Chitosan-oligosaccharide bio-fertilizer and preparation method thereof

Abstract

The application relates to the technical field of agricultural fertilizers, and provides an amino sugar-oligosaccharide biofertilizer and a preparation method thereof. The amino sugar-oligosaccharide biofertilizer comprises 3-4 parts of amino sugar, 12-20 parts of seaweed oligosaccharide preparation liquid, 0.5-1 part of bacillus subtilis, 1-2 parts of trichoderma viride, 1-2 parts of aspergillus niger and 70-83 parts of base material. The seaweed oligosaccharide preparation liquid contained in the amino sugar-oligosaccharide biofertilizer is prepared by an acid enzymolysis-alkaline enzymolysis-biological fermentation three-step method, the content of seaweed oligosaccharide with a stable polymerization degree of 2-5 is high, the content of salicylic acid in crops can be effectively improved, the immune induction effect is obvious, the biocontrol and growth promotion effects of amino sugar and microorganisms are combined, the defense ability of crops against diseases is improved, and especially the growth and reproduction of pathogenic bacteria Pseudomonas syringae pv. tomato can be effectively inhibited, and the occurrence rate of bacterial spot disease of tomatoes can be reduced.

Description

Technical Field

[0001] This invention relates to the field of agricultural fertilizer technology, and in particular to an glucosamine-oligosaccharide bio-fertilizer for the prevention and control of bacterial spot disease in tomatoes and its preparation method. Background Technology

[0002] Glucosamine is widely found in the cell walls of fungi and the exoskeletons of shrimp and crabs. It is a component of chitin and chitosan, possessing extremely high natural activity and rapid translocation. It can be directly transported or absorbed within crops, promoting the absorption and utilization of macro-, meso-, and micronutrients, and enriching cellular nutrition. Currently, research on agricultural inputs using glucosamine-enhanced technology on crops is still relatively limited.

[0003] The specific effects of glucosamine on crop growth are as follows: (1) Glucosamine is ionized in the plant body and directly acts on the synthesis of polysaccharide proteins, thereby polymerizing cellulose, accelerating the division of xylem and phloem cells, and promoting the growth of plant stems; (2) Glucosamine has natural immune function. In the process of energy conversion, it complexes with the organic nutrients of the plant body to synthesize a variety of plant antibiotics, accelerates the detoxification of toxins and decomposes its own organic matter to release growth energy, supplies the required end-nutrient absorption, and enhances the ability to resist disease and stress; (3) Glucosamine can promote plant metabolism, strengthen photosynthesis, promote the accumulation of dry matter, effectively stimulate the growth of new tissues, repair wounds and have strong healing ability, regulate the balance between reproductive growth and vegetative growth, and delay plant aging.

[0004] Alginate is one of the sugars that has been proven to activate the plant immune system. It is currently mainly produced by one-step enzymatic hydrolysis or chemical hydrolysis. However, the structure and degree of polymerization are different. They are mostly alginic acid, sodium alginate or fucoidan. The yield of alginate is not high, especially the yield of alginate with a specific degree of polymerization (2-5). Summary of the Invention

[0005] This invention addresses the problem of bacterial spot disease in tomatoes. Through research and analysis, it utilizes functional substances such as glucosamine, oligosaccharides, and microorganisms that can prevent and control the disease. It also innovates the preparation process of seaweed oligosaccharides, determines specific raw materials and ratios, and ensures the long-lasting, uniform, and stable effects of the product.

[0006] In a first aspect, the present invention provides a seaweed oligosaccharide preparation solution for the prevention and control of bacterial spot disease in tomatoes. The preparation method of the seaweed oligosaccharide preparation solution includes: enzymatically hydrolyzing kelp with acidic cellulase, acidic pectinase, amylase and xylanase to obtain an acidic enzymatic hydrolysate; enzymatically hydrolyzing the acidic enzymatic hydrolysate with alkaline cellulase and alkaline protease to obtain a compound enzymatic hydrolysate; using Stenotrophomonas maltophilia as the fermentation strain and a culture medium containing the compound enzymatic hydrolysate as the fermentation medium for fermentation, with an inoculum size of 5%-10%; the fermentation temperature is 30-37℃ and the fermentation time is 22-26h to obtain the seaweed oligosaccharide preparation solution.

[0007] In preparing the seaweed oligosaccharide preparation solution, the proportions of acidic cellulase, acidic pectinase, amylase, and xylanase in the dry weight of kelp are 1.20-1.25% and 0.20-0.25% respectively; the proportions of alkaline cellulase and alkaline protease are 0.02-0.03% and 0.20-0.25% respectively.

[0008] More specifically, the method for preparing the seaweed oligosaccharide preparation solution provided by the present invention includes:

[0009] (1) Take dried kelp, cut and soak it, then wash and remove impurities. Add water at a ratio of 1:20. Control the temperature at 51-56℃ and adjust the pH to 4.8-5.2.

[0010] (2) Add acidic cellulase at a ratio of 1.20-1.25% of the dry weight of kelp, acidic pectinase at a ratio of 0.20%-0.25%, amylase at a ratio of 0.02-0.03%, and xylanase at a ratio of 0.20%-0.25%, stir; keep the temperature at 51-56℃ and enzymatically hydrolyze for 12-16 hours to obtain acidic enzymatic hydrolysate for later use;

[0011] (3) Adjust the pH of the acidic enzymatic hydrolysate to 8.0-8.5, add alkaline cellulase and alkaline protease at a ratio of 0.20-0.25% of the dry weight of kelp, and stir; the enzymatic hydrolysis temperature is 50-55℃, and the enzymatic hydrolysis is carried out for 1.0-1.5h to obtain a compound enzymatic hydrolysate for later use;

[0012] (4) Inoculate the activated Stenotrophomonas maltophilia into the fermentation culture medium, which contains yeast extract, sodium chloride and the compound enzymatic hydrolysate obtained in step (3); the inoculation amount is 5%-10%;

[0013] (5) Fermentation temperature 30-37℃, rotation speed 180-220 r / min, time 22-26h, to obtain fermentation broth;

[0014] (6) Heat the fermentation broth to 50-55℃, stir, and maintain for 8-12 hours to obtain the seaweed oligosaccharide preparation solution.

[0015] This invention discovers that microbial fermentation based on acid-base two-step enzymatic hydrolysis yields high yields of seaweed oligosaccharides. Furthermore, raising the fermentation broth to 50-55°C and maintaining it for 8-12 hours can significantly improve the yield of seaweed oligosaccharides.

[0016] Furthermore, the seaweed oligosaccharides obtained by this invention have a stable degree of polymerization (2-5), and the immune induction effect of seaweed oligosaccharides with this degree of polymerization has been proven to be superior to that of the enzymatic hydrolysis products.

[0017] Using the preparation method of the present invention, the seaweed oligosaccharide preparation solution obtained has a seaweed oligosaccharide content of more than 8.5% and a degree of polymerization of seaweed oligosaccharides of 2-5.

[0018] Secondly, the present invention provides an glucosamine-oligosaccharide bio-fertilizer, comprising the above-mentioned seaweed oligosaccharide preparation solution, and also including glucosamine, Bacillus subtilis, Trichoderma viride, Aspergillus niger and basic materials.

[0019] The glucosamine-oligosaccharide bio-fertilizer provided by this invention comprises the following components in parts by weight: 3-4 parts glucosamine, 12-20 parts seaweed oligosaccharide preparation solution, 0.5-1 part Bacillus subtilis, 1-2 parts Trichoderma viride, 1-2 parts Aspergillus niger, and 70-83 parts basic materials.

[0020] In the glucosamine-oligosaccharide bio-fertilizer provided by the present invention, the pH range of the basic material is 6.0-6.5, and the moisture content is not higher than 5.0%; the basic material contains 40-45 parts corn residue, 15-20 parts fish bone meal, and 15-20 parts mushroom residue.

[0021] In the glucosamine-oligosaccharide bio-fertilizer provided by the present invention, the glucosamine is a mixture of D-glucosamine and glucosamine hydrochloride, the content of D-glucosamine or glucosamine hydrochloride is above 80g / L, and the pH is 6.0-7.0.

[0022] Thirdly, the present invention provides a method for preparing the above-mentioned glucosamine-oligosaccharide bio-fertilizer, comprising the following steps:

[0023] (1) Mix the basic materials according to 40-45 parts corn residue, 15-20 parts fish bone powder and 15-20 parts mushroom residue, stir evenly, control the pH value of the basic material mixture between 6.0 and 6.5, and control the moisture content below 5.0%;

[0024] (2) Add 0.5-1 part of Bacillus subtilis, 1-2 parts of Trichoderma viride, and 1-2 parts of Aspergillus niger to the basic material mixture, stir evenly, and obtain the mixture;

[0025] (3) Spray 12-20 parts of seaweed oligosaccharide preparation solution and 3-4 parts of glucosamine solution into the mixture obtained in step (2), stir evenly, and obtain glucosamine-oligosaccharide bio-fertilizer.

[0026] Fourthly, the present invention provides the application of the above-mentioned seaweed oligosaccharide preparation solution or the above-mentioned glucosamine-oligosaccharide bio-fertilizer in the prevention and control of bacterial spot disease in tomatoes and / or the promotion of plant root development.

[0027] The beneficial effects of this invention are at least as follows:

[0028] This invention enhances crop immunity and disease resistance through the combined action of glucosamine and oligosaccharides. It adds beneficial microorganisms that can antagonize the pathogen of bacterial spot disease in tomatoes and scientifically formulates the basic materials to ensure suitable pH, carbon-nitrogen ratio, and moisture ratio. This creates a material environment conducive to the survival and reproduction of microorganisms and the absorption of glucosamine and oligosaccharides, amplifying the effects of each functional adjuvant and truly achieving the goal of disease prevention and control.

[0029] The scientific nature of the material combination in this invention is reflected in:

[0030] (1) A reasonable combination of organic materials and nitrogen can provide a suitable carbon-nitrogen ratio (around 25:1), pH, and moisture environment for microorganisms to decompose organic materials and grow and reproduce. This basic environment can ensure the effective reproduction of beneficial microorganisms in the soil micro-zone after fertilizer application.

[0031] (2) Glucosamine and oligosaccharides can be absorbed directly or decomposed in a suitable slightly acidic environment. The pH range of the materials in this invention is 6.0-6.5, which is conducive to the performance of glucosamine-oligosaccharide.

[0032] (3) All three microorganisms have strong antagonistic ability against the pathogenic strain of Pseudomonas syringae in tomatoes. At the same time, Aspergillus niger can produce amylase, which is beneficial to the continuous decomposition and utilization of organic materials. Trichoderma viride has strong cellulose decomposition ability, and Bacillus subtilis has strong competitive and broad-spectrum antibacterial properties. There is no antagonism between the three, and they can work together to exert their effects and effectively prevent the growth of pathogens.

[0033] (4) Oligosaccharides and glucosamine are both small molecule polymers that are easily absorbed by crops. Combined with the competitive and bacteriolytic effects of microorganisms on pathogens, the preventive and therapeutic effects of diseases are taken into account, and the disease prevention and growth promotion effects of crops are taken into account, resulting in synergistic effects. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0035] Figure 1 This is a flowchart illustrating the preparation process of the glucosamine-oligosaccharide bio-fertilizer of the present invention.

[0036] Figure 2 This is a diagram of a potted tomato experiment according to the present invention. Figure 2 The top left is processing group 1; Figure 2 The upper right corner represents processing group 2; Figure 2 The bottom left is processing group 3; Figure 2 The bottom right is processing group 4.

[0037] Figure 3 This invention relates to a potted rapeseed growth-promoting experiment. Figure 3 The left side shows the treatment group using commercially available fertilizer products; the middle side shows the treatment group from Example 3; and the right side shows the control group using clean water.

[0038] Figure 4 This is a diagram illustrating the root development experiment of potted rapeseed according to the present invention. Figure 4 The left side shows the treatment group using commercially available fertilizer products; the middle side shows the treatment group using Example 3; and the right side shows the control group using clean water. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0040] The glucosamine described in this invention was purchased from Shandong Runde Biotechnology Co., Ltd. Technical specifications: liquid, density 1.2 g / cm³. -3 The glucosamine content is 85 g / L, glucosamine is 88 g / L, and ammonium sulfate is 45 g / L.

[0041] The seaweed oligosaccharide preparation solution described in this invention is prepared in-house using an enzymatic hydrolysis-microbial fermentation method. The enzymes and microorganisms used were provided by Qilu University of Technology; the fermentation materials were purchased from Yantai Development Zone Huada Medical Device Co., Ltd.

[0042] The mushroom residue, corn residue, and fish bone meal used in the basic materials of this invention were purchased from Yantai Taoyang Agricultural Development Co., Ltd. The mushroom residue contained 45% organic matter, the corn residue 42% organic matter, and the fish bone meal 80% organic matter. The organic matter content was tested according to the standard NY / T 525-2021 "Organic Fertilizer".

[0043] The Bacillus subtilis, Trichoderma viride, and Aspergillus niger described in this invention were purchased from Qingdao Weilan Biotechnology. The technical specifications are: Bacillus subtilis effective viable count ≥100 billion / g, Trichoderma viride effective viable count ≥10 billion / g, and Aspergillus niger effective viable count ≥10 billion / g.

[0044] Example 1: Method for preparing seaweed oligosaccharides in glucosamine-oligosaccharide biofertilizer

[0045] This embodiment provides a method for preparing seaweed oligosaccharides in glucosamine-oligosaccharide biofertilizer, including the following steps:

[0046] Acidic enzymatic hydrolysis steps: Take 30kg of dried kelp and cut it into small pieces with a diameter of 2-5cm; soak the cut dried kelp, wash and remove impurities, put it into a reaction vessel, and add water at a material-to-water ratio of 1:20; control the reaction vessel temperature at a constant 55℃ and adjust the pH to 5.0; add 375g of acidic cellulase, 75g of acidic pectinase, 9g of amylase, and 75g of xylanase, and stir evenly; hydrolyze at a constant temperature for 12 hours; the acidic enzymatic hydrolysate is ready for use.

[0047] Alkaline enzymatic hydrolysis steps: Adjust the pH of the acidic enzymatic hydrolysate to 8.0, add 70g of alkaline cellulase and 70g of alkaline protease, and stir well; keep the temperature at 55℃ and enzymatically hydrolyze for 1 hour; the compound enzymatic hydrolysate is ready for use.

[0048] Biological fermentation steps:

[0049] (1) Prepare a culture medium for activating the bacterial strain (sodium alginate 10g / L, yeast extract 5g / L, sodium chloride 5g / L, glucose 2.5g / L), sterilize at 121℃ for 30min; inoculate Stenotrophomonas maltophilia into the culture medium under aseptic conditions, and activate and culture at 37℃ and 180r / min for 18h.

[0050] (2) Prepare fermentation culture medium (300g yeast extract, 200g NaCl, 65L seaweed compound enzyme hydrolysate) in a fermenter (100L, 2 / 3 of the liquid volume), with an initial pH of 6.5; inoculate the activated bacteria into the fermentation culture medium, with an inoculation volume of 5L;

[0051] (3) Fermentation temperature 37℃, rotation speed 200 r / min, fermentation time 24h; after fermentation, the fermentation liquid was heated to 51℃, stirred, and maintained for 10h. Seaweed oligosaccharide fermentation liquid (also known as seaweed oligosaccharide preparation liquid) was obtained. The seaweed oligosaccharide content was tested to be 8.9% (commissioned to Qilu University of Technology for testing).

[0052] Example 2: Glucosamine-Oligosaccharide Biofertilizer

[0053] This embodiment provides a method for preparing glucosamine-oligosaccharide bio-fertilizer. The preparation flowchart is shown below. Figure 1 The steps are as follows:

[0054] (1) Mix the basic materials according to the ratio of 43 parts corn residue, 16 parts fish bone powder and 18 parts mushroom residue, stir evenly, the pH value is 6.4 and the moisture content is 4.4% to obtain the following:

[0055] (2) Add 1 part of Bacillus subtilis, 1.5 parts of Trichoderma viride and 1.5 parts of Aspergillus niger to the mixture and stir well;

[0056] (3) Spray 4 parts of glucosamine and 15 parts of seaweed oligosaccharide preparation liquid (seaweed oligosaccharide fermentation liquid obtained in Example 1) into the mixture, stir evenly, and obtain glucosamine-oligosaccharide bio-fertilizer.

[0057] Example 3: Glucosamine-Oligosaccharide Biofertilizer

[0058] This embodiment provides a method for preparing glucosamine-oligosaccharide bio-fertilizer, the steps of which are as follows:

[0059] (1) Mix the basic materials according to the ratio of 45 parts corn residue, 19 parts fish bone meal, and 18 parts mushroom residue, stir evenly, with a pH value of 6.5 and a moisture content of 4.1%;

[0060] (2) Add 0.5 parts of Bacillus subtilis, 0.75 parts of Trichoderma viride, and 0.75 parts of Aspergillus niger to the mixture and stir well;

[0061] (3) Spray 4 parts of glucosamine and 12 parts of seaweed oligosaccharide preparation solution (seaweed oligosaccharide fermentation broth obtained in Example 1) into the mixture and stir evenly.

[0062] Example 4

[0063] This embodiment provides a method for preparing glucosamine-oligosaccharide bio-fertilizer, the steps of which are as follows:

[0064] (1) Mix the basic materials according to the ratio of 40 parts corn residue, 16 parts fish bone meal, 15 parts mushroom residue and 1 part ammonium sulfate, stir evenly, the pH value is 6.1 and the moisture content is 3.9%;

[0065] (2) Add 1 part of Bacillus subtilis, 2 parts of Trichoderma viride, and 2 parts of Aspergillus niger to the mixture and stir well;

[0066] (3) Spray 4 parts of glucosamine and 20 parts of seaweed oligosaccharide preparation solution (seaweed oligosaccharide fermentation broth obtained in Example 1) into the mixture and stir evenly.

[0067] Experimental Example 1

[0068] Experimental subject: Field tomatoes

[0069] Experimental fertilizer:

[0070] (1) Control group: No fertilization treatment, but pathogen inoculation treatment.

[0071] (2) Ordinary biological organic fertilizer (produced by Shandong Yisheng Biological Fertilizer Technology Co., Ltd., technical indicators: organic matter ≥45%, nitrogen, phosphorus and potassium ≥5%, effective live bacteria ≥0.2 billion / g, strains are Bacillus, yeast and lactic acid bacteria);

[0072] (3) The mixture of basic materials in Example 2;

[0073] (4) The composite enzymatic hydrolysate obtained in Example 1;

[0074] (5) The seaweed oligosaccharide preparation solution obtained in Example 1;

[0075] (6) Bacillus subtilis, Trichoderma viride and Aspergillus niger in Example 2.

[0076] (7) The glucosamine-oligosaccharide bio-fertilizer obtained in Example 2;

[0077] Experimental Methods: Tomato seedlings at the 6-7 leaf stage were selected. This experiment included 7 treatment groups, with 10 seedlings in each group. Each treatment group was isolated to prevent cross-infection between different groups. The pathogen (*Pseudomonas syringae* DC3000, a pathogenic tomato strain provided by Qilu University of Technology) was inoculated as follows: 50 μL of the pathogen suspension (concentration: 0.002 μg / mL) was injected into the stem using a sterile 1.5 ml syringe. Gentle squeezing was applied during injection to avoid damaging the tomato stem.

[0078] The processing for each group is as follows:

[0079] (1) Treatment group 1: Control. Pathogen inoculation and watering were performed. The timing was the same as other treatment groups.

[0080] (2) Treatment group 2: Ordinary biological organic fertilizer. The amount of organic fertilizer applied was 10g / plant; the application method was to bury it shallowly near the roots of the plant and then water it. One day after treatment, the plant was inoculated with pathogens.

[0081] (3) Treatment group 3: The basic material mixture in Example 2. The basic material mixture was applied at a rate of 10g per plant. The application method was to bury the mixture near the roots of the plant and then water it. One day after treatment, the plant was inoculated with pathogens.

[0082] (4) Treatment group 4: The compound enzymatic hydrolysis preparation solution of Example 1. The root zone was completely irrigated with the diluted compound enzymatic hydrolysis preparation solution (1.5 mL of seaweed enzymatic hydrolysis solution was added to 100 mL of water). One day after treatment, the roots were inoculated with pathogens.

[0083] (5) Treatment Group 5: Alginate preparation solution from Example 1. The roots were drenched with a diluted alginate solution (1.5 mL of alginate preparation solution was added to 100 mL of water). One day after treatment, the roots were inoculated with pathogens.

[0084] (6) Treatment Group 6: Bacillus subtilis, Trichoderma viride, and Aspergillus niger from Example 2. Dosage: 0.1g of Bacillus subtilis, 0.15g of Trichoderma viride, and 0.15g of Aspergillus niger; Application method: Weigh 1.0g, 1.5g, and 1.5g of each of the three bacteria, add 1000mL of water, stir well, and then measure 100mL for root irrigation. One day after treatment, inoculate with pathogens.

[0085] (7) Treatment Group 7: Glucosamine-Oligosaccharide Bio-fertilizer as described in Example 2. Glucosamine-Oligosaccharide bio-fertilizer was applied at a rate of 10g per plant. The application method was to bury the fertilizer near the roots of the plant and then water it. One day after treatment, the plant was inoculated with pathogens.

[0086] For all the above treatments, the amount of water applied each time was the same, and the disease status of the plants was observed every two days (the day of inoculation with the pathogen was recorded as day 1). Other field management measures were the same.

[0087] Incidence rate = (Number of diseased plants / Number of experimental plants) × 100%.

[0088] The experimental results are shown in Table 1:

[0089] Table 1. Control efficacy test of bacterial spot disease in field tomatoes

[0090] Days Processing Group 1 Processing Group 2 Processing Group 3 Processing Group 4 Processing Group 5 Processing Group 6 Processing Group 7 Incidence on day 2 10 0 10 0 0 0 0 Incidence rate on day 4 40 10 30 10 0 10 0 Incidence on day 6 80 30 60 30 10 30 0 Incidence rate on day 8 100 50 60 30 20 30 10 Incidence rate on day 10 100 60 80 40 20 40 10 Incidence rate on day 12 100 60 80 40 30 40 10

[0091] Treatments 1, 2, and 3 showed symptoms starting 2 days after inoculation with the pathogen. The rate of disease progression was rapid between 2 and 8 days, and slowed down after 8 days. The incidence rates during the observation period were 100%, 60%, and 80%, respectively. Treatments 4, 5, and 6 showed lower incidence rates. The seaweed oligosaccharide treatment group (group 5) was superior to the enzymatic hydrolysis treatment group and the microbial treatment group (group 6). Group 7 had the lowest incidence rate.

[0092] The above results indicate that plants without disease prevention treatment have no resistance, while plants treated with fertilizer or adjuvants showed varying degrees of improvement in disease incidence. Commercially available products (group 2) contain beneficial bacteria and exhibit stronger disease resistance than the group treated with ordinary organic materials (group 3). The seaweed oligosaccharide treatment group (group 5) showed superior disease prevention effects compared to the seaweed enzymatic hydrolysate treatment group (group 4) and the microbial treatment group; the product of this invention treatment group (group 7) showed the best disease resistance, with an incidence rate of only 10%.

[0093] Conclusion: Basic materials alone have virtually no disease-preventing effect; the microorganisms selected in this invention exhibit superior disease resistance; seaweed oligosaccharides have a significant disease-preventing effect, more pronounced than seaweed enzymatic hydrolysate; the patented fertilizer, formulated with seaweed oligosaccharides, basic materials, and microorganisms, demonstrates stronger resistance than a single seaweed oligosaccharide, basic material, or compound microbial strain, with the combined effect being superior to that of a single material. This indicates that the optimal disease-preventing effect comes from the organic combination of basic materials, seaweed oligosaccharides, glucosamine, and microorganisms.

[0094] Experimental Example 2

[0095] Experimental subject: potted tomatoes

[0096] Experimental fertilizer:

[0097] (1) Blank control group: No fertilization treatment or pathogen inoculation treatment was performed.

[0098] (2) Disease control group: No fertilization treatment, but pathogen inoculation treatment.

[0099] (3) Preparation solution of seaweed oligosaccharides in Example 1;

[0100] (4) Example 4: Glucosamine-Oligosaccharide Bio-fertilizer.

[0101] Experimental method: Tomato seedlings at the 6-7 leaf stage were selected. This experiment was divided into 4 treatment groups, with 15 seedlings in each group. The pathogen (Pseudomonas syringae, a pathogenic tomato strain DC3000 provided by Qilu University of Technology) was inoculated by spraying the pathogen suspension (concentration: 0.002 μg / mL) onto the leaf surface using a spray bottle.

[0102] The processing for each group is as follows:

[0103] (1) Treatment group 1: blank control. Watering treatment only. Time is the same as other treatment groups.

[0104] (2) Treatment group 2: Disease control. Inoculated with pathogens. The inoculation time was the same as other treatment groups.

[0105] (3) Treatment Group 3: Alginate preparation solution from Example 1. The roots were irrigated with a diluted alginate solution (1.5 mL of alginate preparation solution was added to 100 mL of water). One day after treatment, the roots were inoculated with pathogens.

[0106] (4) Treatment Group 4: Glucosamine-Oligosaccharide Bio-fertilizer as described in Example 4. Glucosamine-Oligosaccharide bio-fertilizer was applied at a rate of 10g per plant. The application method was to bury the fertilizer shallowly near the roots of the plant and then water it. One day after treatment, the plant was inoculated with pathogens.

[0107] For all the above treatments, the amount of water applied each time was the same, and the disease status of the plants was observed every two days (the day of inoculation with the pathogen was recorded as day 1). Other management measures were the same.

[0108] Incidence rate = (Number of diseased plants / Number of experimental plants) × 100%.

[0109] The experimental results are shown in Table 2, and the experimental diagrams of potted tomatoes are shown in [the table]. Figure 2 , Figure 2 The top left is processing group 1; Figure 2 The upper right corner represents processing group 2; Figure 2 The bottom left is processing group 3; Figure 2 The bottom right is processing group 4.

[0110] Table 2. Control efficacy test of bacterial spot disease in potted tomatoes

[0111] Incidence rate (%) Processing Group 1 Processing Group 2 Processing Group 3 Processing Group 4 Day 2 0 26.6 0 0 Day 4 6.7 53.3 0 0 Day 6 20 66.7 6.7 0 Day 8 20 80.0 6.7 0

[0112] In treatment group 1 (blank control), 3 plants developed the disease during the experimental period, with an incidence rate of 20.0%; in treatment group 2, no control measures were taken after inoculation with the pathogen, and the incidence rate was 80.0%; treatment groups 3 and 4 showed significant control effects on the disease, and no disease was observed in the treatment group treated with the product of this invention.

[0113] The above experimental results show that both the seaweed oligosaccharide prepared in this invention and the fertilizer treatment group of this invention have good control effects on diseases. The control effect of the fertilizer treatment group of this invention is better than that of the seaweed oligosaccharide treatment group alone.

[0114] Experimental Example 3

[0115] Experimental subject: Potted rapeseed (growth promotion experiment)

[0116] Experimental method: After germinating the rapeseed seeds, disperse them in flower pots with soil, cover them evenly with a layer of fine soil, place them in an environment with a room temperature of 20℃, and after the seedlings emerge, thin them out, leaving 7 seedlings in each pot, and then apply fertilizer.

[0117] Fertilizer treatments were as follows: (1) Control group: watered with clean water, no fertilizer was applied; (2) Control group 1 (patent application number: 202111243933.X) treatment group (the patent product used in control group 1 was diluted 600 times and 100 mL was applied to the roots); (3) This Example 3 treatment group: 10 g of the product of Example 3 of this invention was weighed, 100 mL of clean water was added, and after stirring evenly, it was poured into the flower pot.

[0118] Root irrigation was performed every 4 days. After 10 days of cultivation, the development of leaves and roots was observed. Leaf results are shown in […]. Figure 3 Root development results are shown in Figure 4 .

[0119] The above experimental results show that the rapeseed in control group 1 and Example 3 treatments grew significantly better than the blank control. The rapeseed in Example 3 treatment was the best, with large leaves, tall plants, long roots, many fibrous roots, and obvious growth-promoting effect.

[0120] Comparative Example 1: Investigation of the preparation method used for seaweed oligosaccharide preparation solution

[0121] 1. Different proportions of acidic cellulase, acidic pectinase, amylase, and xylanase used.

[0122] Experiments and conclusions on acidic enzymatic hydrolysis conditions:

[0123] (1) Determination of the dosage of acidic enzyme

[0124] Single-factor experiments were conducted using acid cellulase at concentrations of 0, 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, and 1.70%; acid pectinase at concentrations of 0, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, and 0.40%; amylase at concentrations of 0, 0.01, 0.02, 0.03, 0.04, 0.05, and 0.06%; and xylanase at concentrations of 0, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, and 0.40%. As the amount of enzyme added increased, the extraction yield of alginic acid gradually increased. When the amounts of acidic cellulase, acidic pectinase, amylase, and xylanase added reached 1.25%, 0.25%, 0.03%, and 0.25%, respectively, the extraction yield of alginic acid no longer increased.

[0125] (2) Enzymatic hydrolysis temperature: As the enzymatic hydrolysis temperature increases (45℃-55℃), the extraction yield of alginate increases rapidly (3.5%-5.0%). When the enzymatic hydrolysis temperature reaches 55℃, the extraction yield of alginate no longer increases. The suitable temperature for acidic compound enzymatic hydrolysis is between 51-55℃.

[0126] (3) Enzymatic hydrolysis pH: As the enzymatic hydrolysis pH increases (4.3-5.2), the extraction yield of alginic acid rises rapidly (3.5%-4.7%). When the enzymatic hydrolysis pH reaches 5.2, the extraction yield of alginic acid decreases rapidly. The suitable pH for acidic compound enzymatic hydrolysis is between 4.8 and 5.2.

[0127] (4) Enzymatic hydrolysis time: With the extension of enzymatic hydrolysis time (8h-16h), the extraction yield of alginic acid increases rapidly (3.1%-4.7%). After 16h of enzymatic hydrolysis, the extraction yield of alginic acid no longer increases. The suitable time for acidic compound enzymatic hydrolysis is between 12-20h.

[0128] The basic acidic complex enzymatic hydrolysis system consists of: acidic cellulase 1.0-1.25%, acidic pectinase 0.20-0.25%, amylase 0.02-0.03%, xylanase 0.20-0.25%, hydrolysis temperature 51-55℃, hydrolysis pH 4.8-5.2, and hydrolysis time 12-20h.

[0129] 2. Conditions and conclusions regarding alkaline enzymatic hydrolysis:

[0130] (1) Single-factor experiments were conducted with alkaline cellulase additions of 0, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, and 0.40%; single-factor experiments were conducted with alkaline pectinase additions of 0, 0.01, 0.02, 0.03, 0.04, 0.05, and 0.06%; and single-factor experiments were conducted with alkaline protease additions of 0, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, and 0.40%.

[0131] With increasing addition of alkaline pectinase, the extraction yield of alginic acid did not change significantly; therefore, alkaline pectinase was not chosen as a component of the alkaline complex enzyme system for kelp extraction. Both alkaline cellulase and alkaline protease increased the yield of alginic acid with increasing addition, but when the addition amount reached 0.25%, the yield of alginic acid no longer increased.

[0132] (2) Enzymatic hydrolysis temperature: As the enzymatic hydrolysis temperature increases (45℃-53℃), the extraction yield of alginic acid increases rapidly (5.7%-7.9%). When the enzymatic hydrolysis temperature reaches 53℃, the extraction yield of alginic acid no longer increases. The suitable temperature for alkaline compound enzymatic hydrolysis is between 50-55℃.

[0133] (3) Enzymatic hydrolysis pH: As the enzymatic hydrolysis pH increases (7.5-9.0), the extraction yield of alginic acid rises rapidly (5.7%-7.9%). When the enzymatic hydrolysis pH reaches 8.5, the extraction yield of alginic acid begins to decline rapidly. The suitable pH for alkaline compound enzymatic hydrolysis is between 8.0 and 8.5.

[0134] (4) Enzymatic hydrolysis time: With the extension of enzymatic hydrolysis time (0.5h-1.5h), the extraction yield of alginic acid increased rapidly (6.8%-8.9%). After 1.5h of enzymatic hydrolysis, the extraction yield of alginic acid no longer increased. The suitable time for alkaline compound enzymatic hydrolysis is between 1.0-1.5h.

[0135] Basic alkaline enzymatic hydrolysis system: alkaline cellulase 0.20-0.30%, alkaline protease 0.20-0.25%, hydrolysis temperature 50-55℃, hydrolysis pH 8.0-8.5, hydrolysis time 1.0-1.5h.

[0136] After enzymatic hydrolysis using the existing technology (patent application number: 201610061793.7), the yield of alginate was 7.2%. Another existing technology (Jin Yijing, Li Lan, Liu Qiuping, et al. Optimization of process for extracting sodium alginate from kelp by ultrasonic-compound enzymatic hydrolysis [J]. Food Industry Technology, 2020(42):132-137.) yielded a slightly higher alginate yield of 11.5%. The purpose of these two existing technologies is to improve the yield of sodium alginate.

[0137] The purpose of this invention is to improve the yield of alginic acid. After combining acidic and alkaline enzymatic hydrolysis, the yield of alginic acid is 18.9-23.5%.

[0138] 3. Determination of the fermentation strains used and the fermentation endpoint.

[0139] The fermentation strain of this invention was used to track and record the changes in enzyme activity over a 48-hour fermentation cycle. It was found that when the fermentation reached 24 hours, the alginate lyase activity had reached a relatively stable peak (18.5 μ / mL); therefore, fermentation was terminated at 24 hours.

[0140] Meanwhile, the fermentation strains Bacillus licheniformis, Saccharomyces cerevisiae, and Trichoderma viride, as described in patent application number 202111243933.X, were used as fermentation strains. However, the fermentation cycle of the patented strain is longer, and the main fermentation product is alginic acid, not alginic oligosaccharides. This differs from the purpose of this invention.

[0141] Furthermore, under the same fermentation conditions as this invention, the fermentation strains Bacillus licheniformis, Saccharomyces cerevisiae, and Trichoderma viride of patent application number 202111243933.X showed a maximum alginate lyase activity of only 6.4 μ / mL within a 36-hour fermentation cycle.

[0142] 4. An investigation into whether the temperature should be raised after fermentation is complete.

[0143] After fermentation, a constant temperature of 51°C was maintained to allow the alginate lyase to function more effectively, breaking down alginate into seaweed polysaccharides and increasing the yield of seaweed oligosaccharides. After the heating operation, the yield of seaweed oligosaccharides was 8.9%. The difference between this comparative treatment group 5 and Example 1 is that, during the fermentation stage, no temperature maintenance was performed after fermentation; the results showed that the yield of seaweed oligosaccharides was only 5.8%.

[0144] Comparative Example 2: Microbial Activity Detection

[0145] The difference between this comparative treatment group 1 and Example 2 is that the seaweed oligosaccharide preparation solution is omitted, and only glucosamine, basic materials, Bacillus subtilis, Trichoderma viride, and Aspergillus niger are used as bio-fertilizers.

[0146] Compared with the glucosamine-oligosaccharide bio-fertilizer obtained in Example 2, the glucosamine bio-fertilizer in Comparative Treatment Group 1 showed significant differences in microbial activity. The glucosamine-oligosaccharide bio-fertilizer could form a dominant microbial community in the near-root soil in about 4 days. However, the glucosamine bio-fertilizer showed a slight increase in microbial activity within 4 days, but its ability to form a dominant microbial community was weaker.

[0147] Glucosamine-oligosaccharide bio-fertilizers can achieve a disease control effect of up to 90%, while glucosamine bio-fertilizers have a control effect of 60%.

[0148] The difference between this comparative treatment group 2 and Example 2 is that glucosamine is omitted, and only seaweed oligosaccharide preparation solution, basic materials, Bacillus subtilis, Trichoderma viride, and Aspergillus niger are used as bio-fertilizers.

[0149] Compared with the glucosamine-oligosaccharide bio-fertilizer obtained in Example 2, the glucosamine bio-fertilizer in Comparative Treatment Group 2 showed little difference in microbial activity, and both formed dominant microbial communities in the soil near the roots in about 4 days. The disease control effect of glucosamine-oligosaccharide bio-fertilizer reached 90%, while that of oligosaccharide bio-fertilizer was 80%.

[0150] The difference between this comparative treatment group 3 and Example 2 is that the basic materials are omitted, and only seaweed oligosaccharide preparation solution, glucosamine, Bacillus subtilis, Trichoderma viride, and Aspergillus niger are used as bio-fertilizer. The bio-fertilizer obtained in this comparative treatment group 3 has a significant impact on microbial activity during use; without the basic materials, the biological activity rapidly declines within 2 days after application. The glucosamine-oligosaccharide bio-fertilizer achieves a disease control effect of up to 90%, while the bio-fertilizer without basic materials only achieves a control effect of 40%.

[0151] This comparative study also explored the effects of different microbial agents combined with glucosamine and seaweed oligosaccharide preparation solutions as biofertilizers, and the results are recorded in Table 3.

[0152] Table 3. Effects of different microbial inoculants combined with glucosamine and seaweed oligosaccharide preparation solutions as biofertilizers.

[0153] strain name Prevention and control effect Bacillus subtilis, Bacillus licheniformis, yeast 60% Yeast, Bacillus amyloliquefaciens, Trichoderma viride 70% Bacillus polymyxa, Bacillus megaterium, Aspergillus niger 60% gelatinous spore-forming bacteria, Trichoderma viride, Aspergillus niger 70%

[0154] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A seaweed oligosaccharide preparation solution, characterized in that, The preparation method of the seaweed oligosaccharide preparation solution includes: (1) Take dried kelp, cut and soak it, then wash and remove impurities. Add water at a ratio of 1:

20. Control the temperature at 51-56℃ and adjust the pH to 4.8-5.

2. (2) Add acidic cellulase at a ratio of 1.20-1.25% of the dry weight of kelp, acidic pectinase at a ratio of 0.20%-0.25%, amylase at a ratio of 0.02-0.03%, and xylanase at a ratio of 0.20%-0.25%, stir; keep the temperature at 51-56℃ and enzymatically hydrolyze for 12-16 hours to obtain acidic enzymatic hydrolysate for later use; (3) Adjust the pH of the acidic enzymatic hydrolysate to 8.0-8.5, add alkaline cellulase and alkaline protease at a ratio of 0.20-0.25% of the dry weight of kelp, and stir; the enzymatic hydrolysis temperature is 50-55℃, and the enzymatic hydrolysis is carried out for 1.0-1.5h to obtain a compound enzymatic hydrolysate for later use; (4) Inoculate the activated Stenotrophomonas maltophilia into the fermentation culture medium, which contains yeast extract, sodium chloride and the compound enzymatic hydrolysate obtained in step (3); the inoculation amount is 5%-10%; (5) Fermentation temperature 30-37℃, rotation speed 180-220 r / min, time 22-26h, to obtain fermentation broth; (6) Heat the fermentation broth to 50-55℃, stir, and maintain for 8-12 hours to obtain seaweed oligosaccharide preparation solution.

2. The seaweed oligosaccharide preparation solution according to claim 1, characterized in that, The content of seaweed oligosaccharides is above 8.5%, and the degree of polymerization of seaweed oligosaccharides is 2-5.

3. A glucosamine-oligosaccharide bio-fertilizer, characterized in that, The preparation solution containing the seaweed oligosaccharide as described in claim 1 or 2 further includes glucosamine, Bacillus subtilis, Trichoderma viride, Aspergillus niger, and basic materials.

4. The glucosamine-oligosaccharide bio-fertilizer according to claim 3, characterized in that, It includes the following components: 3-4 parts glucosamine, 12-20 parts seaweed oligosaccharide preparation solution, 0.5-1 part Bacillus subtilis, 1-2 parts Trichoderma viride, 1-2 parts Aspergillus niger, and 70-83 parts basic materials.

5. The glucosamine-oligosaccharide bio-fertilizer according to claim 4, characterized in that, The pH range of the base material is 6.0-6.5, and the moisture content is not higher than 5.0%; the base material contains 40-45 parts corn residue, 15-20 parts fish bone meal, and 15-20 parts mushroom residue.

6. The glucosamine-oligosaccharide bio-fertilizer according to claim 5, characterized in that, The glucosamine is a mixture of D-glucosamine and glucosamine hydrochloride, with the content of both D-glucosamine and glucosamine hydrochloride both above 80 g / L, and the pH is 6.0-7.

0.

7. The method for preparing the glucosamine-oligosaccharide bio-fertilizer according to any one of claims 3-6, characterized in that, Includes the following steps: (1) Mix the basic materials according to 40-45 parts corn residue, 15-20 parts fish bone powder and 15-20 parts mushroom residue, stir evenly, control the pH value of the basic material mixture between 6.0 and 6.5, and control the moisture content below 5.0%; (2) Add 0.5-1 part of Bacillus subtilis, 1-2 parts of Trichoderma viride, and 1-2 parts of Aspergillus niger to the basic material mixture, stir evenly, and obtain the mixture; (3) Spray 12-20 parts of seaweed oligosaccharide preparation solution and 3-4 parts of glucosamine solution into the mixture obtained in step (2), stir evenly, and obtain glucosamine-oligosaccharide bio-fertilizer.

8. The application of the seaweed oligosaccharide preparation solution according to claim 1 or 2 or the glucosamine-oligosaccharide bio-fertilizer according to any one of claims 3-6 in the prevention and control of bacterial spot disease in tomatoes and / or the promotion of plant root development.

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