mL each time).

[0072] (5) Freeze drying: Pre-freezing: Freeze in a -80°C refrigerator for 4 hours (cooling rate 1°C / min); Freeze drying: cold trap temperature -55°C, vacuum degree 10Pa, 24 hours; Finished product inspection: Particle size distribution (laser particle size analyzer): 150-250μm accounts for ≥90%.

[0073] 3. Preparation of Modified Diatomaceous Earth

[0074] (1) Pickling and pore expansion: Raw materials: natural diatomaceous earth (SiO2 ≥ 85%, D50 = 15 μm) 100.0 g; acid treatment: 2 M HCl solution 500 mL (solid-liquid ratio 1:5), 60.0 ± 0.5 ° C water bath; stirring: mechanical stirring (paddle type, diameter 5 cm, 300 rpm) 120 min; centrifugation: 8000 rpm, 10 min (supernatant pH detected to 7.00 ± 0.05).

[0075] (2) Calcination modification:

[0076] Mixing: Grind and mix the impurity-removed diatomaceous earth and Na2CO3 at a ratio of 1:0.3 (i.e. 30.0g); Calcination: Program the temperature in a muffle furnace (5℃ / min→550℃, hold for 120min→natural cooling); Acid washing: Wash with 0.5M HNO3 solution until the conductivity is <10μS / cm.

[0077] (3) Amination: APTES solution: 2.00 mL APTES, 98 mL ethanol / water (95:5); Reaction: 70.0±0.5°C oil bath reflux, condenser circulating water temperature 20°C; Drying: Vacuum drying oven (80°C, 10 kPa) to constant weight.

[0078] (4) Biomodification: citrus phloem extract: fresh phloem: water = 1:10, extraction at 80°C for 2 h, filtration and concentration to 50°Brix; soaking: solid-liquid ratio 1:5 (W / V), shaking at 25°C (50 rpm) for 120 min; final drying: vacuum drying at 50°C to a moisture content of ≤3% (determined by Karl Fischer method).

[0079] 4. Preparation of microbial fertilizer

[0080] (1) Pretreatment: 350 g modified diatomaceous earth: dried in a hot air circulation oven at 80.0 ± 0.5 ° C for 120 min; sieved: 200 mesh stainless steel sieve (pore size 75 μm), sieve residue ≤ 1%; nano zinc oxide: 0.100 g sodium citrate and 100 mL water, ultrasonic treatment (40 kHz, 300 W) for 10.0 min.

[0081] (2) Dry mixing: Equipment: three-dimensional motion mixer (volume 50 L); order of addition: modified diatomaceous earth 350 g, nano zinc oxide 40 g and humic acid chelated boron 30 g; parameters: speed 15 rpm, mixing time 30.0 min, temperature ≤ 30 °C.

[0082] (3) Wet mixing: dissolution of hesperidin: 70.00 g hesperidin, 140 mL 5% ethanol (V / V), ultrasonic-assisted dissolution at 40 °C; spraying: using a peristaltic pump (flow rate 10 mL / min) and a two-fluid nozzle (compressed air pressure 0.2 MPa); adding 450 g sodium alginate-chitosan microspheres: temperature-controlled mixing (jacket water temperature 35.0 ± 0.5 °C), mixing time 20 min.

[0083] (4) Final adjustment: 30.0 g of sucrose fatty acid ester was pre-dissolved in 50 mL of 60°C water and added while hot; Moisture adjustment: diatomaceous earth was added (5 g increments each time) until the moisture meter displayed 6.0 ± 0.5%; Sieving: All the samples were passed through a 60-mesh sieve (pore size 250 μm).

[0084] (5) Packaging: Heat-sealed aluminum foil bag (thickness 0.1 mm); nitrogen-filled for protection (residual oxygen content <3%); Storage: Store at 4°C away from light.

[0085] Example 2

[0086] The specific implementation method is the same as that of Example 1, except that the raw material formula is as follows: modified diatomaceous earth: 300 g; sodium alginate-chitosan microspheres: 330 g; hesperidin: 50 g; nano zinc oxide: 30 g; and the microsphere particle size is 100-150 μm.

[0087] Example 3

[0088] The specific implementation method is the same as Example 1, except that the raw material formula is: modified diatomaceous earth: 400g; sodium alginate-chitosan microspheres: 500g; hesperidin: 80g; difference: plasma-activated diatomaceous earth (non-chemically modified) is used; 5% citrus essential oil is added to the microspheres.

[0089] Comparative Example 1

[0090] The specific implementation method is the same as that of Example 1, except that ordinary diatomaceous earth: 350 g (not acidified and pore-expanded).

[0091] Comparative Example 2

[0092] The specific implementation method is the same as that of Example 1, except that only sodium alginate microspheres (without chitosan coating) are used.

[0093] Comparative Example 3

[0094] The specific implementation method is the same as that of Example 1, except that only Trichoderma is contained (without Bacillus and Actinomycetes).

[0095] 3. Performance Testing

[0096] 1. Pathogen inhibition rate test

[0097] 1) Inoculation treatment: Citrus seedlings infected with the Huanglongbing pathogen were selected, with 10 plants per group. 5g of microbial fertilizer was applied to the roots, while the control group was treated with only water. Pathogen load detection: Leaf sampling: The 3rd to 5th leaves from the top of each plant were taken, quickly frozen in liquid nitrogen, and stored at -80°C. DNA extraction: Phloem tissue DNA was extracted using the CTAB method, and primers were designed to target the 16S rRNA gene of the pathogen (forward: 5'-GCGCTTATTTTTAATAGGAGC-3'; reverse: 5'-AGCCTCACCGTTACAGCG-3'). qPCR quantification: SYBR Green method, cycling conditions: 95°C pre-denaturation for 5min, 40 cycles (95°C for 30s, 58°C for 30s, 72°C for 30s).

[0098] 2) Calculation of disease index

[0099] Leaf yellowing grading standards: Level 0: no yellowing; Level 1: yellowing area ≤ 25%; Level 2: 25% < yellowing ≤ 50%; Level 3: 50% < yellowing ≤ 75%; Level 4: yellowing > 75%;

[0100] The calculation formula is: disease index = [(1×number of leaves with level 1 disease)+(2×number of leaves with level 2 disease)+(3×number of leaves with level 3 disease)+(4×number of leaves with level 4 disease)]÷[total number of leaves surveyed×highest disease level].

[0101] 2. Phloem colonization ability test

[0102] Fluorescent labeling of strains: The GFP plasmid was introduced into microbial strains (Trichoderma, Bacillus, Actinomycetes) by electroporation, and the stable fluorescent expression strains were screened. Microscopic observation and quantification: Phloem sections: Citrus stem sections were taken 7 / 14 / 30 days after fertilization, and 30 μm frozen sections were made. Fluorescence microscopy was used for detection: the excitation wavelength was 488 nm, the emission wavelength was 510 nm, and the number of fluorescent spots per square centimeter (CFU / cm 2 ).

[0103] 3. Survival time test

[0104] Soil sampling and treatment: Time point: 0 / 15 / 30 / 60 days, randomly sample rhizosphere soil (depth 0-20cm) at each stage, mix and pass through a 2mm sieve. Gradient dilution: take 1g of soil and add 9mL sterile saline, vortex and shake, then serially dilute to 10 -6 .

[0105] Plate count method: Bacterial culture: Spread the diluted solution onto LB agar plates (PDA medium for Trichoderma) and incubate at 30°C for 48 hours. Spore count: Inactivate vegetative cells by treating in a 70°C water bath for 10 minutes before spreading.

[0106] 4. Sustained release performance test

[0107] Simulated Release System: Release medium: pH 5.8 phosphate buffer (containing 0.1% Tween-80) to simulate the citrus phloem environment. Control conditions: Constant temperature and shaking at 37°C (100 rpm), protected from light. HPLC detection: Hesperidin extraction: Samples were taken at regular intervals (0 / 2 / 6 / 12 / 24 / 48 h) and filtered through a 0.22 μm filter. Chromatographic conditions: Column: C18 (4.6 × 250 mm, 5 μm); Mobile phase: acetonitrile-0.1% phosphoric acid (25:75); Detection wavelength: 283 nm, Flow rate: 1.0 mL / min.

[0108] (2) Test results:

[0109] Table 1: Test results of various embodiments and comparative examples

[0110]

[0111] As can be seen from Table 1, the present invention successfully solves the key problem in the prevention and control of citrus Huanglongbing through innovative microbial microsphere encapsulation technology and modified diatomaceous earth targeted delivery system. In terms of prevention and control effect, the pathogen inhibition rate of the example group reached 82.3%-85.0%, significantly higher than the 38.9%-60.7% of the control group. This is due to the synergistic antibacterial effect of hesperidin and nano-zinc oxide and the targeted delivery ability of the diatomaceous earth carrier. In order to solve the problem of strain colonization difficulty, the test data showed that the colonization density of the example group in the citrus phloem reached 7.2-10.5×10 4 CFU / cm 2, which is 2-3 times that of the control example, verifying the effectiveness of citrus phloem modification on the surface of diatomaceous earth. In terms of survival time, the number of live bacteria in the embodiment group was maintained for more than 50 days, which was significantly better than the 45 days of the control example, which was attributed to the sustained-release protection effect of the sodium alginate-chitosan microsphere structure. At the same time, the 28-30 day sustained-release cycle perfectly matches the incubation period of Huanglongbing, and the 24-hour release rate is controlled within 30%, which effectively avoids the problem of sudden drug release and achieves precise targeted therapeutic effects. These data fully demonstrate that the technical solution of the present invention systematically solves the core problems of the prior art, such as unsatisfactory prevention and control effects, difficult colonization, short survival time, and lack of targeted sustained-release function.

[0112] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. A microbial fertilizer for treating citrus Huanglongbing, characterized in that: In terms of mass percentage, the raw materials include: Modified diatomaceous earth: 30-40%; Sodium alginate-chitosan microspheres: 33-50%; Hesperidin: 5-8%; Nano zinc oxide: 3-5%; Humic acid chelated boron: 2-4%; Sucrose fatty acid esters: 2-4%; Diatomaceous earth: 2-4%; The sodium alginate-chitosan microsphere preparation steps include: mixing sodium alginate with a microbial suspension, dripping it into a calcium chloride solution with a syringe, and letting it stand to solidify to form a microsphere core; immersing the solidified microspheres in a chitosan solution with a pH of 6.0, taking them out and freeze-drying them to obtain finished microspheres; wherein the microbial suspension includes Trichoderma, Bacillus and Actinomycetes.

2. The microbial fertilizer for treating citrus Huanglongbing according to claim 1, wherein The preparation steps of the microbial suspension include: culturing Trichoderma, Bacillus and Actinomycetes in LB liquid medium at 30°C and 180rpm to OD600=1.2; collecting the cells by centrifugation and resuspending them in sterile saline; mixing Trichoderma: Bacillus: Actinomycetes in a ratio of 3:2:1; adjusting the final concentration to 10 8 CFU / mL.

3. The microbial fertilizer for treating citrus Huanglongbing according to claim 1, wherein The mass volume ratio concentration of the calcium chloride solution is 3%; the mass volume ratio concentration of the chitosan solution is 1%; and the particle size of the finished microspheres is 100-300 μm.

4. The microbial fertilizer for treating citrus Huanglongbing according to claim 1, wherein The preparation steps of the modified diatomite include: contacting natural diatomite with a 2M HCl solution to obtain a mixture; centrifuging the mixture and washing it with deionized water to a pH of 7.0; drying it at 105°C for 12 hours, crushing it to D50=5μm, and obtaining impurity-removed diatomite; mixing the impurity-removed diatomite with Na2CO3, and calcining it at 550°C in a muffle furnace for 2 hours; washing it with a 0.5M HNO3 solution to remove residual alkali; obtaining pore-enlarged diatomite; dissolving 3-aminopropyltriethoxysilane in an ethanol / water mixture at a volume ratio of 2%, adding the pore-enlarged diatomite, wherein the solid-liquid ratio is 1:10; refluxing at 70°C for 4 hours, washing it with ethanol three times, and vacuum drying it at 80°C to obtain primary modified diatomite; soaking the primary modified diatomite in a citrus phloem extract for 2 hours; and vacuum drying it at 50°C to obtain the modified diatomite.

5. The microbial fertilizer for treating citrus Huanglongbing according to claim 4, characterized in that, The contact conditions of natural diatomaceous earth and HCl solution are: stirring at 60°C for 2 hours; the solid-liquid ratio of natural diatomaceous earth and HCl solution is 1:5; the impurity-removed diatomaceous earth and Na2CO3 are mixed in a mass ratio of 1:0.3; the pore diameter of the expanded diatomaceous earth is 10-20 nm.

6. The preparation method of the microbial fertilizer for treating citrus Huanglongbing according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1, removing moisture from the modified diatomaceous earth and passing the mixture through a 200-mesh sieve, then mixing the modified diatomaceous earth, nano zinc oxide, and humic acid chelated boron to obtain a mixture; S2, hesperidin was dissolved in 5% ethanol and then sprayed into the mixture, and then sodium alginate-chitosan microspheres were mixed with the mixture; S3, finally adding sucrose fatty acid ester and diatomaceous earth to prepare microbial fertilizer.

7. The preparation method according to claim 6, characterized in that In step S1, the modified diatomaceous earth is dried at 80° C. for 2 hours to remove moisture.

8. The preparation method according to claim 6, characterized in that In step S2, after adding sodium alginate-chitosan microspheres, the temperature is controlled to be ≤35°C.

9. The preparation method according to claim 6, characterized in that In step S3, diatomaceous earth is added to make the water content of the microbial fertilizer reach 5-8%.

10. The preparation method according to claim 6, characterized in that Also includes: Nano-zinc oxide was ultrasonically dispersed in 0.1% sodium citrate solution for 10 min before mixing.

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