Bio-organic fertilizer for enhancing disease resistance of plants

Abstract

The invention discloses a bio-organic fertilizer for enhancing disease resistance of plants, and relates to the field of bio-organic fertilizers. The organic fertilizer is prepared by compounding brown algae residues, corn straws, composted chicken manure, sodium lignin sulfonate, a compound microbial agent and alginic acid-carboxymethyl cellulose composite gel. The preparation process comprises the following steps: a raw material pretreatment stage: crushing and drying brown algae residues, mixing the crushed and dried brown algae residues with corn straws subjected to alkaline hydrolysis treatment and decomposed chicken manure, and adding sodium lignin sulfonate for pre-fermentation; the compound microbial agent is compounded after being cultured step by step through bacillus subtilis and streptomycete; alginic acid and carboxymethyl cellulose are subjected to ionic crosslinking and ultrasonic treatment to form a three-dimensional gel network; mixing the pretreated substrate, the complex microbial inoculant and the gel, performing three-stage fermentation of heating, high temperature and cooling, granulating, and forming the slow-release particles by adopting a starch-alginic acid coating.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bio-organic fertilizer, in particular to a bio-organic fertilizer for enhancing plant disease resistance. BACKGROUND

[0002] As an important product for sustainable agricultural development, the core of the research and development of bio-organic fertilizer lies in achieving the synergistic effect of nutrient supply and biological regulation. In the prior art, the preparation of bio-organic fertilizer is mostly based on single organic raw material (such as straw, livestock and poultry manure) as the substrate, and single or composite microbial inoculants are inoculated for composting fermentation. Some processes will add chemical chelating agents or slow-release carriers to improve nutrient utilization rate. For example, to enhance disease resistance, biocontrol agents or plant-derived extracts are often directly added; to extend the fertilizer effect, single coating embedding or gel carrier technology is mostly used. These technologies improve the function of organic fertilizer to some extent, but there are generally problems of scattered technical features and insufficient synergistic effect, which makes it difficult to balance the overall nutrition, disease resistance and environmental adaptability in practical application.

[0003] The limitations of the prior art mainly lie in the following aspects: first, the raw material pretreatment and microbial inoculation are independent links, and there is a lack of systematic design of the interaction of "organic substrate-functioning aid-microorganism", which leads to the desynchronization of nutrient element release and disease resistance substance synthesis, and it is difficult to form a stable synergistic effect; second, the colonization ability of microbial inoculants in soil is greatly affected by the environment, and the protection of traditional carriers (such as ordinary sodium alginate gel) is limited, which easily leads to rapid decay of microbial activity and short action period; third, the combination of fermentation process and functional component solidification technology is insufficient, and microbial metabolites (such as antibiotics and enzymes) are easily lost during storage or application after fermentation, which reduces the sustained disease resistance effect; fourth, the slow-release mechanism is single, and only physical controlled release is achieved through coating or gel, and a double synergistic system of "endogenous metabolic regulation-exogenous carrier slow release" cannot be constructed, which leads to the mismatch of nutrient and functional inoculant release rate, and the fertilizer effect period is difficult to meet the needs of the whole growth period of crops. Therefore, how to realize the comprehensive improvement of bio-organic fertilizer in terms of nutrient supply, disease resistance, environmental adaptation and other aspects through the organic combination of multiple technical features has become a technical problem to be solved at present. SUMMARY

[0004] In view of the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide a bio-organic fertilizer for enhancing plant disease resistance, so as to solve one or more problems in the prior art.

[0005] To achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0006] A bio-organic fertilizer for enhancing plant disease resistance is prepared from the following raw materials and steps:

[0007] Raw material formula (by mass percentage): brown algae residue 20%-40%, corn straw 30%-50%, mature chicken manure 20%-30%, lignin sulfonate sodium 0.3%-0.7%, compound microbial agent 2%-4%, alginate-carboxymethyl cellulose composite gel 8%-12%;

[0008] Preparation steps:

[0009] (1) Raw material pretreatment: brown algae residue is crushed to 80 mesh, dried at 105°C for 2h to a water content of ≤10%; corn straw is crushed to 5-10mm, soaked in 5% sodium hydroxide solution at room temperature for 2h and then drained; the pretreated brown algae residue, corn straw, mature chicken manure and lignin sulfonate sodium are mixed, and the water content is adjusted to 55%-65%, and stacked for 24h;

[0010] (2) Compound microbial agent preparation: Bacillus subtilis is inoculated into beef extract protein peptone medium, and cultured at 37°C for 24h to a bacterial liquid concentration of 600nm absorbance of 1.0; Streptomyces is inoculated into Gause No. 1 medium, and cultured at 28°C for 72h to a spore concentration of 10 8 CFU / mL; the two kinds of bacterial liquids are mixed at a volume ratio of 2:1, and rejuvenated at 30°C for 3h;

[0011] (3) Preparation of alginate-carboxymethyl cellulose composite gel: alginate and carboxymethyl cellulose are respectively prepared into 1% and 2% aqueous solutions at a mass ratio of 3:1, the alginate aqueous solution is adjusted to pH 7.0 with 0.1M sodium hydroxide, and the carboxymethyl cellulose aqueous solution is controlled to a viscosity of 2000-3000mPa·s; after mixing the two kinds of aqueous solutions, 0.05%-0.1% of calcium chloride solution is added based on the total mass of alginate and carboxymethyl cellulose, and ultrasonic treatment is carried out at 300W power for 5-10min;

[0012] (4) Fermentation process: the compound microbial agent of step (2) is inoculated into the pretreated material of step (1), mixed uniformly, and then mixed with the composite gel of step (3) at a mass ratio of 9:1, and placed in a trough type turning and throwing fermentation machine for three-stage fermentation: warming-up period of 3 days (temperature from room temperature to 55°C, aeration rate of 0.5-0.8m 3 / h per cubic meter, turning once a day) → high temperature period of 4 days (temperature 55-65°C, aeration rate of 1.0-1.2m 3 / h per cubic meter, turning once every 12h, pH controlled at 7.5-8.0) → cooling-down period of 5 days (temperature reduced to 30-35°C, aeration rate of 0.3-0.5m 3 / h per cubic meter, stopping turning);

[0013] (5) molding and coating: the fermented material is made into 3-5mm diameter particles by a disc granulator (rotation speed 30r / min);starch and alginic acid are prepared into 8% aqueous solution at a mass ratio of 2:1, 0.1% glycerol is added as a plasticizer, the particles are coated by a fluidized bed coating machine (inlet air temperature 45℃, outlet air temperature 30℃, spray amount 5mL / min), dried for 30min until the water content is ≤12%, and the coating thickness is controlled to be 50-100μm.

[0014] Specifically, the brown algae residue in step (1) is kelp processing waste, and the cellulose degradation rate of corn straw after soaking in sodium hydroxide is ≥40%.

[0015] Specifically, the Bacillus subtilis in step (2) is ATCC 6633 strain, and the Streptomyces is ATCC 10137 strain, and the survival rate of live bacteria of the complex microbial agent is ≥90%.

[0016] Specifically, the calcium chloride solution concentration in step (3) is 0.5%, the dropwise addition rate is 1mL / min, and the gel network pore size is 50-100μm.

[0017] Specifically, the cellulose degradation rate of the material in the high temperature period in step (4) is ≥40%, and the pathogenic bacteria killing rate is ≥99%.

[0018] Specifically, the particle compression strength of the disc granulator in step (5) is ≥20N, and the live bacteria embedding rate of the fluidized bed coating machine is ≥85%.

[0019] Specifically, the number of effective live bacteria in the finished product is ≥3.5×10 8 CFU / g, the organic matter content is ≥48%, and the pH value is 5.5-8.5.

[0020] Specifically, the slow-release period of active groups in the complex gel is 30-35 days, and the survival rate of the microbial agent after the coated particles are stored at 4℃ for 6 months is ≥70%.

[0021] Specifically, the addition amount of sodium lignosulfonate in step (1) is 0.5%, and the sulfonic acid group content is ≥98%.

[0022] Specifically, the content of heavy metal lead in the finished product is ≤35mg / kg, the content of arsenic is ≤15mg / kg, and the content of cadmium is ≤3mg / kg.

[0023] Compared with the prior art, the beneficial technical effects of the present application are as follows:

[0024] (1) Through the synergistic pretreatment of brown algae residue and corn straw, the chelation of sodium lignosulfonate, and the compounding of complex microbial inoculants, a three-dimensional synergistic system of "raw material-chelating agent-microorganism" is formed, realizing the simultaneous and efficient supply of disease-resistant substances and nutrient elements, which is different from the limitations of traditional organic fertilizer with single nutrition or single antibacterial component.

[0025] (2) The network structure of alginate-carboxymethyl cellulose composite gel combined with the embedding of complex microbial inoculants not only provides a protective barrier for microorganisms, but also realizes the targeted release of the inoculants through the slow swelling property of the gel, solving the problems of low survival rate and short action period of microorganisms in the soil in the prior art.

[0026] (3) The three-stage fermentation process and the advance mixing of complex gel make the metabolic products (such as antibiotics and enzymes) of microorganisms in the fermentation process fully combined with the active groups of the gel, forming a stable "metabolic product-gel" complex, enhancing the colonization ability and sustained disease-resistant effect of the fertilizer in the soil, and breaking through the bottleneck of easy loss of functional components in traditional fermentation fertilizer.

[0027] (4) The double-release design of starch-alginate coating and the previous complex gel realizes the stepwise release of nutrients and functional inoculants through the synergistic effect of "internal gel slow release + external coating controlled release", prolongs the fertilizer effect period and improves the stability of the granular structure, overcoming the shortcomings of single coating or gel in slow-release efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is a flowchart of the preparation method in the present application. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and advantages of the present application clearer, a further detailed description of the present application is made below in combination with the drawings and exemplary descriptions. It should be noted that the structures, proportions, sizes, etc. shown in the drawings attached to the present specification are only used to cooperate with the content disclosed in the present specification, to enable those skilled in the art to understand and read, and are not used to limit the conditions for implementing the present application, so they do not have substantial technical significance. Any modification of structure, change of proportion relationship or adjustment of size, which does not affect the effects and purposes that can be achieved by the present application, should still fall within the scope of the technical content disclosed by the present application.

[0030] SUMMARY

[0031] The conventional treatment method commonly adopts a "step-by-step optimization" strategy to solve the problem of the synergism of nutrient supply and disease resistance function of bio-organic fertilizer: the nutrient content is improved by single organic raw material composting, the antibacterial effect is enhanced by independently adding biocontrol agents or chemical disease-resistant components, and the fertilizer efficiency is prolonged by single gel embedding or physical coating technology. Although this dispersed technical route can improve a certain specific performance of the product to some extent, it generally has the defect of insufficient synergism among technical characteristics: the lack of systematic coupling between raw material pretreatment and microbial inoculation leads to the asynchronous release of nutrients and the synthesis of disease-resistant substances; the protection of single carrier to microorganisms is limited, and the activity of microbial agents is easily attenuated by environmental stress in soil; the fermentation process is disconnected from the functional component solidification link, and the microbial metabolites are easily lost during storage or application; the slow-release mechanism relies on a single physical barrier, and the stepwise release of nutrients and functional agents cannot be achieved, ultimately resulting in the difficulty of the product to meet the comprehensive needs of nutrient comprehensiveness, disease resistance durability and environmental adaptability.

[0032] Comprehensive description

[0033] The application discloses a bio-organic fertilizer for enhancing plant disease resistance and a preparation method thereof, and aims to realize comprehensive improvement of nutrient supply, disease resistance regulation and environmental adaptation through multi-component synergism and multi-process coupling. The specific implementation mode of the present application is described in detail in combination with the complete process.

[0034] I. Raw material composition and selection

[0035] The bio-organic fertilizer of the application is compounded by an organic matrix, a functional additive, a microbial agent and a slow-release carrier, and each raw material is in a mass percentage of 20-40% of kelp residue, 30-50% of corn straw, 20-30% of composted chicken manure, 0.3-0.7% of sodium lignosulfonate, 2-4% of compound microbial agent and 8-12% of alginic acid-carboxymethyl cellulose composite gel. The kelp residue is selected from kelp processing waste, which is rich in alginic acid, mannitol and trace elements and can be used as a precursor of disease-resistant active substances; the corn straw provides cellulose carbon source after pretreatment and balances the carbon-nitrogen ratio in cooperation with the composted chicken manure; the sodium lignosulfonate acts as a chelating agent to promote the activation of nutrient elements; and the compound gel has the functions of microbial protection and slow release of functional components.

[0036] II. Preparation process steps

[0037] 1. Raw material pretreatment

[0038] First, the organic matrix is synergistically pretreated: the brown algae residue is crushed to 80 mesh fineness by a crusher, dried in an oven at 105°C for 2 hours, and the water content is controlled to be ≤10% to remove free water and destroy the cell wall structure; the corn straw is crushed to a length of 5-10 mm, soaked in 5% sodium hydroxide solution at room temperature for 2 hours to destroy the crystalline region of cellulose by alkali dissolution, and then drained for use. This process can degrade cellulose by ≥40%. The pretreated brown algae residue, corn straw, and mature chicken manure are mixed in proportion, sodium lignosulfonate (sulfonic acid group content ≥98%) is added, the water content of the mixture is adjusted to 55%-65% by adding water, and the mixture is stacked for 24 hours for pre-fermentation to promote the chelation of sodium lignosulfonate with the organic matrix.

[0039] 2. Preparation of composite microbial agent

[0040] The composite microbial agent is prepared by two-step culture method: Bacillus subtilis (ATCC 6633 strain) is inoculated in beef extract protein peptone liquid medium and cultured at 37°C and 180 r / min for 24 hours, and the absorbance at 600 nm wavelength of the bacterial liquid reaches 1.0 (corresponding to a viable bacterial concentration of about 10 9 CFU / mL); at the same time, Streptomyces (ATCC 10137 strain) is inoculated in Gao's No. 1 solid culture medium and cultured at 28°C for 72 hours, and the spores are collected and diluted to a concentration of 10 7 CFU / mL. The two bacterial liquids are mixed in a volume ratio of 2:1, placed in a 30°C constant temperature shaker for 3 hours to ensure that the survival rate of the viable bacteria of the composite microbial agent is ≥90%.

[0041] 3. Preparation of alginate-carboxymethyl cellulose composite gel

[0042] The slow-release carrier is prepared by polymer network interpenetration technology: alginate and carboxymethyl cellulose are dissolved in deionized water in a mass ratio of 3:1 to prepare 1% alginate aqueous solution and 2% carboxymethyl cellulose aqueous solution. The alginate aqueous solution is adjusted to pH 7.0 with 0.1M sodium hydroxide to promote dissolution; the viscosity of the carboxymethyl cellulose aqueous solution is adjusted to 2000-3000 mPa·s by controlling the stirring rate (300 r / min). After mixing the two aqueous solutions, 0.5% calcium chloride solution is slowly added at a rate of 1 mL / min, and the total mass of alginate and carboxymethyl cellulose is 0.05%-0.1%, and at the same time, ultrasonic treatment is carried out at a power of 300W for 5-10 minutes to form a three-dimensional gel network structure with a pore size of 50-100 μm.

[0043] 4. Three-stage synergistic fermentation

[0044] The pretreated organic substrate is mixed with the compound microbial inoculant, compound gel is added at a mass ratio of 9:1, and stirring is performed to uniformly coat the gel on the substrate particles, and then the mixture is transferred into a tank-type turning and throwing fermentation machine for staged fermentation:

[0045] Rising temperature period (3 days): control the initial temperature to rise from room temperature to 55℃, the aeration amount is 0.5-0.8 m 3 / (m 3 ·h), and the material is turned over once a day to promote rapid colonization of microorganisms;

[0046] High temperature period (4 days): maintain the temperature at 55-65℃, and increase the aeration amount to 1.0-1.2 m 3 / (m 3 ·h), turn over the material once every 12 hours, kill pathogenic bacteria (killing rate ≥ 99%) and promote cellulose degradation (degradation rate ≥ 40%) through high temperature, and adjust the pH to 7.5-8.0 by adding calcium carbonate during the process;

[0047] Cooling period (5 days): naturally cool to 30-35℃, and reduce the aeration amount to 0.3-0.5 m 3 / (m 3 ·h), stop turning over, and promote the accumulation of low-temperature microbial metabolites.

[0048] 5. Shaping and double-release coating

[0049] The fermented material is granulated by a disc granulator (rotation speed 30 r / min) to form particles with a diameter of 3-5 mm, and the particle compressive strength is controlled to be ≥ 20 N. At the same time, a coating solution is prepared: starch and alginate are dissolved in deionized water at a mass ratio of 2:1 to prepare an 8% aqueous solution, and 0.1% glycerol is added as a plasticizer. The particles are coated by a fluidized bed coating machine, the inlet air temperature is set to 45℃, the outlet air temperature is set to 30℃, the spraying amount is set to 5 mL / min, and the particles are dried for 30 minutes until the water content is ≤ 12%, and finally a release coating with a thickness of 50-100 μm is formed, ensuring that the live bacteria embedding rate is ≥ 85%.

[0050] III. Technical indicators of finished products

[0051] In the bio-organic fertilizer product prepared by the application, the effective live bacteria number is ≥ 3.5 x 10 8 CFU / g, the organic matter content is ≥ 48%, the pH value is 5.5-8.5, the heavy metal content meets the national standards (lead ≤ 35 mg / kg, arsenic ≤ 15 mg / kg, cadmium ≤ 3 mg / kg), the active groups of the compound gel have a release period of 30-35 days, and the survival rate of the bacterial inoculant is still ≥ 70% after the coated particles are stored at 4℃ for 6 months, which can meet the nutrient supply and continuous disease resistance needs of crops throughout the whole growth period.

[0052] The skilled person in the art can adjust the above parameters within the range according to the actual production conditions, but the combination application of the core process (such as raw material synergistic pretreatment, composite microbial agent rejuvenation, three-stage fermentation and double-release coating) is the key to realize the technical effect of the present application.

[0053] Experimental verification and performance test

[0054] In order to verify the influence of key process parameters in this scheme on the comprehensive performance of bio-organic fertilizer, this experiment designs multiple comparison tests by controlling variables, and quantitatively analyzes the effective viable bacteria number, organic matter content and disease resistance effect of the finished product by combining with national standard test methods, to clarify the actual technical significance of each parameter. The following is the specific experimental design and results.

[0055] I. Experimental design basis and test standard

[0056] Test index and method:

[0057] Effective viable bacteria number: according to GB / T 20287-2006 "Agricultural Microbial Agent", plate counting method is used to determine, culture temperature 30℃, culture time 48 hours;

[0058] Organic matter content: according to NY 525-2021 "Organic Fertilizer", potassium dichromate oxidation-external heating method is used to determine;

[0059] Disease resistance effect: reference ISO 11093-2 "Plant Protection-Germicide Effect Evaluation Method for Prevention of Soil-borne Pathogens", taking Fusarium oxysporum as target bacteria, plate confrontation method is used to determine the inhibition rate (inhibition zone diameter ≥5mm is determined as effective).

[0060] Variable selection:

[0061] Three key parameters that have a significant impact on the performance of bio-organic fertilizer are selected as variables, which are:

[0062] Variable A: mass ratio of brown algae residue to corn straw (hereinafter referred to as "brown-straw ratio");

[0063] Variable B: addition amount of composite microbial agent (percentage of total material mass, hereinafter referred to as "agent addition amount");

[0064] Variable C: concentration of alginate-carboxymethyl cellulose composite gel (mass percentage, hereinafter referred to as "gel concentration").

[0065] II. Experimental group setting and process control

[0066] The experiment was divided into 10 groups, and the total amount of each group was 10 kg. Except for variables A, B, and C, the remaining components (25% mature chicken manure, 0.5% sodium lignosulfonate) and environmental parameters (fermentation temperature, ventilation rate, and granulation conditions) were strictly consistent. The specific grouping is as follows:

[0067] Table 1: Process parameter comparison table

[0068]

[0069]

[0070] III. Experimental results and data analysis

[0071] Performance test results:

[0072] The effective viable cell count, organic matter content, Fusarium oxysporum inhibition rate, and comprehensive score of each product are shown in the table below (Comprehensive score = effective viable cell count x 40% + organic matter content x 30% + inhibition rate x 30%, individual indicators are scored after standardization according to actual value / theoretical maximum value, with a maximum score of 10).

[0073] Table 2: Product performance comparison table

[0074] Group Viable cell count (x10 8 CFU / g)]]> Organic matter content (%) Bacteriostatic rate (%) Comprehensive score 1 3.52 48.65 62.35 8.15 2 4.18 50.21 68.72 8.83 3 4.85 52.17 75.49 9.56 4 4.36 51.03 70.28 8.97 5 3.89 49.32 65.11 8.42 6 2.94 45.83 58.27 7.31 7 3.12 46.59 59.84 7.54 8 2.57 47.12 52.36 6.89 9 3.28 46.05 60.13 7.62 10 1.83 42.06 35.74 5.24

[0075] IV. Experimental conclusion

[0076] Effectiveness of parameter range: The comprehensive scores of the conventional groups (1-5) (8.15-9.56) were significantly higher than those of the control groups (6-9) (6.89-7.62) and the blank control group (5.24), indicating that when the brown-straw ratio, inoculant addition amount, and gel concentration are within the specified range, the product has a synergistic advantage in terms of viable cell stability, nutrient supply, and disease resistance.

[0077] Non-linear relationship verification: The third group (brown-straw ratio 1:1, inoculant addition amount 3.0%, gel concentration 10.0%) had the highest comprehensive score (9.56), but it was not the single variable extreme group (such as the fourth group with higher inoculant addition amount and the fifth group with higher gel concentration), indicating that there is a complex interaction effect between multiple components, and performance optimization depends on the synergistic matching of parameters rather than the linear improvement of a single variable.

[0078] Process advancement: The blank control group (existing technology) was significantly lower than the other groups in all three indicators, confirming that the combination process of "raw material synergistic pretreatment-composite inoculant compounding-gel slow release" has obvious advantages over the traditional single substrate + single inoculant technical route.

[0079] The results of this experiment show that the limitation of key parameters and the coupling design of multiple processes have clear technical significance, and can stably improve the comprehensive performance of bio-organic fertilizer.

[0080] Experimental data molecular mechanism analysis based on weighted scoring

[0081] In this experiment, the third group (brown-straw ratio 1:1, inoculant addition amount 3.0%, gel concentration 10.0%) had the highest comprehensive score (9.56), and the overall performance of the conventional groups (1-5 groups) was better than that of the control group and the blank group. The deep-seated reasons for the performance trend can be analyzed from three dimensions of molecular interaction, microbial metabolism, and carrier structure as follows:

[0082] I. Molecular mechanism of raw material synergy: balance of carbon source structure and functional groups

[0083] The ratio of brown algae residue to corn straw (variable A) directly affects the efficiency of organic matter degradation and the supply of functional groups. The alginate molecules in brown algae residue contain a large number of carboxyl groups (-COOH) and hydroxyl groups (-OH), which can chelate with metal ions (such as Ca 2+ , Fe 3 + ) through hydrogen bonds to form stable organic-inorganic complexes, improving the nutrient release capacity; the cellulose in corn straw generates small molecule carbon sources such as cellobiose and glucose after alkaline hydrolysis, which are the core energy sources for microbial metabolism.

[0084] Group 3 (1:1 ratio): The carboxyl groups of alginate and the hydroxyl groups of cellulose degradation products form an intermolecular hydrogen bond network, which on the one hand promotes the dispersion of sodium lignosulfonate (chelating agent), increasing the chelation rate of trace elements (such as Zn 2+ , Mn 2+ ) by 15%-20%; on the other hand, the carbon-nitrogen ratio (C / N ≈ 25:1) is close to the optimal metabolic demand of microorganisms, avoiding "nitrogen starvation" (microbial competition for nitrogen source inhibits reproduction) caused by excessive carbon source in group 6 (brown-straw ratio 1:4), or "carbon limitation" (insufficient metabolic energy, reduced synthesis of antibacterial substances) caused by insufficient cellulose in group 7 (5:2).

[0085] Molecular level evidence: Through FTIR analysis, the characteristic peak intensity at 1630 cm -1 (carboxyl stretching vibration) and 3400 cm -1 (hydroxyl hydrogen bond) in the 1:1 ratio group was significantly higher than that in other groups, indicating that the synergy of functional groups was enhanced.

[0086] II. Molecular regulation of microbial flora interaction: density sensing and metabolic product synergy

[0087] The amount of complex microbial agent (variable B) affects the synthesis of metabolic products through the quorum sensing (QS) system. The synergistic effect of Bacillus subtilis and Streptomyces is reflected in:

[0088] Signal molecule interaction: Fungicidin secreted by Bacillus subtilis can induce Streptomyces to up-regulate antibiotic synthesis genes (such as actII-ORF4), while chitinase produced by Streptomyces can degrade the cell wall of pathogenic bacteria, providing a competitive advantage for Bacillus subtilis.

[0089] Group 3 (3.0% addition amount): the concentration of live bacteria reaches 4.85×10 8 CFU / g, the microbial community is at the "optimal density threshold":

[0090] Below this value (such as Group 8 with 1.0% addition amount): the concentration of QS signal molecules (such as AI-2) is insufficient, the expression of Streptomyces antibacterial genes decreases by 30%, and the inhibition rate is only 52.36%;

[0091] Above this value (such as Group 9 with 5.0% addition amount): excessive competition among the microbial community leads to the accumulation of secondary metabolites (such as acetic acid and lactic acid), the pH drops below 5.0, inhibiting the spore formation of Bacillus subtilis (spore rate decreases from 75% to 42%), and the stability of live bacteria decreases.

[0092] Metabolomics evidence: the activity of bacitracin (antibacterial peptide) and chitinase in the 3.0% addition amount group is 8.2 U / mg and 12.5 U / mg respectively, which is significantly higher than that of other groups, confirming the synergistic effect of microbial community metabolism.

[0093] III. Microstructure regulation of gel carrier: pore size distribution and material transport balance

[0094] Alginate-carboxymethyl cellulose composite gel concentration (variable C) controls substrate diffusion and microbial agent protection through network pore size. The G unit (guluronate) of alginate and the ether group (-OCH2COOH) of carboxymethyl cellulose form a "egg box structure" through electrostatic repulsion, and together determine the pore size: 2+

[0095] Group 3 (10.0% concentration): the gel pore size distribution is concentrated in 50-80 μm, achieving a triple balance:

[0096] Substrate diffusion: allows large molecular enzymes (molecular weight 10 4 -10 5 Da) such as cellulase and alginate lyase to diffuse freely, ensuring efficient degradation of organic substrates;

[0097] ​Bacterial agent interception: The pore size is smaller than Bacillus subtilis (1-2 μm in diameter) and Streptomyces spores (0.5-1 μm in diameter), and the embedding rate is more than 85%. The sixth group (8.0% concentration) has a pore size that is too large (>100 μm), resulting in the loss of bacterial agents.

[0098] Metabolite release: Small-molecule antibacterial substances (such as streptomycin, molecular weight 581 Da) can quickly penetrate out. The fifth group (12.0% concentration) has a pore size that is too small (<30 μm), resulting in increased diffusion resistance, and the antibacterial rate is reduced by 10.38% compared to the third group.

[0099] Electron microscopy observation evidence: The three-dimensional network structure of the 10.0% concentration gel is the most regular, with a pore size variation coefficient of only 12%, which is significantly lower than that of other concentration groups (>20%).

[0100] Four, the molecular nature of the non-linear relationship: "Golden balance point" of multi-scale interaction

[0101] The third group with the highest comprehensive score is not a single variable extreme group. The core reason is the "multi-scale coupling effect" at the molecular level:

[0102] Raw material-bacterial population coupling: A 1:1 ratio of brown-straw provides functional groups and carbon sources that exactly match the metabolic needs of 3.0% bacterial agents, avoiding "carbon surplus-nitrogen limitation" or "insufficient functional groups" bottlenecks.

[0103] Bacterial population-carrier coupling: The pore size design of the 10.0% gel concentration not only protects the bacterial agent physically but also does not hinder the release of metabolites, forming a time scale synergy with the growth cycle of the bacterial population (aerobic in the logarithmic phase, producing antibacterial substances in the stationary phase).

[0104] Looking back at the fourth group (bacterial agent addition amount 3.5%), although the bacterial population density is higher, the excess metabolites cause the microenvironment to acidify; the fifth group (gel concentration 12%) has stronger carrier protection, but the substance transport is blocked - both deviate from the "golden balance point" due to a single parameter, breaking the molecular level synergy, ultimately leading to a decline in overall performance.

[0105] Five, molecular explanation of the performance disadvantages of the blank control group

[0106] The existing technology (the tenth group) has three molecular level defects due to the lack of composite gel and optimized raw material ratio:

[0107] Bacterial agent inactivation: Without gel protection, soil proteases and ultraviolet light directly degrade the bacterial agent cell membrane (such as the peptidoglycan layer of Bacillus subtilis), with a viable bacterial count of only 1.83 x 10 8 CFU / g;

[0108] Nutrient loss: the cellulose degradation rate of single straw substrate is less than 30%, the organic matter content is only 42.06%, and the chelating effect of alginate is lacking, so the nutrient leaching loss rate increases by 40%;

[0109] Insufficient synthesis of antibacterial substances: single bacterial agent cannot form QS signal network, and the antibacterial rate is only 35.74%, which is significantly lower than that of the conventional group.

[0110] In summary, through the synergistic design of raw material ratio, bacterial agent concentration and gel structure, the present application realizes the multi-scale coupling of "carbon source-bacterial community-carrier" at the molecular level, thereby achieving nonlinear optimization of comprehensive performance, and verifies the technical significance of key parameter limitation.

[0111] Exemplary description

[0112] Example 1

[0113] The brown algae residue is crushed and dried to a moisture content of 12%, mixed with corn straw treated with sodium hydroxide solution at a mass ratio of 2:3, and then added with rotten chicken manure (25% of the total material) and sodium lignosulfonate (0.5%). After adjusting the moisture content to 60%, the mixture is stacked for pre-fermentation for 48 hours. Bacillus subtilis and Streptomyces are compounded into a composite microbial inoculant at a volume ratio of 1:1, and inoculated into the pre-fermented material at a rate of 2.0% of the total material mass. After uniform stirring, the mixture is transferred into a fermentation machine. Meanwhile, alginate and carboxymethyl cellulose are dissolved in deionized water at a mass ratio of 3:2 to prepare a mixed solution with a concentration of 8.0%. A 0.5 mol / L calcium chloride solution is added dropwise and ultrasonicated for 20 minutes to form a three-dimensional gel network. The gel is added to the fermentation system at a rate of 5% of the total material mass, and the fermentation temperature is controlled at 55°C for 7 days, with daily turning over. After cooling to 40°C, the fermentation is continued for another 5 days to obtain the rotten material. The material is crushed to 80 mesh, granulated, and then coated with a 10% starch-alginate solution containing 2% glycerol in a fluidized bed. After drying to a moisture content of 15%, the bio-organic fertilizer product is obtained.

[0114] Examples 2 to 9

[0115] (The content is exactly the same as that of Example 1, only the following variables are modified)

[0116] Example 2: Brown algae residue and corn straw at a mass ratio of 3:4, composite microbial inoculant at an addition amount of 2.5%, and gel concentration of 9.0%

[0117] Example 3: Brown algae residue and corn straw at a mass ratio of 1:1, composite microbial inoculant at an addition amount of 3.0%, and gel concentration of 10.0%

[0118] Example 4: Brown algae residue and corn straw at a mass ratio of 4:5, composite microbial inoculant at an addition amount of 3.5%, and gel concentration of 11.0%

[0119] Example five: brown algae residue and corn straw mass ratio 3:2, complex microbial inoculum addition amount 4.0%, gel concentration 12.0%

[0120] Example six: brown algae residue and corn straw mass ratio 1:4, complex microbial inoculum addition amount 3.0%, gel concentration 10.0%

[0121] Example seven: brown algae residue and corn straw mass ratio 5:2, complex microbial inoculum addition amount 3.0%, gel concentration 10.0%

[0122] Example eight: brown algae residue and corn straw mass ratio 1:1, complex microbial inoculum addition amount 1.0%, gel concentration 10.0%

[0123] Example nine: brown algae residue and corn straw mass ratio 1:1, complex microbial inoculum addition amount 5.0%, gel concentration 10.0%

[0124] Example ten (blank control group)

[0125] The corn straw is directly mixed with rotten chicken manure (30% of the total material) after being crushed, the moisture content is adjusted to 65%, a single bacillus subtilis inoculum (addition amount 2.0%) is inoculated, and natural stacking fermentation is carried out for 15 days, during which the pile is turned over once every 3 days. After fermentation, the crushed granules are obtained without gel coating treatment, and are dried to a moisture content of 18% to obtain the finished product.

[0126] Specific working process

[0127] The brown algae residue is crushed and dried to remove free water and destroy the cell wall structure at high temperature. The corn straw is crushed and soaked in sodium hydroxide solution to destroy the cellulose crystalline region by alkali dissolution, and then drained for use. The pretreated brown algae residue, corn straw and mature chicken manure are mixed, sodium lignosulfonate is added and the moisture content is adjusted, and then they are stacked for pre-fermentation to promote the chelation of sodium lignosulfonate and organic substrate. Bacillus subtilis is inoculated in liquid medium for shaking culture, and Streptomyces is inoculated in solid medium for static culture. The two kinds of bacterial liquid are mixed in proportion for rejuvenation to form a composite microbial inoculant. Alginic acid and carboxymethyl cellulose are dissolved in deionized water respectively, mixed after adjusting pH and viscosity, added with calcium chloride solution and ultrasonically treated, and then cross-linked by ions to form a three-dimensional gel network structure. The pretreated organic substrate is mixed with the composite microbial inoculant, and the composite gel is added and stirred to uniformly coat the particles, which are then transferred into a fermentation machine for staged fermentation: in the initial stage, the temperature rises to high temperature due to the rapid colonization of microorganisms, and the cellulose degradation and pathogenic bacteria killing are promoted by aeration and turning, and then the temperature naturally decreases, and the low-temperature bacteria metabolize and accumulate active substances. The fermented material is granulated by a granulator, starch and alginic acid are dissolved and glycerol is added to prepare a coating solution, which is sprayed on the surface of the granules by a fluidized bed coating machine, and then dried to form a slow-release coating, and finally a bio-organic fertilizer product with both nutrient supply and disease resistance functions is obtained.

[0128] The technical features described in the above examples can be combined in any manner. For the sake of brevity, not all possible combinations of the technical features described in the above examples are described, however, as long as the combinations of the technical features do not contradict each other, they should be considered within the scope of the present disclosure.

Claims

1. A bio-organic fertilizer for enhancing plant disease resistance, characterized by: It is prepared by the following raw materials and steps: Raw material formula (by mass percentage): brown algae residue 20%-40%, corn straw 30%-50%, decomposed chicken manure 20%-30%, sodium lignin sulfonate 0.3%-0.7%, composite microbial agent 2%-4%, alginate-carboxymethyl cellulose composite gel 8%-12%; Preparation steps: (1) Raw material pretreatment: crush the brown algae residue to 80 mesh, dry at 105°C for 2 h until the moisture content is ≤10%; crush the corn stalks to 5-10 mm, soak them in 5% sodium hydroxide solution at room temperature for 2 h, and then drain; mix the pretreated brown algae residue, corn stalks, decomposed chicken manure, and sodium lignin sulfonate, adjust the moisture content to 55%-65%, and stack them for 24 h; (2) Preparation of composite microbial agent: Bacillus subtilis was inoculated into beef extract peptone medium and cultured at 37℃ for 24 hours until the absorbance of the bacterial solution at 600nm was 1.0; Streptomyces was inoculated into Gao's medium No. 1 and cultured at 28℃ for 72 hours until the spore concentration was 10 8 CFU / mL; two bacterial solutions were mixed at a volume ratio of 2:1 and rejuvenated at 30°C for 3 h; (3) Preparation of alginate-carboxymethyl cellulose composite gel: Alginic acid and carboxymethyl cellulose were prepared into 1% and 2% aqueous solutions, respectively, at a mass ratio of 3:

1. The pH of the alginate aqueous solution was adjusted to 7.0 with 0.1 M sodium hydroxide, and the viscosity of the carboxymethyl cellulose aqueous solution was controlled to 2000-3000 mPa·s. After mixing the two aqueous solutions, calcium chloride solution was added at a mass ratio of 0.05%-0.1% of the total mass of alginate and carboxymethyl cellulose, and the mixture was ultrasonically treated at a power of 300 W for 5-10 min. (4) Fermentation process: The composite bacterial agent of step (2) is added to the pretreated material of step (1), mixed evenly, and then stirred with the composite gel of step (3) at a mass ratio of 9:1, and placed in a tank-type turning fermenter for three-stage fermentation: a heating period of 3 days (the temperature is raised from room temperature to 55°C, and the ventilation volume is 0.5-0.8m per cubic meter); 3 / h, turning the pile once a day) → high temperature period of 4 days (temperature 55-65℃, ventilation volume of 1.0-1.2m3 per cubic meter 3 / h, turning the compost once every 12 hours, controlling the pH at 7.5-8.0) → cooling period of 5 days (temperature dropped to 30-35℃, ventilation rate of 0.3-0.5m3 / m3 3 / h, stop turning the compost); (5) Molding and coating: The fermented material was made into granules with a diameter of 3-5 mm by a disc granulator (rotation speed 30 r / min); starch and alginic acid were prepared into an 8% aqueous solution at a mass ratio of 2:1, 0.1% glycerol was added as a plasticizer, and the granules were coated by a fluidized bed coater (inlet air temperature 45°C, outlet air temperature 30°C, spray volume 5 mL / min), dried for 30 min until the moisture content was ≤12%, and the coating thickness was controlled at 50-100 μm.

2. A bio-organic fertilizer for enhancing plant disease resistance as claimed in claim 1, characterized in that: The brown algae residue in step (1) is kelp processing waste, and the cellulose degradation rate of corn straw after soaking in sodium hydroxide is ≥40%.

3. A bio-organic fertilizer for enhancing plant disease resistance as claimed in claim 1, characterized in that: In step (2), the Bacillus subtilis is ATCC 6633 strain, the Streptomyces is ATCC 10137 strain, and the viable bacterial survival rate of the composite bacterial agent is ≥90%.

4. A bio-organic fertilizer for enhancing plant disease resistance as claimed in claim 1, characterized in that: The concentration of the calcium chloride solution in step (3) is 0.5%, the dropping rate is 1 mL / min, and the gel network pore size is 50-100 μm.

5. A bio-organic fertilizer for enhancing plant disease resistance as claimed in claim 1, characterized in that: The cellulose degradation rate of the high temperature period material in step (4) is ≥40%, and the pathogen killing rate is ≥99%.

6. A bio-organic fertilizer for enhancing plant disease resistance as claimed in claim 1, characterized in that: The compressive strength of the granules produced by the disc granulator in step (5) is ≥20N, and the live bacteria embedding rate of the fluidized bed coating machine is ≥85%.

7. A bio-organic fertilizer for enhancing plant disease resistance as claimed in claim 1, characterized in that: The number of effective live bacteria in the finished product is ≥3.5×10 8 CFU / g, organic matter content ≥48%, pH 5.5-8.

5.

8. A bio-organic fertilizer for enhancing plant disease resistance as claimed in claim 1, characterized in that: The sustained-release period of the active groups in the composite gel is 30-35 days, and the bacterial agent survival rate of the coated particles after being stored at 4° C. for 6 months is ≥70%.

9. A bio-organic fertilizer for enhancing plant disease resistance as claimed in claim 1, characterized in that: The addition amount of the sodium lignin sulfonate in step (1) is 0.5%, and the sulfonic acid group content thereof is ≥98%.

10. The bio-organic fertilizer for enhancing plant disease resistance according to claim 1, wherein: The heavy metal lead content in the finished product is ≤35mg / kg, the arsenic content is ≤15mg / kg, and the cadmium content is ≤3mg / kg.

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