Preparation method of trichoderma particles

Abstract

The invention relates to the technical field of biocontrol microbial agents, and provides a preparation method of trichoderma particles. Comprising the following steps: inoculating and culturing trichoderma in a PDB culture medium, centrifugally collecting trichoderma thalli, embedding the trichoderma by using sodium alginate powder as a wall material, carrying out microencapsulation treatment on the trichoderma to obtain a microencapsulated trichoderma agent, drying the microencapsulated trichoderma agent at low temperature, and mixing and granulating an organic matter base fertilizer and the microencapsulated trichoderma agent to obtain the trichoderma particles. The trichoderma is subjected to microencapsulation treatment, so that the release time can be delayed, the influence on the activity of the trichoderma in the germination process of the coated corn seeds is avoided, and meanwhile, the trichoderma can be slowly released in the subsequent corn growth process to achieve a continuous biocontrol effect; the trichoderma particles prepared by the method are high in stability, a relatively high viable count of trichoderma can be still kept within a relatively long storage time, the storage time of the trichoderma particles is greatly prolonged, and the actually required long-time storage requirement is met.

Description

Technical Field

[0001] The present invention relates to the technical field of biocontrol agents, and specifically, to a preparation method of Trichoderma granules. Background Art

[0002] Trichoderma is a biocontrol microorganism widely used internationally. As an antagonistic fungus with important biocontrol value, its antagonistic effect on plant pathogenic fungi was discovered as early as the 1930s. Its action mechanisms include antibiotic production, hyperparasitism, bacteriolysis, competition, etc.; corn stalk rot is a soil-borne disease that seriously threatens corn production, causing serious lodging and yield losses. At present, the main method for controlling this disease is to dress seeds with chemical pesticides, but the control effect is generally poor, and most of the control effects are only about 30%. On the other hand, with the increasing demand for green prevention and control of crop pests and diseases in China, the long-term use of chemical pesticides has caused varying degrees of adverse effects on the ecological environment. Trichoderma is a biocontrol fungus rich in resources widely present in the soil. It can not only be widely used to control various soil-borne diseases of crops, but also promote plant growth, increase yields, and reduce soil agrochemical pollution, with broad application prospects. Therefore, developing a biocontrol Trichoderma agent with good biocontrol and yield-increasing effects is of great significance for the green prevention and control of soil-borne diseases.

[0003] After retrieval, the "Preparation method of a Trichoderma biological granule agent" disclosed in Chinese Patent (Publication No.: CN104788175B) specifically includes the following steps: 1. Plate strain: Inoculate Trichoderma asperellum ZJSX5003 on a PDA medium petri dish and place it in an incubator at 28°C; incubate it upside down for 2 - 3 days; PDA medium: Cut 200 grams of potatoes into small pieces of 1 cm³, boil them over low heat for 30 minutes, filter with four layers of gauze, and retain the juice; add 20 grams of glucose, 20 grams of agar powder, make up the volume to 1 liter with distilled water, and sterilize it at 121°C under high pressure steam for 30 minutes; 2. Preparation of secondary strain: Take 200 grams of potatoes, 20 grams of glucose, and 1 liter of water, prepare a secondary fermentation culture solution, adjust the pH value to 6 - 8, dispense 100 milliliters into 250 - milliliter Erlenmeyer flasks, sterilize at 121°C for 30 minutes, inoculate the plate strain, set the shaker speed at 180 revolutions per minute, fermentation temperature at 28°C, and fermentation time at 3 days to obtain a large amount of divergent flocculent mycelia and dispersed conidial liquid strain; 3. Preparation of Trichoderma liquid fermentation broth: The components of the fermentation medium are: 10 kilograms of corn flour, 210 kilograms of water, 766 grams of potassium dihydrogen phosphate, 100 grams of magnesium sulfate, 0.5 grams of manganese sulfate, 0.4 grams of zinc sulfate, 284 grams of sodium nitrate, 220 grams of ammonium sulfate, 200 grams of sodium chloride. First, dissolve the inorganic salts in water, then fully mix the culture materials with water to make a solid fermentation medium. Load the solid fermentation medium into a 300 - liter fermentation tank, sterilize at 121°C for 30 - 60 minutes, inoculate the secondary strain at a weight ratio of 0.5%, control the fermentation temperature at 28°C, and fermentation time at 7 days until the culture materials turn green, which is the Trichoderma liquid fermentation broth; 4. Preparation of Trichoderma granule agent: 40% of Trichoderma liquid fermentation broth, 5.2% of corn flour, 40% of diatomaceous earth, 13% of wheat bran, 1% of zinc sulfate fertilizer, 0.4% of humic acid, 0.4% of nitrogen - phosphorus - potassium compound fertilizer are stirred evenly with a stirrer, extruded and granulated with a granulator, and then dried at 43 - 50°C for 1 - 2 hours. The finished product is the Trichoderma granule agent.

[0004] However, in the process of implementing the related technology, there are certain technical defects in this patent:

[0005] 1. In the preparation process of this patent, the Trichoderma liquid fermentation broth is mainly mixed and granulated with organic fertilizer and nitrogen - phosphorus - potassium compound fertilizer. Although the Trichoderma granule agent prepared by this preparation process has a certain slow - release effect, the germination period of corn is 15 - 20 days, and the Trichoderma in the Trichoderma granule agent is completely released during the corn germination period. The un - germinated coated corn seeds will still affect the activity of Trichoderma spores.

[0006] 2. During the storage process of the patented Trichoderma granule, the solution is affected by the storage environment. Its disintegration rate is high in a high-temperature and high-humidity environment, making it unsuitable for long-term storage. As a result, the shelf life of this type of product is short and cannot meet the actual long-term storage requirements, presenting certain market limitations. In view of this, the present invention proposes a method for preparing Trichoderma granules. Summary of the Invention

[0007] The present invention proposes a method for preparing Trichoderma granules, which solves the problem that the activity of Trichoderma conidia is easily reduced when the wettable powder of Trichoderma is used for coating maize seeds in the prior art.

[0008] The technical solution of the present invention is as follows: A method for preparing Trichoderma granules, comprising the following steps:

[0009] S1. Cultivation of Trichoderma: Inoculate Trichoderma into a PDB medium and shake-culture it at 26 - 30 °C for 3 - 4 days to obtain a high-concentration spore suspension;

[0010] S2. Collection of thalli: Centrifuge the spore suspension obtained in S1 to collect the thallus precipitate, and then wash away the metabolites with sterile water or buffer solution;

[0011] S3. Preparation of core material: Mix the thalli collected in S2 with a protective agent at a ratio of 1:1, and adjust the viable cell concentration of the bacterial suspension to 10 8 -10 9 CFU / mL, and perform homogenization treatment to make it evenly dispersed to obtain a bacterial suspension;

[0012] S4. Preparation of wall material: Dissolve sodium alginate powder in deionized water, then heat it to 60 - 70 °C and stir until completely dissolved, and then filter it through a sieve to remove undissolved particles to obtain a sodium alginate solution with a concentration of 3 - 4% (w / v);

[0013] S5. Microencapsulation treatment: Mix the bacterial suspension in S3 with the sodium alginate solution in S4, form an aqueous phase (W / O emulsion) through high-pressure homogenization, and then drop a 3 - 5% w / v CaCl2 solution into the emulsion. Sodium alginate crosslinks with Ca 2+ to form gel microcapsules. After crosslinking for 25 - 30 minutes, filter and collect the microcapsules, and wash them with sterile water to remove the residual CaCl2 to obtain microencapsulated Trichoderma agent;

[0014] S6. Low-temperature drying: Place the microencapsulated Trichoderma agent in S5 in a freeze-drying device for low-temperature drying to retain the spore activity;

[0015] S7. Mixed granulation: Add organic matter base fertilizer, microencapsulated Trichoderma agent, and binder into a mixer, stir at low speed until it becomes evenly viscous, then perform disk granulation, and then dry it through a fluidized bed dryer until the moisture content ≤ 10%, and finally remove oversized or undersized particles through a vibrating screen, retaining finished particles of 2 - 4 mm.

[0016] Preferably, in S1, the preparation process of the PDB medium is as follows:

[0017] (1). Peel and cut potatoes into pieces, boil for 30 minutes, and filter to obtain the juice;

[0018] (2). Add glucose and water to the above potato juice, heat and dissolve it, and then dispense it into conical flasks;

[0019] (3). Autoclave the above solution at 115 °C for 20 minutes, and cool it to room temperature after sterilization to obtain a liquid medium.

[0020] Preferably, in S1, the specific implementation steps of the inoculation process of Trichoderma are as follows:

[0021] (1). Take the preserved Trichoderma strain, pick a small amount of spores or hyphae with an inoculation loop in a laminar flow hood, and then streak inoculate it onto a PDA plate, and incubate it upside down at 25 - 28 °C for 3 - 5 days until a green spore layer is formed;

[0022] (2). Pick the spores on the solid medium, suspend them in 5 mL of sterile water, and vortex to make a spore suspension;

[0023] (3). Transfer it to the PDB medium according to an inoculation amount of 1 - 5%, and shake culture on a shaker to obtain a seed solution;

[0024] (4). Add the above seed solution to a fresh PDB medium according to an inoculation amount of 5 - 10%, and shake culture at 26 - 30 °C on a shaker for 3 - 4 days to form a high - concentration spore suspension.

[0025] Preferably, in S2, the specific implementation process of the centrifugation treatment of the spore suspension is as follows:

[0026] (1). Dispense the high - concentration spore suspension in S1 into centrifuge tubes, and the liquid filling amount is less than 80% of the tube capacity to prevent overflow;

[0027] (2). Start a low - speed centrifuge, with a centrifugation speed of 3500 - 5000 rpm and a centrifugation time of 10 - 15 minutes, and the spores settle to form a precipitate;

[0028] (3). Add pre - cooled buffer solution to the precipitate, and vortex or pipette to resuspend the spores;

[0029] (4). Repeat centrifugation 2 - 3 times until the supernatant is clear;

[0030] (5) Transfer the precipitate to a sterile container using a sterile spatula or pipette for collection.

[0031] Preferably, the buffer solution is a phosphate buffer solution, and the specific preparation process is as follows:

[0032] (1) Dissolve sodium chloride, potassium dihydrogen phosphate, and disodium hydrogen phosphate in deionized water in sequence, and stir until completely dissolved;

[0033] (2) Adjust the pH to 6.5 - 7.0;

[0034] (3) Make up the volume to 1 L, and autoclave (121 °C) for 20 - 25 minutes.

[0035] Preferably, in S3, the cryoprotectant includes 5 - 10% (w / v) trehalose and 0.5 - 1% (v / v) glycerol, and the balance is phosphate buffer solution.

[0036] Preferably, the specific preparation process of the cryoprotectant is as follows:

[0037] (1) Heat 80 - 85% of the volume of deionized water of the cryoprotectant to 60 - 70 °C, and then slowly add trehalose powder, stirring while adding until the trehalose powder is completely dissolved to obtain a trehalose solution;

[0038] (2) When the trehalose solution cools below 40 °C, add the corresponding proportion of glycerol and mix well;

[0039] (3) Add phosphate buffer solution to adjust the pH to 6.5 - 7.0;

[0040] (4) Make up the volume to the target volume with deionized water, and autoclave (121 °C) for 15 - 20 minutes.

[0041] Preferably, in S6, the specific implementation steps of the low-temperature drying process of the microencapsulated Trichoderma viride agent are as follows:

[0042] (1) Spread the microencapsulated Trichoderma viride agent particles evenly on the freeze-drying tray with a thickness ≤ 1 cm, and then place them in a -80 °C ultra-low temperature refrigerator for rapid freezing to obtain a pre-frozen sample;

[0043] (2) Transfer the above pre-frozen sample to a freeze-dryer, set the vacuum degree to 20 - 50 Pa, and the cold trap temperature below -50 °C;

[0044] (3) Slowly heat up to -30 °C and maintain for 16 - 24 hours to directly sublimate the ice crystals.

[0045] Preferably, in S7, the mass ratio of the organic matter base fertilizer, the microencapsulated Trichoderma viride agent, and the binder is 8:1:0.5.

[0046] Preferably, the organic matter base fertilizer is a mixture of compost, humic acid and soybean meal, and the binder is a CMC solution.

[0047] The working principle and beneficial effects of the present invention are as follows:

[0048] 1. The Trichoderma granule prepared by the present invention has a long slow-release period. The slow-release characteristics of the organic matter base fertilizer continuously improve the nutrition and germination rate during the germination process of the coated corn seeds. Moreover, after the Trichoderma is microencapsulated, the release time can be delayed and the release time is after the corn germination period, avoiding the influence on the activity of Trichoderma during the germination process of the coated corn seeds. At the same time, Trichoderma can be slowly released during the subsequent growth process of corn to achieve a continuous biocontrol effect. At the same time, the organic matter base fertilizer can improve the survival rate of Trichoderma, which can further enhance the biocontrol effect of Trichoderma;

[0049] 2. The Trichoderma granule prepared by the present invention has high stability and can maintain a high number of viable Trichoderma bacteria during a long storage time, greatly improving the storage time of the Trichoderma granule and meeting the actual long-term storage requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The present invention will be further described in detail below with reference to the drawings and specific embodiments.

[0051] Figure 1 It is a table of the number of viable bacteria after breaking the wall of each group of Trichoderma granule in Test Example 1 of the present invention;

[0052] Figure 2 It is a table of the disintegration rate of each group of Trichoderma granule in Test Example 2 of the present invention;

[0053] Figure 3 It is a line graph of the number of viable bacteria increasing with time of each group of Trichoderma granule in Test Example 3 of the present invention under the environment of 4°C;

[0054] Figure 4 It is a line graph of the number of viable bacteria increasing with time of each group of Trichoderma granule in Test Example 3 of the present invention under the environment of 25°C;

[0055] Figure 5 It is a line graph of the germination rate of corn seeds changing with time after applying different Trichoderma granules in Test Example 4 of the present invention;

[0056] Figure 6 It is a line graph of the number of viable bacteria in the soil of corn seeds changing with time after applying different Trichoderma granules in Test Example 4 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0057] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0058] Embodiment 1:

[0059] This embodiment provides a method for preparing Trichoderma granules, which includes the following steps:

[0060] S1. Cultivation of Trichoderma: The cultivation process is as follows:

[0061] (1) Take 200 g of peeled and cut potatoes, boil for 30 minutes, and filter to obtain the juice;

[0062] (2) Add 20 g of glucose and 1 L of water to the above potato juice, heat and dissolve, and then dispense into conical flasks;

[0063] (3) Sterilize the above solution at 115 °C under high pressure for 20 minutes. After sterilization, cool it to room temperature to obtain a liquid medium, and then inoculate Trichoderma into the above liquid medium and shake culture at 26 °C for 4 days to obtain a high-concentration spore suspension;

[0064] S2. Collection of bacteria: Centrifuge the high-concentration spore suspension obtained in S1 and collect the bacterial cell precipitate. The specific implementation process of the centrifugation treatment is as follows:

[0065] (1) Dispense the high-concentration spore suspension in S1 into centrifuge tubes, and the liquid filling volume is less than 80% of the tube capacity to prevent overflow;

[0066] (2) Start a low-speed centrifuge (Thermo ST16R), with a centrifugation speed of 3500 rpm and a centrifugation time of 15 minutes, and the spores sediment to form a precipitate;

[0067] (3) Add pre-cooled buffer solution to the precipitate, and vortex or pipette to resuspend the spores;

[0068] (4) Repeat centrifugation 3 times until the supernatant is clear;

[0069] (5) Use a sterile spatula or pipette to transfer the precipitate to a sterile container for collection, and then wash with sterile water or buffer solution to remove metabolites;

[0070] S3. Preparation of core material: Mix the bacteria collected in S2 with a protective agent (trehalose 10% (w / v), glycerol 1% (v / v), and the balance is phosphate buffer solution) at a ratio of 1:1, and adjust the viable cell concentration of the bacterial suspension to 10 9CFU / mL. Homogenization treatment was performed to achieve uniform dispersion and obtain a bacterial suspension;

[0071] S4. Preparation of wall material: Sodium alginate powder was dissolved in deionized water, then heated to 60 °C and stirred until completely dissolved. It was then filtered through a sieve to remove undissolved particles, obtaining a 3% (w / v) sodium alginate solution;

[0072] S5. Microencapsulation treatment: The bacterial suspension in S3 was mixed with the sodium alginate solution in S4, and a water phase (W / O emulsion) was formed by high-pressure homogenization. Then, a 3% w / v CaCl2 solution was dropped into the emulsion. Sodium alginate and Ca 2+ crosslinked to form gel microcapsules. After 25 minutes of crosslinking, the microcapsules were collected by filtration, and the residual CaCl2 was removed by washing with sterile water to obtain a microencapsulated Trichoderma agent;

[0073] S6. Low-temperature drying: The microencapsulated Trichoderma agent in S5 was placed in a freeze-drying device for low-temperature drying to retain spore activity;

[0074] S7. Mixing and granulation: Organic matter base fertilizer, microencapsulated Trichoderma agent, and CMC solution were added to a mixer in a mass ratio of 8:1:0.5, stirred at low speed until uniformly viscous, then subjected to disk granulation, and dried by a fluidized bed dryer until the moisture content ≤ 10%. Finally, oversized or undersized particles were removed by a vibrating screen, and 2 mm finished particles were retained.

[0075] Example 2:

[0076] This example presents a method for preparing Trichoderma granules, including the following steps:

[0077] S1. Cultivation of Trichoderma: The cultivation process is as follows:

[0078] (1). Take 200 g of peeled and cut potatoes, boil for 30 minutes, and filter to obtain the juice;

[0079] (2). Add 20 g of glucose and 1 L of water to the above potato juice, heat and dissolve, and then dispense into conical flasks;

[0080] (3). Autoclave the above solution at 115 °C for 20 minutes. After sterilization, cool to room temperature to obtain a liquid medium. Then, inoculate Trichoderma into the above liquid medium and shake culture at 26 °C for 4 days to obtain a high-concentration spore suspension;

[0081] S2. Collection of bacterial cells: The high-concentration spore suspension obtained in S1 was subjected to centrifugation to collect the bacterial cell precipitate. The specific implementation process of the centrifugation is as follows:

[0082] (1). Dispense the high-concentration spore suspension in S1 into centrifuge tubes, with the liquid volume less than 80% of the tube capacity to prevent overflow;

[0083] (2), Start the low-speed centrifuge (Thermo ST16R), with a centrifugation speed of 3500 rpm and a centrifugation time of 15 minutes, so that the spores settle to form a precipitate;

[0084] (3), Add pre-cooled buffer to the precipitate, and vortex or pipette to resuspend the spores;

[0085] (4), Repeat centrifugation 3 times until the supernatant is clear;

[0086] (5), Use a sterile spatula or pipette to transfer the precipitate to a sterile container for collection, and then wash it with sterile water or buffer to remove metabolites;

[0087] S3, Prepare the core material: Mix the bacteria collected in S2 with the protectant (10% (w / v) trehalose, 1% (v / v) glycerol, and the balance is phosphate buffer) in a ratio of 1:1, and adjust the viable bacteria concentration of the bacterial suspension to 10 9 CFU / mL, and homogenize to make it evenly dispersed to obtain a bacterial suspension;

[0088] S4, Prepare the wall material: Dissolve sodium alginate powder in deionized water, then heat to 60°C and stir until completely dissolved, and then filter through a sieve to remove undissolved particles to obtain a 3.5% (w / v) sodium alginate solution;

[0089] S5, Microencapsulation treatment: Mix the bacterial suspension in S3 with the sodium alginate solution in S4, form an aqueous phase (W / O emulsion) through high-pressure homogenization, and then drop 3% w / v CaCl2 solution into the emulsion. Sodium alginate and Ca 2+ crosslink to form gel microcapsules. After crosslinking for 25 minutes, filter and collect the microcapsules, and wash them with sterile water to remove residual CaCl2 to obtain the microcapsule Trichoderma agent;

[0090] S6, Low-temperature drying: Place the microcapsule Trichoderma agent in S5 in a freeze-drying device for low-temperature drying to retain the spore activity;

[0091] S7, Mixing and granulation: Add organic matter base fertilizer, microencapsulated Trichoderma agent, and CMC solution to the mixer according to a mass ratio of 8:1:0.5, stir at low speed until evenly viscous, then perform disk granulation, and then dry through a fluidized bed dryer until the moisture content ≤ 10%, and finally remove oversized or undersized particles through a vibrating sieve to retain 2 mm finished particles.

[0092] Example 3:

[0093] This example proposes a method for preparing Trichoderma granules, including the following steps:

[0094] S1, Cultivation of Trichoderma: The cultivation process is as follows:

[0095] (1) Take 200 g of peeled and cut potatoes, boil for 30 minutes, and filter to obtain the juice.

[0096] (2) Add 20 g of glucose and 1 L of water to the above potato juice, heat and dissolve, and then dispense into conical flasks.

[0097] (3) Autoclave the above solution at 115 °C for 20 minutes. After sterilization, cool to room temperature to obtain a liquid medium. Then inoculate Trichoderma into the above liquid medium and shake culture at 26 °C for 4 days to obtain a high-concentration spore suspension.

[0098] S2. Collect the thalli: Centrifuge the high-concentration spore suspension obtained in S1 and collect the thallus precipitate. The specific implementation process of the centrifugation is as follows:

[0099] (1) Dispense the high-concentration spore suspension in S1 into centrifuge tubes, and the liquid filling volume is less than 80% of the tube capacity to prevent overflow.

[0100] (2) Start a low-speed centrifuge (Thermo ST16R), with a centrifugation speed of 3500 rpm and a centrifugation time of 15 minutes, and the spores settle to form a precipitate.

[0101] (3) Add pre-cooled buffer solution to the precipitate, and vortex or pipette to resuspend the spores.

[0102] (4) Repeat centrifugation 3 times until the supernatant is clear.

[0103] (5) Use a sterile spatula or pipette to transfer the precipitate to a sterile container for collection, and then wash with sterile water or buffer solution to remove metabolites.

[0104] S3. Prepare the core material: Mix the thalli collected in S2 with a protective agent (10% (w / v) trehalose, 1% (v / v) glycerol, and the balance is phosphate buffer solution) in a ratio of 1:1, and adjust the viable concentration of the bacterial suspension to 10 9 CFU / mL, and homogenize to make it evenly dispersed to obtain a bacterial suspension.

[0105] S4. Prepare the wall material: Dissolve sodium alginate powder in deionized water, then heat to 60 °C and stir until completely dissolved, and then filter through a sieve to remove undissolved particles to obtain a 4% (w / v) sodium alginate solution.

[0106] S5. Microencapsulation treatment: Mix the bacterial suspension in S3 with the sodium alginate solution in S4, form an aqueous phase (W / O emulsion) by high-pressure homogenization, and then drop 3% w / v CaCl2 solution into the emulsion. Sodium alginate crosslinks with Ca 2+ to form gel microcapsules. After crosslinking for 25 minutes, filter and collect the microcapsules, and wash with sterile water to remove residual CaCl2 to obtain the microencapsulated Trichoderma agent.

[0107] S6, Low-temperature drying: Place the microencapsulated Trichoderma agent in S5 into a freeze-drying device for low-temperature drying to retain spore activity;

[0108] S7, Mixing and granulation: Add organic matter base fertilizer, microencapsulated Trichoderma agent, and CMC solution to the mixer according to a mass ratio of 8:1:0.5, stir at low speed until it becomes uniformly viscous, then perform disk granulation, and then dry it through a fluidized bed dryer until the moisture content ≤ 10%, and finally remove oversized or undersized particles through a vibrating sieve to retain 2-mm finished particles.

[0109] Example 4:

[0110] This example proposes a method for preparing Trichoderma granules, which includes the following steps:

[0111] S1, Cultivation of Trichoderma: The cultivation process is as follows:

[0112] (1). Take 200 g of peeled and cut potatoes, boil for 30 minutes, and filter to obtain the juice;

[0113] (2). Add 20 g of glucose and 1 L of water to the above potato juice, heat and dissolve it, and then dispense it into conical flasks;

[0114] (3). Autoclave the above solution at 115 °C for 20 minutes, cool it to room temperature after sterilization to obtain a liquid medium, and then inoculate Trichoderma into the above liquid medium, and shake and culture it on a shaker at 26 °C for 4 days to obtain a high-concentration spore suspension;

[0115] S2, Collection of bacterial cells: Centrifuge the high-concentration spore suspension obtained in S1 and collect the bacterial cell precipitate. The specific implementation process of the centrifugation treatment is as follows:

[0116] (1). Dispense the high-concentration spore suspension in S1 into centrifuge tubes, and the liquid filling volume is less than 80% of the tube capacity to prevent overflow;

[0117] (2). Start a low-speed centrifuge (Thermo ST16R), the centrifugation speed is 3500 rpm, and the centrifugation time is 15 minutes, and the spores settle to form a precipitate;

[0118] (3). Add pre-cooled buffer solution to the precipitate, and vortex or pipette to resuspend the spores;

[0119] (4). Repeat centrifugation 3 times until the supernatant is clear;

[0120] (5). Use a sterile spatula or pipette to transfer the precipitate to a sterile container for collection, and then wash it with sterile water or buffer solution to remove metabolites;

[0121] S3. Preparation of core material: Mix the bacterial cells collected in S2 with a protective agent (10% (w / v) trehalose, 1% (v / v) glycerol, and the balance being phosphate buffer) at a ratio of 1:1, and adjust the viable cell concentration of the bacterial suspension to 10 9 CFU / mL, and perform homogenization treatment to make it evenly dispersed to obtain a bacterial suspension;

[0122] S4. Preparation of wall material: Dissolve sodium alginate powder in deionized water, then heat it to 60 °C and stir until completely dissolved, and then filter through a sieve to remove undissolved particles to obtain a 4% (w / v) sodium alginate solution;

[0123] S5. Microencapsulation treatment: Mix the bacterial suspension in S3 with the sodium alginate solution in S4, form an aqueous phase (W / O emulsion) through high-pressure homogenization, and then drop 3% w / v CaCl2 solution into the emulsion. Sodium alginate cross-links with Ca 2+ to form gel microcapsules. After cross-linking for 25 minutes, filter and collect the microcapsules, and wash them with sterile water to remove residual CaCl2 to obtain a microencapsulated Trichoderma agent;

[0124] S6. Low-temperature drying: Place the microencapsulated Trichoderma agent in S5 in a freeze-drying device for low-temperature drying to retain spore activity;

[0125] S7. Mixing and granulation: Add organic matter base fertilizer, microencapsulated Trichoderma agent, and CMC solution to a mixer at a mass ratio of 8:1:0.5, stir at low speed until it becomes evenly viscous, then perform disk granulation, and then dry it through a fluidized bed dryer until the moisture content ≤ 10%, and finally remove oversized or undersized particles through a vibrating sieve to retain 3 mm finished particles.

[0126] Example 5:

[0127] This example proposes a method for preparing Trichoderma granules, which includes the following steps:

[0128] S1. Cultivation of Trichoderma: The cultivation process is as follows:

[0129] (1). Take 200 g of peeled and cut potatoes, boil for 30 minutes, and filter to obtain the juice;

[0130] (2). Add 20 g of glucose and 1 L of water to the above potato juice, heat and dissolve it, and then dispense it into conical flasks;

[0131] (3). Autoclave the above solution at 115 °C for 20 minutes, cool it to room temperature after sterilization to obtain a liquid medium, and then inoculate Trichoderma into the above liquid medium, and shake and culture it on a shaker at 26 °C for 4 days to obtain a high-concentration spore suspension;

[0132] S2. Collect Bacteria: Centrifuge the high-concentration spore suspension obtained in S1 and collect the bacterial precipitate. The specific implementation process of the centrifugation is as follows:

[0133] (1). Dispense the high-concentration spore suspension in S1 into centrifuge tubes, with the liquid volume less than 80% of the tube capacity to prevent overflow;

[0134] (2). Start a low-speed centrifuge (Thermo ST16R), with a centrifugation speed of 3500 rpm and a centrifugation time of 15 minutes, so that the spores settle to form a precipitate;

[0135] (3). Add pre-cooled buffer to the precipitate, and vortex or pipette to resuspend the spores;

[0136] (4). Repeat centrifugation 3 times until the supernatant is clear;

[0137] (5). Use a sterile spatula or pipette to transfer the precipitate to a sterile container for collection, and then wash with sterile water or buffer to remove metabolites;

[0138] S3. Prepare Core Material: Mix the bacteria collected in S2 with a protective agent (10% (w / v) trehalose, 1% (v / v) glycerol, and the balance is phosphate buffer) in a ratio of 1:1, and adjust the viable bacteria concentration of the bacterial suspension to 10 9 CFU / mL, and homogenize to make it evenly dispersed to obtain a bacterial suspension;

[0139] S4. Prepare Wall Material: Dissolve sodium alginate powder in deionized water, then heat to 60°C and stir until completely dissolved, and then filter through a sieve to remove undissolved particles to obtain a 4% (w / v) sodium alginate solution;

[0140] S5. Microencapsulation Treatment: Mix the bacterial suspension in S3 with the sodium alginate solution in S4, form an aqueous phase (W / O emulsion) through high-pressure homogenization, and then drop 3% w / v CaCl2 solution into the emulsion. Sodium alginate and Ca 2+ crosslink to form gel microcapsules. After crosslinking for 25 minutes, filter and collect the microcapsules, and wash with sterile water to remove residual CaCl2 to obtain a microencapsulated Trichoderma agent;

[0141] S6. Low-temperature Drying: Place the microencapsulated Trichoderma agent in S5 in a freeze-drying device for low-temperature drying to retain spore activity;

[0142] S7. Mixing and Granulation: Add organic matter base fertilizer, microencapsulated Trichoderma agent, and CMC solution to the mixer in a mass ratio of 8:1:0.5, stir at low speed until evenly viscous, then perform disk granulation, and then dry through a fluidized bed dryer until the moisture content ≤ 10%, and finally remove oversized or undersized particles through a vibrating screen to retain 4 mm finished particles.

[0143] Comparative Example 1:

[0144] This comparative example presents a preparation method for Trichoderma granules. The preparation steps are basically the same as those in Example 1, with the only difference being that no protective agent is added in S3;

[0145] Comparative Example 2: This comparative example presents a preparation method for Trichoderma granules. The preparation steps are basically the same as those in Example 1, with the only difference being that compound fertilizer is used to replace the organic matter base fertilizer in S7.

[0146] Test Example 1:

[0147] This test example is used to perform cyst-breaking treatment on the microencapsulated Trichoderma granules prepared in Example 1, Example 2, and Example 3, and calculate the viable bacteria count of Trichoderma after the wall-breaking of each microencapsulated Trichoderma granule by the plate counting method, calculated by the following formula:

[0148] The calculation results are as Figure 1 shown;

[0149] It can be Figure 1 seen that as the concentration of the sodium alginate solution increases, the effective viable bacteria count inside the microencapsulated Trichoderma agent first increases and then decreases. From this, the optimal concentration range of the sodium alginate solution can be determined to be 3.5 - 4% (w / v).

[0150] Test Example 2:

[0151] This test example is used to measure the particle disintegration rate of the microencapsulated Trichoderma granules prepared in Example 1, Example 4, and Example 5.

[0152] (1) Take 0.5 g of microencapsulated Trichoderma granules from Example 1, Example 4, and Example 5 respectively:

[0153] (2) Place each microencapsulated Trichoderma granule in water for 30 minutes, then take out the granule and weigh it. Calculate the disintegration rate of each group by the following formula:

[0154] The calculation results are as Figure 2 shown;

[0155] It can be Figure 2 seen that as the particle size of the microencapsulated Trichoderma granules increases, the disintegration rate of the microencapsulated Trichoderma granules in water first decreases and then increases. This shows that the slow-release effect of the microencapsulated Trichoderma granules is the best when the particle size is 2 - 3 mm.

[0156] Test Example 3:

[0157] This test example is used to store the microencapsulated Trichoderma particles prepared in Example 1 and Comparative Example 1 at 4°C and 25°C respectively, and detect the viable bacteria count at 10, 20, 30, and 40 days respectively. The viable bacteria count of Trichoderma after breaking the wall of each microencapsulated Trichoderma particle is mainly calculated by the plate counting method, and is calculated by the following formula:

[0158] The calculation results are as Figure 3 and Figure 4 shown.

[0159] From Figure 3 and Figure 4 it can be seen that regardless of whether it is in a low-temperature or normal-temperature environment, the viable bacteria count inside the microencapsulated Trichoderma particles in Example 1 remains at a relatively high level after long-term storage. This shows that the addition of the protective agent can greatly improve the stability of the microencapsulated Trichoderma particles during storage.

[0160] Test Example 4:

[0161] This test example is used to detect the influence of the microencapsulated Trichoderma particles prepared in Example 1 and Comparative Example 2 on the germination of coated corn seeds. Experimental location: Shijiazhuang City, Hebei Province. The terrain of this plot is flat. After sowing the coated corn seeds, the microencapsulated Trichoderma particles prepared in Example 1 and Comparative Example 2 are applied to two sowing areas respectively. Then, every 5, 10, 15, and 20 days, calculate the germination rate of the corn seeds in each area and the viable bacteria count of Trichoderma in the soil. The germination rate is calculated by the following formula:

[0162] The calculation results are as Figure 5 shown;

[0163] The formula for calculating the viable bacteria count of Trichoderma in the soil is as follows: The calculation results are as Figure 6 shown;

[0164] From Figure 5 it can be seen that during the 5 - 20-day germination period of the corn seeds, after applying the microencapsulated Trichoderma particles of Example 1, a relatively high germination rate growth can be maintained, while after applying the microencapsulated Trichoderma particles of Comparative Example 2, the germination growth rate significantly decreases after 15 days. Therefore, adding organic matter base fertilizer to the microencapsulated Trichoderma particles prepared in Example 1 can maintain a relatively high corn seed germination growth rate for a long time, and in Comparative Example 2, a relatively high corn seed germination rate can be achieved in a short period. This shows that the organic matter base fertilizer has a good slow-release effect and can continuously provide nutrients for the germination of corn seeds, thereby greatly improving the germination rate of corn.

[0165] From Figure 6It can be seen that a large amount of Trichoderma is released from the microencapsulated Trichoderma granules after 15 days, that is, after the germination period of corn, indicating that the microencapsulated Trichoderma granules will not affect the germination of corn; from Figure 6 It can be seen that the number of Trichoderma in the sowing soil with the microencapsulated Trichoderma granules of Example 1 applied is larger, indicating that the organic matter base fertilizer can effectively improve the survival rate of Trichoderma.

[0166] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A preparation method of Trichoderma granules, characterized in that, It includes the following steps: S1. Cultivation of Trichoderma: Inoculate Trichoderma into PDB medium and culture it on a shaker at 26 - 30 °C for 3 - 4 days to obtain a high - concentration spore suspension; S2. Collection of thalli: Centrifuge the spore suspension obtained in S1 and collect the thallus precipitate, then wash it with sterile water or buffer to remove metabolites; S3. Preparation of the core material: Mix the bacterial cells collected in S2 with the protective agent at a ratio of 1:1, and adjust the viable cell concentration of the bacterial suspension to 10 8 -10 9 CFU / mL, and perform homogenization treatment to make it evenly dispersed to obtain a bacterial suspension; S4. Preparation of wall material: Dissolve sodium alginate powder in deionized water, then heat it to 60 - 70 °C and stir until completely dissolved, and then filter it through a sieve to remove undissolved particles to obtain a 3 - 4% (w / v) sodium alginate solution; S5. Microencapsulation treatment: Mix the bacterial suspension in S3 with the sodium alginate solution in S4, and form an aqueous phase (W / O emulsion) through high-pressure homogenization. Then, drop a 3-5% w / v CaCl2 solution into the emulsion. Sodium alginate reacts with Ca 2+ to crosslink and form gel microcapsules. After crosslinking for 25-30 minutes, filter and collect the microcapsules, and wash them with sterile water to remove the residual CaCl2 to obtain the microencapsulated Trichoderma agent; S6. Low - temperature drying: Place the microencapsulated Trichoderma agent in S5 in a freeze - drying device for low - temperature drying to retain spore activity; S7. Mixing and granulation: Add organic matter base fertilizer, microencapsulated Trichoderma agent, and binder into a mixer, stir at low speed until it becomes uniformly viscous, then perform disk granulation, and then dry it in a fluidized - bed dryer until the moisture content ≤ 10%, and finally remove oversized or undersized particles through a vibrating sieve to retain finished particles of 2 - 4 mm.

2. The preparation method of a trichoderma granule according to claim 1, characterized in that, In S1, the preparation process of the PDB medium is as follows: (1). Peel and cut potatoes into pieces, boil for 30 minutes, and filter to obtain the juice; (2). Add glucose and water to the above potato juice, heat and dissolve it, and then dispense it into conical flasks; (3). Autoclave the above solution at 115 °C for 20 minutes, and cool it to room temperature after sterilization to obtain a liquid medium.

3. The preparation method of a trichoderma granule according to claim 2, characterized in that, In S1, the specific implementation steps of the inoculation process of Trichoderma are as follows: (1). Take the preserved Trichoderma strain, pick a small amount of spores or hyphae with an inoculation loop in a laminar flow hood, and then streak - inoculate it onto a PDA plate, and culture it upside - down at 25 - 28 °C for 3 - 5 days until a green spore layer is formed; (2). Pick the spores on the solid medium, suspend them in 5 mL of sterile water, and vortex - oscillate to make a spore suspension; (3). Transfer it to the PDB medium according to an inoculation amount of 1 - 5%, and culture it on a shaker to obtain a seed solution; (4). Add the above seed solution to fresh PDB medium according to an inoculation amount of 5 - 10%, and culture it on a shaker at 26 - 30 °C for 3 - 4 days to form a high - concentration spore suspension.

4. The preparation method of a trichoderma granule according to claim 3, characterized in that, In S2, the specific implementation process of the centrifugation of the spore suspension is as follows: (1). Dispense the high - concentration spore suspension in S1 into centrifuge tubes, and the liquid filling volume is less than 80% of the tube capacity to prevent overflow; (2). Start a low - speed centrifuge, with a centrifugation speed of 3500 - 5000 rpm and a centrifugation time of 10 - 15 minutes, so that the spores settle to form a precipitate; (3). Add pre - cooled buffer to the precipitate, and vortex - oscillate or pipette to resuspend the spores; (4). Repeat centrifugation 2 - 3 times until the supernatant is clear; (5). Use a sterile spatula or pipette to transfer the precipitate to a sterile container for collection.

5. The preparation method of a trichoderma granule according to claim 4, wherein, The buffer is a phosphate buffer, and its specific preparation process is as follows: (1). Dissolve sodium chloride, potassium dihydrogen phosphate, and disodium hydrogen phosphate in deionized water in sequence, and stir until completely dissolved; (2). Adjust the pH to 6.5 - 7.0; (3). Make up the volume to 1 L, and autoclave (121 °C) for 20 - 25 minutes.

6. The preparation method of a Trichoderma granule according to claim 1, characterized in that, In S3, the protective agent comprises 5-10% (w / v) of trehalose and 0.5-1% (v / v) of glycerol, and the balance is phosphate buffer solution.

7. The preparation method of a trichoderma granule according to claim 6, wherein, The specific preparation process of the protective agent is as follows: (1) Take ionized water accounting for 80-85% of the volume of the protective agent, heat it to 60-70 °C, and then slowly add trehalose powder, stirring while adding until the trehalose powder is completely dissolved to obtain a trehalose solution; (2) Wait until the temperature of the trehalose solution drops below 40 °C, add glycerol in the corresponding proportion and mix well; (3) Add phosphate buffer solution to adjust the pH to 6.5-7.0; (4) Make up deionized water to the target volume and sterilize it under high pressure (121 °C) for 15-20 minutes.

8. The preparation method of a trichoderma granule according to claim 1, characterized in that In S6, the specific implementation steps of the low-temperature drying process of the microencapsulated Trichoderma agent are as follows: (1) Spread the microencapsulated Trichoderma agent flat on a freeze-drying tray with a thickness ≤ 1 cm, and then place it in a -80 °C ultra-low temperature refrigerator for rapid freezing to obtain a pre-frozen sample; (2) Transfer the above pre-frozen sample to a freeze dryer, set the vacuum degree to 20-50 Pa, and the cold trap temperature below -50 °C; (3) Slowly raise the temperature to -30 °C and maintain it for 16-24 hours to directly sublimate the ice crystals.

9. The preparation method of a trichoderma granule according to claim 1, wherein, In S7, the mass ratio of the organic matter base fertilizer, the microencapsulated Trichoderma agent, and the binder is 8:1:0.

5.

10. The preparation method of a trichoderma granule according to claim 9, characterized in that, The organic matter base fertilizer is a mixture of compost, humic acid, and soybean meal, and the binder is a CMC solution.

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