Composite microbial bactericide and preparation method thereof

Abstract

The invention discloses a compound microorganism bactericide and a preparation method thereof, and relates to the technical field of agricultural microorganism application. Comprising the following components: 10 to 15 parts of a core strain, 3 to 5 parts of humic acid, 4 to 6 parts of plant polysaccharide, 8 to 12 parts of a protective agent, 2 to 4 parts of a nutritional ingredient, 1 to 3 parts of adenosine, 2 to 4 parts of nano-scale hydroxyapatite, 1 to 2 parts of graphene quantum dots, 0.8 to 1.5 parts of glutamine and 1 to 3 parts of carboxymethyl-beta-cyclodextrin. The core strains comprise paecilomyces lilacinus, bacillus amyloliquefaciens, trichoderma harzianum, trichoderma viride, bacillus subtilis, pseudomonas fluorescens, bacillus licheniformis, brevibacillus laterosporus and candida utilis. The bactericide is remarkable in sterilizing effect, sufficient in stability and good in field control effect.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial application technology, and in particular to a compound microbial bactericide and its preparation method. Background Technology

[0002] In agricultural production, the long-term use of traditional chemical fungicides has led to a series of problems. Chemical fungicides not only easily cause pathogens to develop resistance, reducing their effectiveness, but also leave residues in agricultural products, harming human health and causing serious pollution to the soil and water ecosystems. It is under these circumstances that microbial fungicides have emerged. How to develop and correctly use microbial fungicides is a primary issue in the development of organic and green agriculture.

[0003] Traditional microbial fungicides mostly use single strains, resulting in unstable control effects, weak colonization ability, single target, and narrow antibacterial spectrum, making them unable to cope with complex and diverse pathogen invasions. Most existing compound microbial fungicides simply mix strains without fully exploring the potential for synergistic effects between strains, leading to species incompatibility, short-lasting effects, poor environmental tolerance during storage and use, and the fungicidal efficacy still needs further improvement.

[0004] To address the aforementioned problems, Chinese invention patent CN102964178B discloses a compound microbial fungicide, its preparation method, and its application. The compound microbial fungicide, by weight, comprises 10-20 parts of a compound microbial agent, 10-20 parts of nutrients, 4-6 parts of enzyme-active substances, and 54-78 parts of a fungicide carrier. The compound microbial agent contains biocontrol-promoting bacteria and protein-decomposing bacteria; the biocontrol-promoting bacteria are *Trichoderma harzianum*, *Trichoderma viride*, and *Bacillus licheniformis*; the protein-decomposing bacteria are *Bacillus subtilis*. This invention also provides a method for preparing the above-mentioned compound microbial fungicide. The compound microbial fungicide provided by this invention can rapidly colonize and grow around the plant rhizosphere, forming a "protective shield" to prevent infection by root pathogenic fungi. During interaction and hyperparasitism with pathogens, it produces antibiotics and plant growth-promoting substances, significantly preventing, antagonizing, or directly killing various crop diseases. Simultaneously, it promotes root and shoot growth, fostering healthy crop growth, thereby increasing yield and improving quality. However, its bactericidal effect and bactericidal stability still need further improvement.

[0005] It is evident that there is still a need in this field to develop a compound microbial fungicide with significant bactericidal effect, sufficient stability, and good field control efficacy, as well as its preparation method. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a compound microbial fungicide with significant bactericidal effect, good stability, and good field control efficacy, as well as its preparation method.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is: a compound microbial bactericide, comprising the following components by weight: 10-15 parts of core bacterial strain, 3-5 parts of humic acid, 4-6 parts of plant polysaccharides, 8-12 parts of protective agent, 2-4 parts of nutrients, 1-3 parts of adenosine, 2-4 parts of nano-grade hydroxyapatite, 1-2 parts of graphene quantum dots, 0.8-1.5 parts of glutamine, and 1-3 parts of carboxymethyl-β-cyclodextrin; wherein the core bacterial strain includes Paecilomyces lilacinus, Bacillus amyloliquefaciens, Trichoderma harzianum, Trichoderma viride, Bacillus subtilis, Pseudomonas fluorescens, Bacillus licheniformis, Bacillus laterosporus, and Candida utilis.

[0008] Preferably, the viable cell ratio of Paecilomyces lilacinus, Bacillus amyloliquefaciens, Trichoderma harzianum, Trichoderma viride, Bacillus subtilis, Pseudomonas fluorescens, Bacillus licheniformis, Bacillus brevis, and Candida utilis is (8-12):(15-23):(10-18):(10-18):(18-28):(8-12):(15-23):(8-15):(5-8).

[0009] Preferably, the plant polysaccharide is at least one of ginkgo leaf polysaccharide, tea polysaccharide, and seaweed polysaccharide.

[0010] Preferably, the protective agent includes sodium alginate and chitosan.

[0011] Preferably, the nutrient is a mixture of ammonium nitrate and potassium dihydrogen phosphate in a mass ratio of 1:(0.8-1.2).

[0012] Preferably, the average particle size of the nano-sized hydroxyapatite is 10-70 nm.

[0013] Preferably, the graphene quantum dots have 1-5 layers, a thickness of 1-2 nm, and a lateral dimension of 5-15 nm.

[0014] Preferably, the degree of substitution of the carboxymethyl-β-cyclodextrin is 7, and the molecular weight is 1540 Da.

[0015] Another object of the present invention is to provide a method for preparing the aforementioned composite microbial bactericide, comprising the following steps: Step S1, Strain Selection and Modification: Highly active original strains were screened from different ecological environments. Bacillus subtilis was genetically engineered to overexpress genes related to antimicrobial peptide synthesis. Candida utilis was mutagenized to screen mutant strains with polysaccharide secretion increased by more than 30%. Step S2, Strain Culture: Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus brevis, and Pseudomonas fluorescens were inoculated into LB medium and cultured at 32℃ and 180 rpm for 22 h; Trichoderma harzianum and Trichoderma viride were inoculated into PDA medium and cultured at 28℃ and 180 rpm for 6 days; Paecilomyces lilacinus was inoculated into PDB medium and cultured with shaking at 28℃ and 180 rpm for 72 h; Candida utilis was inoculated into YM medium and cultured at 26℃ and 150 rpm for 48 h. Step S3, Cell Preparation and Processing: Centrifuge the cultured Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus laterosporus brevis, and Pseudomonas fluorescens fermentation broths at 4℃ and 6000-8000 r / min for 10-15 min, and collect the cell precipitate; rinse the colony surfaces of the cultured Trichoderma harzianum, Trichoderma viride, and Paecilomyces lilacinus with sterile water, then scrape the mycelium with an inoculation loop, grind it in a sterile mortar, and add an appropriate amount of sterile water to prepare a mycelial suspension; concentrate the fermentation broth of Candida utilis by membrane filtration to achieve a cell concentration of 10. 9 CFU / mL; Step S4, Microencapsulation: Sodium alginate and chitosan are prepared into 2% and 1% aqueous solutions, respectively, and mixed at a volume ratio of 2:1. Other components are added, and a mixture is formed under stirring at 30℃ and 120r / min. The mixture is sprayed into a 0.2mol / L calcium chloride solution through a high-voltage electrostatic spraying device and solidified for 30min to form a microencapsulated composite microbial bactericide primary product. Step S5, Drying and Post-processing: Vacuum freeze drying is performed to obtain a powdered finished product, which is then packaged with nitrogen for protection.

[0016] Preferably, the voltage of the high-voltage electrostatic spray device in step S4 is 15kV and the nozzle orifice diameter is 0.8mm.

[0017] Preferably, the pre-freezing temperature of the vacuum freeze-drying is -40℃, the vacuum degree is 10-30Pa, and the drying time is 22-25h.

[0018] Another object of the present invention is to provide an application of the aforementioned compound microbial bactericide in crop production.

[0019] Due to the application of the above technical solution, the present invention has the following beneficial effects: (1) The preparation method of the compound microbial bactericide disclosed in this invention is simple and easy to implement, convenient to operate and control, and has high efficiency and high finished product qualification rate. It is suitable for continuous large-scale production and has high promotion and application value.

[0020] (2) The compound microbial fungicide disclosed in this invention comprises, by weight, the following components: 10-15 parts of core microbial strain, 3-5 parts of humic acid, 4-6 parts of plant polysaccharides, 8-12 parts of protectant, 2-4 parts of nutrients, 1-3 parts of adenosine, 2-4 parts of nano-grade hydroxyapatite, 1-2 parts of graphene quantum dots, 0.8-1.5 parts of glutamine, and 1-3 parts of carboxymethyl-β-cyclodextrin; the core microbial strain includes Paecilomyces lilacinus, Bacillus amyloliquefaciens, Trichoderma harzianum, Trichoderma viride, Bacillus subtilis, Pseudomonas fluorescens, Bacillus licheniformis, Bacillus brevis, and Candida utilis. Through the synergistic effect of the various components, the prepared compound microbial fungicide exhibits significant fungicidal effect, sufficient stability, and good field control efficacy. The core microbial strains include nine species with different characteristics, such as *Paecilomyces lilacinus*, *Bacillus amyloliquefaciens*, and *Trichoderma harzianum*, offering a richer variety and quantity of strains compared to common compound microbial fungicides. *Paecilomyces lilacinus* has parasitic and inhibitory effects on harmful organisms such as root-knot nematodes; *Bacillus amyloliquefaciens* produces various antibacterial proteins and enzymes; and *Trichoderma harzianum* and *Trichoderma viride* can parasitize and inhibit various fungi. The combination of multiple strains not only effectively controls bacterial and fungal diseases but also manages harmful organisms such as nematodes, significantly broadening the antibacterial spectrum and far exceeding traditional single-strain or limited-strain fungicides. Different strains have different functions in the soil ecosystem. *Bacillus subtilis* and *Pseudomonas fluorescens* can improve the soil microecological environment and promote the growth of beneficial microorganisms; *Bacillus licheniformis* and *Bacillus laterosporus* can decompose insoluble substances in the soil, improving soil fertility. Candida utilis can produce a variety of nutrients. These microorganisms work together to achieve comprehensive regulation of the soil ecology while killing bacteria, creating a favorable environment for crop growth. This is something that traditional single-function fungicides cannot achieve.

[0021] (3) The compound microbial bactericide disclosed in this invention incorporates nano-sized hydroxyapatite and graphene quantum dots in its formulation. Nano-sized hydroxyapatite has good biocompatibility and adsorption properties, enabling it to adsorb pathogens while slowly releasing nutrients such as calcium ions, thus enhancing crop disease resistance. Graphene quantum dots possess excellent photocatalytic activity and antibacterial properties, generating free radicals to kill pathogens under light conditions. The application of these two nanomaterials endows the microbial bactericide with new bactericidal mechanisms and functions, which are not found in traditional formulations. The combination of adenosine, glutamine, and carboxymethyl-β-cyclodextrin has unique effects. Adenosine participates in cellular energy metabolism, enhancing the activity and reproductive capacity of microorganisms. Glutamine is an important nitrogen source for microbial growth and can enhance the stress resistance of microorganisms. Carboxymethyl-β-cyclodextrin can be used as an encapsulation material to protect microorganisms and other active ingredients, improving their stability and slow-release properties. The combined use of these bioactive substances enhances the performance of the bactericide in multiple ways, unlike traditional formulations that only add simple nutrients.

[0022] (4) The compound microbial bactericide disclosed in this invention, through the synergistic effect of humic acid, plant polysaccharides, and protectants, not only provides microorganisms with carbon sources and other nutrients, but also forms a viscous and adsorbent complex that encapsulates microorganisms and other components, providing protection while achieving slow release and prolonging the duration of the bactericidal effect. Moreover, humic acid can improve soil structure and enhance soil water and fertilizer retention capacity, mutually promoting the function of the bactericide and achieving multiple effects. This multifunctional combination is not available in traditional formulations.

[0023] (5) The composite microbial bactericide disclosed in this invention combines sodium alginate-chitosan composite protectant with microencapsulation technology to form a dual protective barrier. It can effectively improve the survival rate of microorganisms and extend their shelf life; thereby improving the shelf life and stability of the product. Detailed Implementation

[0024] The following description is intended to disclose the invention and enable those skilled in the art to implement it. The preferred embodiments described below are merely examples, and other obvious variations will occur to those skilled in the art.

[0025] Example 1 A compound microbial bactericide, by weight, comprises the following components: 10 parts core bacterial strain, 3 parts humic acid, 4 parts plant polysaccharides, 8 parts protective agent, 2 parts nutrients, 1 part adenosine, 2 parts nano-grade hydroxyapatite, 1 part graphene quantum dots, 0.8 parts glutamine, and 1 part carboxymethyl-β-cyclodextrin; wherein the core bacterial strain includes Paecilomyces lilacinus, Bacillus amyloliquefaciens, Trichoderma harzianum, Trichoderma viride, Bacillus subtilis, Pseudomonas fluorescens, Bacillus licheniformis, Bacillus laterosporus, and Candida utilis.

[0026] The viable cell ratio of Paecilomyces lilacinus, Bacillus amyloliquefaciens, Trichoderma harzianum, Trichoderma viride, Bacillus subtilis, Pseudomonas fluorescens, Bacillus licheniformis, Bacillus brevis, and Candida utilis is 8:15:10:10:18:8:15:8:5.

[0027] The plant polysaccharide is ginkgo leaf polysaccharide; the protective agent includes sodium alginate and chitosan; the nutrient is ammonium nitrate and potassium dihydrogen phosphate mixed in a mass ratio of 1:0.8; the average particle size of the nano-sized hydroxyapatite is 10 nm; the number of layers of the graphene quantum dots is 1-5, the thickness is 1-2 nm, and the lateral dimension is 5-15 nm; the degree of substitution of the carboxymethyl-β-cyclodextrin is 7, and the molecular weight is 1540 Da.

[0028] A method for preparing the aforementioned composite microbial bactericide includes the following steps: Step S1, Strain Selection and Modification: Highly active original strains were screened from different ecological environments. Bacillus subtilis was genetically engineered to overexpress genes related to antimicrobial peptide synthesis. Candida utilis was mutagenized to screen mutant strains with polysaccharide secretion increased by more than 30%. Step S2, Strain Culture: Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus brevis, and Pseudomonas fluorescens were inoculated into LB medium and cultured at 32℃ and 180 rpm for 22 h; Trichoderma harzianum and Trichoderma viride were inoculated into PDA medium and cultured at 28℃ and 180 rpm for 6 days; Paecilomyces lilacinus was inoculated into PDB medium and cultured with shaking at 28℃ and 180 rpm for 72 h; Candida utilis was inoculated into YM medium and cultured at 26℃ and 150 rpm for 48 h. Step S3, Cell Preparation and Processing: Centrifuge the cultured Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus laterosporus brevis, and Pseudomonas fluorescens fermentation broths at 4℃ and 6000 r / min for 10 min, and collect the cell precipitate; rinse the colony surfaces of the cultured Trichoderma harzianum, Trichoderma viride, and Paecilomyces lilacinus with sterile water, then scrape the mycelium with an inoculation loop, grind it in a sterile mortar, and add an appropriate amount of sterile water to prepare a mycelial suspension; concentrate the fermentation broth of Candida utilis by membrane filtration to achieve a cell concentration of 10. 9 CFU / mL; Step S4, Microencapsulation: Sodium alginate and chitosan are prepared into 2% and 1% aqueous solutions, respectively, and mixed at a volume ratio of 2:1. Other components are added, and a mixture is formed under stirring at 30℃ and 120r / min. The mixture is sprayed into a 0.2mol / L calcium chloride solution through a high-voltage electrostatic spraying device and solidified for 30min to form a microencapsulated composite microbial bactericide primary product. Step S5, Drying and Post-processing: Vacuum freeze drying is performed to obtain a powdered finished product, which is then packaged with nitrogen for protection.

[0029] In step S4, the voltage of the high-voltage electrostatic spray device is 15kV and the nozzle diameter is 0.8mm; the pre-freezing temperature of the vacuum freeze-drying is -40℃, the vacuum degree is 10Pa, and the drying time is 22h.

[0030] Example 2 A compound microbial bactericide, by weight, comprises the following components: 11 parts core microbial strain, 3.5 parts humic acid, 4.5 parts plant polysaccharides, 9 parts protectant, 2.5 parts nutrients, 1.5 parts adenosine, 2.5 parts nano-hydroxyapatite, 1.2 parts graphene quantum dots, 1 part glutamine, and 1.5 parts carboxymethyl-β-cyclodextrin; wherein the core microbial strain includes Paecilomyces lilacinus, Bacillus amyloliquefaciens, Trichoderma harzianum, Trichoderma viride, Bacillus subtilis, Pseudomonas fluorescens, Bacillus licheniformis, Bacillus laterosporus brevis, and Candida utilis.

[0031] The viable cell ratio of *Paecilomyces lilacinus*, *Bacillus amyloliquefaciens*, *Trichoderma harzianum*, *Trichoderma viride*, *Bacillus subtilis*, *Pseudomonas fluorescens*, *Bacillus licheniformis*, *Bacillus laterosporus*, and *Candida utilis* is 9:17:12:13:20:9:17:10:6; the plant polysaccharide is tea polysaccharide; the protective agent includes sodium alginate and chitosan; the nutrient component is ammonium nitrate and potassium dihydrogen phosphate mixed in a mass ratio of 1:0.9; the average particle size of the nano-hydroxyapatite is 30 nm; the number of layers of the graphene quantum dots is 1-5, the thickness is 1-2 nm, and the lateral dimension is 5-15 nm; the degree of substitution of the carboxymethyl-β-cyclodextrin is 7, and the molecular weight is 1540 Da.

[0032] A method for preparing the aforementioned composite microbial bactericide includes the following steps: Step S1, Strain Selection and Modification: Highly active original strains were screened from different ecological environments. Bacillus subtilis was genetically engineered to overexpress genes related to antimicrobial peptide synthesis. Candida utilis was mutagenized to screen mutant strains with polysaccharide secretion increased by more than 30%. Step S2, Strain Culture: Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus brevis, and Pseudomonas fluorescens were inoculated into LB medium and cultured at 32℃ and 180 rpm for 22 h; Trichoderma harzianum and Trichoderma viride were inoculated into PDA medium and cultured at 28℃ and 180 rpm for 6 days; Paecilomyces lilacinus was inoculated into PDB medium and cultured with shaking at 28℃ and 180 rpm for 72 h; Candida utilis was inoculated into YM medium and cultured at 26℃ and 150 rpm for 48 h. Step S3, Cell Preparation and Processing: The fermentation broths of Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus laterosporus brevis, and Pseudomonas fluorescens were centrifuged at 4℃ and 6500 r / min for 12 min, and the cell pellet was collected. The colony surfaces of the cultured Trichoderma harzianum, Trichoderma viride, and Paecilomyces lilacinus were rinsed with sterile water, and then mycelia were scraped off with an inoculation loop, ground in a sterile mortar, and an appropriate amount of sterile water was added to prepare a mycelial suspension. The fermentation broth of Candida utilis was concentrated by membrane filtration to achieve a cell concentration of 10. 9 CFU / mL; Step S4, Microencapsulation: Sodium alginate and chitosan are prepared into 2% and 1% aqueous solutions, respectively, and mixed at a volume ratio of 2:1. Other components are added, and a mixture is formed under stirring at 30℃ and 120r / min. The mixture is sprayed into a 0.2mol / L calcium chloride solution through a high-voltage electrostatic spraying device and solidified for 30min to form a microencapsulated composite microbial bactericide primary product. Step S5, Drying and Post-processing: Vacuum freeze drying is performed to obtain a powdered finished product, which is then packaged with nitrogen for protection.

[0033] In step S4, the voltage of the high-voltage electrostatic spray device is 15kV and the nozzle diameter is 0.8mm; the pre-freezing temperature of the vacuum freeze-drying is -40℃, the vacuum degree is 15Pa, and the drying time is 23h.

[0034] Example 3 A compound microbial bactericide, by weight, comprises the following components: 13 parts core bacterial strain, 4 parts humic acid, 5 parts plant polysaccharides, 10 parts protectant, 3 parts nutrients, 2 parts adenosine, 3 parts nano-grade hydroxyapatite, 1.5 parts graphene quantum dots, 1.2 parts glutamine, and 2 parts carboxymethyl-β-cyclodextrin; wherein the core bacterial strain includes Paecilomyces lilacinus, Bacillus amyloliquefaciens, Trichoderma harzianum, Trichoderma viride, Bacillus subtilis, Pseudomonas fluorescens, Bacillus licheniformis, Bacillus laterosporus brevis, and Candida utilis.

[0035] The viable cell ratio of Paecilomyces lilacinus, Bacillus amyloliquefaciens, Trichoderma harzianum, Trichoderma viride, Bacillus subtilis, Pseudomonas fluorescens, Bacillus licheniformis, Bacillus brevis, and Candida utilis is 10:21:14:14:23:10:19:12:6.5.

[0036] The plant polysaccharide is a seaweed polysaccharide; the protective agent includes sodium alginate and chitosan; the nutrient is a mixture of ammonium nitrate and potassium dihydrogen phosphate in a mass ratio of 1:1; the average particle size of the nano-sized hydroxyapatite is 50 nm; the number of layers of the graphene quantum dots is 1-5, the thickness is 1-2 nm, and the lateral dimension is 5-15 nm; the degree of substitution of the carboxymethyl-β-cyclodextrin is 7, and the molecular weight is 1540 Da.

[0037] A method for preparing the aforementioned composite microbial bactericide includes the following steps: Step S1, Strain Selection and Modification: Highly active original strains were screened from different ecological environments. Bacillus subtilis was genetically engineered to overexpress genes related to antimicrobial peptide synthesis. Candida utilis was mutagenized to screen mutant strains with polysaccharide secretion increased by more than 30%. Step S2, Strain Culture: Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus brevis, and Pseudomonas fluorescens were inoculated into LB medium and cultured at 32℃ and 180 rpm for 22 h; Trichoderma harzianum and Trichoderma viride were inoculated into PDA medium and cultured at 28℃ and 180 rpm for 6 days; Paecilomyces lilacinus was inoculated into PDB medium and cultured with shaking at 28℃ and 180 rpm for 72 h; Candida utilis was inoculated into YM medium and cultured at 26℃ and 150 rpm for 48 h. Step S3, Cell Preparation and Processing: The cultured Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus laterosporus brevis, and Pseudomonas fluorescens fermentation broths were centrifuged at 4℃ and 7000 r / min for 13 min, and the cell pellet was collected. The cultured Trichoderma harzianum, Trichoderma viride, and Paecilomyces lilacinus colony surfaces were rinsed with sterile water, and then mycelia were scraped off with an inoculation loop, ground in a sterile mortar, and an appropriate amount of sterile water was added to prepare a mycelial suspension. The Candida utilis fermentation broth was concentrated by membrane filtration to achieve a cell concentration of 10-1. 9 CFU / mL; Step S4, Microencapsulation: Sodium alginate and chitosan are prepared into 2% and 1% aqueous solutions, respectively, and mixed at a volume ratio of 2:1. Other components are added, and a mixture is formed under stirring at 30℃ and 120r / min. The mixture is sprayed into a 0.2mol / L calcium chloride solution through a high-voltage electrostatic spraying device and solidified for 30min to form a microencapsulated composite microbial bactericide primary product. Step S5, Drying and Post-processing: Vacuum freeze drying is performed to obtain a powdered finished product, which is then packaged with nitrogen for protection.

[0038] In step S4, the voltage of the high-voltage electrostatic spray device is 15kV and the nozzle diameter is 0.8mm; the pre-freezing temperature of the vacuum freeze-drying is -40℃, the vacuum degree is 20Pa, and the drying time is 24h.

[0039] Example 4 A compound microbial bactericide, by weight, comprises the following components: 14 parts core microorganisms, 4.5 parts humic acid, 5.5 parts plant polysaccharides, 11 parts protectant, 3.5 parts nutrients, 2.5 parts adenosine, 3.5 parts nano-hydroxyapatite, 1.8 parts graphene quantum dots, 1.4 parts glutamine, and 2.5 parts carboxymethyl-β-cyclodextrin; wherein the core microorganisms include Paecilomyces lilacinus, Bacillus amyloliquefaciens, Trichoderma harzianum, Trichoderma viride, Bacillus subtilis, Pseudomonas fluorescens, Bacillus licheniformis, Bacillus laterosporus, and Candida utilis.

[0040] The viable cell ratio of Paecilomyces lilacinus, Bacillus amyloliquefaciens, Trichoderma harzianum, Trichoderma viride, Bacillus subtilis, Pseudomonas fluorescens, Bacillus licheniformis, Bacillus brevis, and Candida utilis is 11:22:17:17:27:11:22:14:7.5.

[0041] The plant polysaccharide is a mixture of ginkgo leaf polysaccharide, tea polysaccharide, and seaweed polysaccharide in a mass ratio of 1:2:1; the protective agent includes sodium alginate and chitosan; the nutrient is a mixture of ammonium nitrate and potassium dihydrogen phosphate in a mass ratio of 1:1.1; the average particle size of the nano-sized hydroxyapatite is 60 nm; the graphene quantum dots have 1-5 layers, a thickness of 1-2 nm, and a lateral dimension of 5-15 nm; the degree of substitution of the carboxymethyl-β-cyclodextrin is 7, and the molecular weight is 1540 Da.

[0042] A method for preparing the aforementioned composite microbial bactericide includes the following steps: Step S1, Strain Selection and Modification: Highly active original strains were screened from different ecological environments. Bacillus subtilis was genetically engineered to overexpress genes related to antimicrobial peptide synthesis. Candida utilis was mutagenized to screen mutant strains with polysaccharide secretion increased by more than 30%. Step S2, Strain Culture: Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus brevis, and Pseudomonas fluorescens were inoculated into LB medium and cultured at 32℃ and 180 rpm for 22 h; Trichoderma harzianum and Trichoderma viride were inoculated into PDA medium and cultured at 28℃ and 180 rpm for 6 days; Paecilomyces lilacinus was inoculated into PDB medium and cultured with shaking at 28℃ and 180 rpm for 72 h; Candida utilis was inoculated into YM medium and cultured at 26℃ and 150 rpm for 48 h. Step S3, Cell Preparation and Processing: Centrifuge the cultured Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus laterosporus brevis, and Pseudomonas fluorescens fermentation broths at 4℃ and 6000-8000 r / min for 10-15 min, and collect the cell precipitate; rinse the colony surfaces of the cultured Trichoderma harzianum, Trichoderma viride, and Paecilomyces lilacinus with sterile water, then scrape the mycelium with an inoculation loop, grind it in a sterile mortar, and add an appropriate amount of sterile water to prepare a mycelial suspension; concentrate the fermentation broth of Candida utilis by membrane filtration to achieve a cell concentration of 10. 9 CFU / mL; Step S4, Microencapsulation: Sodium alginate and chitosan are prepared into 2% and 1% aqueous solutions, respectively, and mixed at a volume ratio of 2:1. Other components are added, and a mixture is formed under stirring at 30℃ and 120r / min. The mixture is sprayed into a 0.2mol / L calcium chloride solution through a high-voltage electrostatic spraying device and solidified for 30min to form a microencapsulated composite microbial bactericide primary product. Step S5, Drying and Post-processing: Vacuum freeze drying is performed to obtain a powdered finished product, which is then packaged with nitrogen for protection.

[0043] In step S4, the voltage of the high-voltage electrostatic spray device is 15kV and the nozzle diameter is 0.8mm; the pre-freezing temperature of the vacuum freeze-drying is -40℃, the vacuum degree is 25Pa, and the drying time is 24h.

[0044] Example 5 A compound microbial bactericide, by weight, comprises the following components: 15 parts core bacterial strain, 5 parts humic acid, 6 parts plant polysaccharides, 12 parts protective agent, 4 parts nutrients, 3 parts adenosine, 4 parts nano-grade hydroxyapatite, 2 parts graphene quantum dots, 1.5 parts glutamine, and 3 parts carboxymethyl-β-cyclodextrin; wherein the core bacterial strain includes Paecilomyces lilacinus, Bacillus amyloliquefaciens, Trichoderma harzianum, Trichoderma viride, Bacillus subtilis, Pseudomonas fluorescens, Bacillus licheniformis, Bacillus laterosporus, and Candida utilis.

[0045] The viable cell ratio of *Paecilomyces lilacinus*, *Bacillus amyloliquefaciens*, *Trichoderma harzianum*, *Trichoderma viride*, *Bacillus subtilis*, *Pseudomonas fluorescens*, *Bacillus licheniformis*, *Bacillus laterosporus*, and *Candida utilis* is 12:23:18:18:28:12:23:15:8; the plant polysaccharide is ginkgo leaf polysaccharide; the protective agent includes sodium alginate and chitosan; the nutrient is ammonium nitrate and potassium dihydrogen phosphate mixed in a mass ratio of 1:1.2; the average particle size of the nano-sized hydroxyapatite is 70 nm; the number of layers of the graphene quantum dots is 1-5, the thickness is 1-2 nm, and the lateral dimension is 5-15 nm; the degree of substitution of the carboxymethyl-β-cyclodextrin is 7, and the molecular weight is 1540 Da.

[0046] A method for preparing the aforementioned composite microbial bactericide includes the following steps: Step S1, Strain Selection and Modification: Highly active original strains were screened from different ecological environments. Bacillus subtilis was genetically engineered to overexpress genes related to antimicrobial peptide synthesis. Candida utilis was mutagenized to screen mutant strains with polysaccharide secretion increased by more than 30%. Step S2, Strain Culture: Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus brevis, and Pseudomonas fluorescens were inoculated into LB medium and cultured at 32℃ and 180 rpm for 22 h; Trichoderma harzianum and Trichoderma viride were inoculated into PDA medium and cultured at 28℃ and 180 rpm for 6 days; Paecilomyces lilacinus was inoculated into PDB medium and cultured with shaking at 28℃ and 180 rpm for 72 h; Candida utilis was inoculated into YM medium and cultured at 26℃ and 150 rpm for 48 h. Step S3, Cell Preparation and Processing: The fermentation broths of Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus laterosporus brevis, and Pseudomonas fluorescens were centrifuged at 4℃ and 8000 r / min for 15 min, and the cell pellet was collected. The colony surfaces of the cultured Trichoderma harzianum, Trichoderma viride, and Paecilomyces lilacinus were rinsed with sterile water, and then the mycelium was scraped off with an inoculation loop, ground in a sterile mortar, and an appropriate amount of sterile water was added to prepare a mycelial suspension. The fermentation broth of Candida utilis was concentrated by membrane filtration to achieve a cell concentration of 10-1. 9 CFU / mL; Step S4, Microencapsulation: Sodium alginate and chitosan are prepared into 2% and 1% aqueous solutions, respectively, and mixed at a volume ratio of 2:1. Other components are added, and a mixture is formed under stirring at 30℃ and 120r / min. The mixture is sprayed into a 0.2mol / L calcium chloride solution through a high-voltage electrostatic spraying device and solidified for 30min to form a microencapsulated composite microbial bactericide primary product. Step S5, Drying and Post-processing: Vacuum freeze drying is performed to obtain a powdered finished product, which is then packaged with nitrogen for protection.

[0047] In step S4, the voltage of the high-voltage electrostatic spray device is 15kV and the nozzle orifice diameter is 0.8mm; the pre-freezing temperature of the vacuum freeze-drying is -40℃, the vacuum degree is 30Pa, and the drying time is 25h.

[0048] Comparative Example 1 This example provides a compound microbial bactericide and its application, which is basically the same as in Example 1, except that adenosine and nano-sized hydroxyapatite are not added.

[0049] Comparative Example 2 This example provides a composite microbial bactericide and its application, which is basically the same as Example 1, except that graphene quantum dots and plant polysaccharides are not added.

[0050] Comparative Example 3 This example provides a compound microbial bactericide and its application, which is basically the same as in Example 1, except that an equal amount of Trichoderma harzianum is used instead of Paecilomyces lilacinus, and an equal amount of Bacillus subtilis is used instead of Candida utilis.

[0051] To further illustrate the beneficial technical effects of the composite microbial bactericides involved in the various embodiments of the present invention, relevant performance tests were conducted on the composite microbial bactericides involved in each example. The test results are shown in Table 1, and the test methods are as follows: (1) Field efficacy test: Field efficacy test was conducted in accordance with GB / T 17980.121-2004, and the control effect was statistically analyzed 7 days after application.

[0052] (2) Stability test: Each bactericide was placed in a high temperature (50℃) and strong acid (pH=2) environment for 7 days. The change in the number of viable microorganisms in the bactericide was measured, and the retention rate of viable microorganisms was statistically calculated. The larger the value, the better the stability.

[0053] Table 1 Performance test results of compound microbial bactericide

[0054] As can be seen from Table 1, the composite microbial bactericides involved in the embodiments of the present invention have better control effects and stability than the comparative products. The combined use of adenosine, nano-sized hydroxyapatite, graphene quantum dots, plant polysaccharides, Paecilomyces lilacinus and Candida utilis is beneficial to improving the above performance.

[0055] The above embodiments are only for illustrating the technical concept and features of the present invention. Their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A compound microbial bactericide, characterized in that, By weight, it includes the following components: 10-15 parts core microorganisms, 3-5 parts humic acid, 4-6 parts plant polysaccharides, 8-12 parts protective agent, 2-4 parts nutrients, 1-3 parts adenosine, 2-4 parts nano-grade hydroxyapatite, 1-2 parts graphene quantum dots, 0.8-1.5 parts glutamine, and 1-3 parts carboxymethyl-β-cyclodextrin; the core microorganisms include Paecilomyces lilacinus, Bacillus amyloliquefaciens, Trichoderma harzianum, Trichoderma viride, Bacillus subtilis, Pseudomonas fluorescens, Bacillus licheniformis, Bacillus laterosporus, and Candida utilis.

2. The compound microbial bactericide according to claim 1, characterized in that, The viable cell ratio of Paecilomyces lilacinus, Bacillus amyloliquefaciens, Trichoderma harzianum, Trichoderma viride, Bacillus subtilis, Pseudomonas fluorescens, Bacillus licheniformis, Bacillus brevis lateralis, and Candida utilis is (8-12):(15-23):(10-18):(10-18):(18-28):(8-12):(15-23):(8-15):(5-8).

3. The compound microbial bactericide according to claim 1, characterized in that, The plant polysaccharide is at least one of ginkgo leaf polysaccharide, tea polysaccharide, and seaweed polysaccharide.

4. The compound microbial bactericide according to claim 1, characterized in that, The protective agent includes sodium alginate and chitosan.

5. The compound microbial bactericide according to claim 1, characterized in that, The nutrient is composed of ammonium nitrate and potassium dihydrogen phosphate mixed in a mass ratio of 1:(0.8-1.2).

6. The compound microbial bactericide according to claim 1, characterized in that, The average particle size of the nano-sized hydroxyapatite is 10-70 nm; the number of layers of the graphene quantum dots is 1-5, the thickness is 1-2 nm, and the lateral dimension is 5-15 nm.

7. The compound microbial bactericide according to claim 1, characterized in that, The degree of substitution of the carboxymethyl-β-cyclodextrin is 7, and the molecular weight is 1540 Da.

8. A method for preparing a composite microbial bactericide according to any one of claims 1-7, characterized in that, Includes the following steps: Step S1, strain selection and modification: Screen high-activity original strains from different ecological environments, genetically modify Bacillus subtilis, and overexpress genes related to antimicrobial peptide synthesis; Mutagenesis was performed on Candida utilis, and mutant strains with polysaccharide secretion increased by more than 30% were screened out. Step S2, Strain Culture: Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus brevis, and Pseudomonas fluorescens were inoculated into LB medium and cultured at 32℃ and 180 rpm for 22 h; Trichoderma harzianum and Trichoderma viride were inoculated into PDA medium and cultured at 28℃ and 180 rpm for 6 days; Paecilomyces lilacinus was inoculated into PDB medium and cultured with shaking at 28℃ and 180 rpm for 72 h; Candida utilis was inoculated into YM medium and cultured at 26℃ and 150 rpm for 48 h. Step S3, Cell Preparation and Processing: Centrifuge the cultured Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus laterosporus brevis, and Pseudomonas fluorescens fermentation broths at 4℃ and 6000-8000 r / min for 10-15 min, and collect the cell precipitate; rinse the colony surfaces of the cultured Trichoderma harzianum, Trichoderma viride, and Paecilomyces lilacinus with sterile water, then scrape the mycelium with an inoculation loop, grind it in a sterile mortar, and add an appropriate amount of sterile water to prepare a mycelial suspension; concentrate the fermentation broth of Candida utilis by membrane filtration to achieve a cell concentration of 10. 9 CFU / mL; Step S4, Microencapsulation: Sodium alginate and chitosan are prepared into 2% and 1% aqueous solutions, respectively, and mixed at a volume ratio of 2:

1. Other components are added, and a mixture is formed under stirring at 30℃ and 120r / min. The mixture is sprayed into a 0.2mol / L calcium chloride solution through a high-voltage electrostatic spraying device and solidified for 30min to form a microencapsulated composite microbial bactericide primary product. Step S5, Drying and Post-processing: Vacuum freeze drying is performed to obtain a powdered finished product, which is then packaged with nitrogen for protection.

9. A method for preparing the composite microbial bactericide according to claim 8, characterized in that, In step S4, the voltage of the high-voltage electrostatic spray device is 15kV and the nozzle diameter is 0.8mm; the pre-freezing temperature of the vacuum freeze-drying is -40℃, the vacuum degree is 10-30Pa, and the drying time is 22-25h.

10. The application of a compound microbial bactericide according to any one of claims 1-7 in crop production.

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