A protective agent and biocontrol agent to enhance the stability of the biocontrol bacterium *Talaromyces kabodanensis*.

Abstract

This invention belongs to the field of strawberry biocontrol technology, and relates to an enhanced biocontrol bacteria. Talaromyces kabodanensis A stability protectant, mainly composed of the following components by weight: 0.5-1.0 parts betaine, 2-3 parts sorbitol, 1-2 parts glycerol, and 2-4 parts sucrose. This invention utilizes the synergistic effect of betaine, sorbitol, glycerol, and sucrose, achieving significantly better results than any single protectant, and can significantly improve the stability of biocontrol bacteria. Talaromyces kabodanensis The product exhibits stability in biocontrol agents and extends their shelf life. After application of the biocontrol agent to strawberries, the biocontrol bacteria... Talaromyces kabodanensis The 7-day survival rate increased from 41.5% to 78.3%, thus ensuring the stable effectiveness of the biocontrol bacteria. The protective agents provided in this invention all use food-grade or pharmaceutical-grade raw materials, meeting the development needs of green and organic agriculture.

Description

Technical Field

[0001] This invention belongs to the field of microbial pesticide technology and relates to a protective agent and a biocontrol agent that enhances the stability of the biocontrol bacterium Talaromyces kabodanensis. Background Technology

[0002] Talamyces spp. is an important endophytic fungus in plants. The inventor's patented biocontrol agent for strawberry diseases, *Talaromyces kabodanensis*, and its application (publication number CN 116179367 A), exhibits significant antagonistic activity against gray mold and anthracnose. However, further research revealed a technical problem of poor stability in the application of this biocontrol agent, specifically in the following aspects:

[0003] 1. Poor storage stability: The viable bacteria rate decreases rapidly under normal temperature conditions, and the shelf life is usually less than 30 days under refrigeration.

[0004] 2. Difficulty in field colonization: Directly applied inoculants face environmental stresses such as ultraviolet radiation and temperature fluctuations. The survival rate is less than 45% 7 days after field application, resulting in unstable biocontrol effects.

[0005] Therefore, developing a protective agent system that can enhance the stability of the biocontrol bacterium Talaromyces kabodanensis is of great significance for promoting the industrial application of this type of biocontrol agent. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a protective agent that enhances the stability of the biocontrol bacterium *Talaromyces kabodanensis*, a biocontrol agent containing the protective agent, and a method for preparing the same.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A protective agent for enhancing the stability of the biocontrol bacterium *Talaromyces kabodanensis*, mainly composed of the following components in parts by weight: 0.5-1.0 parts betaine, 2-3 parts sorbitol, 1-2 parts glycerol, and 2-4 parts sucrose.

[0009] As a preferred embodiment of the present invention, the protective agent is mainly composed of the following components in parts by weight: 1.0 part betaine, 2 parts sorbitol, 1.5 parts glycerin, and 2.5 parts sucrose.

[0010] As another technical solution of the present invention, a biocontrol agent contains a protective agent for enhancing the stability of the biocontrol bacterium Talaromyces kabodanensis as described in claim 1 or 2, and the biocontrol bacterium Talaromyces kabodanensis; the weight ratio of the protective agent to the biocontrol bacterium is 7:93.

[0011] As a preferred biocontrol agent, the biocontrol agent contains a viable count of at least 1 CFU / mL of the biocontrol bacterium *Talaromyces kabodanensis*.

[0012] Furthermore, in the biocontrol agent, the viable count of the biocontrol bacterium *Talaromyces kabodanensis* is not less than 1, and the viable count is not less than CFU / mL.

[0013] Furthermore, the biocontrol agent can be formulated into a form acceptable to microbial pesticides. The formulation includes liquid inoculants, wettable powders, water-dispersible granules, or solid granules.

[0014] As the third technical solution of the present invention, a method for preparing a biocontrol agent includes the following steps:

[0015] (1) Activation of bacterial strain: The biocontrol bacterium Talaromyces kabodanensis was inoculated into PDA medium and cultured in the dark for 3-5 days at a concentration of 25 strains.

[0016] (2) Seed culture preparation: Take mycelial blocks of activated strains, inoculate them into PDB liquid medium, and culture at 25°C and 160 rpm for 5-7 days to obtain seed culture;

[0017] (3) Fermentation culture: Inoculate the seed liquid into the fermentation medium at an inoculation rate of 5-10%, and culture at 160 rpm for 7-10 days to obtain the fermentation broth;

[0018] (4) Collection of bacterial cells: Collect the bacterial cells after centrifugation or filtration of the fermentation broth to obtain bacterial solution;

[0019] (5) Preparation of protective agent: Weigh betaine, sorbitol, glycerin and sucrose according to the weight ratio, dissolve them in sterile water to obtain protective agent solution;

[0020] (6) Forming of bacterial agent: Mix the bacterial liquid with the protective agent solution evenly to obtain liquid bacterial agent.

[0021] As a further improvement to the preparation method of the biocontrol agent of the present invention, in step (3), the fermentation culture medium is composed of: 200 g / L potato starch, 20 g / L maltose, 5 g / L peptone, 3 g / L yeast extract, 1 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, and pH 5.5~6.0.

[0022] Compared with existing technologies, this invention has the following beneficial effects: Through the synergistic effect of betaine, sorbitol, glycerol, and sucrose, the effect is significantly superior to that of any single protectant, significantly improving the stability of the biocontrol bacterium *Talaromyces kabodanensis* in the biocontrol agent product, while also extending the product's shelf life. After application of the biocontrol agent product to strawberries, the 7-day survival rate of *Talaromyces kabodanensis* increased from 41.5% to 78.3%, thus ensuring the stable effectiveness of the biocontrol bacterium.

[0023] The protective agents provided by this invention all use food-grade or pharmaceutical-grade raw materials, which meet the development needs of green agriculture and organic agriculture. Attached Figure Description

[0024] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments will be briefly introduced below.

[0025] Figure 1 Photographs comparing the viability of biocontrol spore suspensions with and without the protective agent, and with the protective agent of this invention (Formula 2), after storage at 4°C for 90 days. The left side represents the suspension without the protective agent, and the right side represents the suspension with the protective agent.

[0026] Figure 2 Photographs comparing the survival rates of the biocontrol agents applied to the rhizosphere of strawberries 7 days after application, with and without the added protectant (Formula 2) of this invention. The left side shows the biocontrol agent without the protectant, and the right side shows the biocontrol agent with the protectant. Detailed Implementation

[0027] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0028] The biocontrol bacterium *Talaromyces kabodanensis* involved in this invention, classified as *Talaromyces kabodanensis*, was deposited on December 8, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO. 40438, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. It was provided by Jiangsu Vocational College of Agriculture and Forestry. The methods for isolation, purification, and molecular biological identification of this strain are described in patent CN116179367B, and will not be repeated here.

[0029] All raw materials used in this invention are commercially available products, specifically:

[0030] Betaine: Food grade, purity ≥ betaine;

[0031] Sorbitol: Food grade, purity ≥ sorbitol;

[0032] Glycerin: pharmaceutical grade, purity ≥ ; Oil: pharmaceutical grade;

[0033] Sucrose: food grade, purity ≥ sugar: food.

[0034] Example 1: Screening of different protective agent ratios

[0035] 1.1 Composite protective agents with different weight ratios were prepared, as shown in Table 1.

[0036] 1.2 Preparation of biocontrol agents

[0037] Prepare the biocontrol agent according to the proportions in Table 1 using the following method:

[0038] (1) Activation of bacterial strain: The biocontrol bacterium Talaromyces kabodanensis was inoculated into PDA medium and cultured in the dark for 3-5 days at a concentration of 25 strains.

[0039] (2) Seed culture preparation: Use a punch (5 mm in diameter) to punch out mycelial blocks of activated strains at the edge of the colony, inoculate them into 200 mL of PDB liquid medium, and culture at 25°C and 160 rpm for 5-7 days to obtain seed culture;

[0040] (3) Fermentation culture: Inoculate the seed liquid into the fermentation medium at an inoculation rate of 5-10%, and culture at 160 rpm for 7-10 days to obtain the fermentation broth; wherein, the composition of the fermentation medium is: potato starch 200 g / L, maltose 20 g / L, peptone 5 g / L, yeast extract 3 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.5 g / L, pH 5.5-6.0;

[0041] (4) Collection of bacterial culture: The fermentation broth was centrifuged at 8000 rpm for 15 min, and the bacterial culture was collected and washed twice with sterile water;

[0042] (5) Preparation of protective agent: Dissolve the protective agent in the above test materials in 100 mL of sterile water to obtain a protective agent solution;

[0043] (6) Forming of bacterial agent: Mix the bacterial solution and the protective agent solution at a ratio of 1:2 (w / v) to obtain liquid bacterial agent. Dispense the liquid bacterial agent into sterile containers.

[0044] 1.3 Determination Method:

[0045] The effect of each formulation on the viability of biocontrol bacteria Talamoyces kabodanensis after 60 days of storage at 25°C was determined using the dilution plating method.

[0046] Viable bacteria rate (%) = (number of viable bacteria after storage / number of viable bacteria before storage) × 100%.

[0047] Table 1. Effects of different formulations on the viability of biocontrol bacteria *Talaromyces kabodanensis* after 60 days of storage at 25°C.

[0048]

[0049] The experimental results showed that Formula 2 had the highest viable bacteria rate, reaching 71.2%, and was determined to be the optimal formula.

[0050] Example 2: Effect of composite protectant on the storage stability of biocontrol bacterium *Talaromyces kabodanensis*

[0051] 2.1 Test Materials

[0052] Biocontrol bacterium Talaromyces kabodanensis;

[0053] Protective agent: Formulation 2 in Example 1.

[0054] 2.2 Preparation of microbial agents

[0055] 2.2.1 Preparation of biocontrol agent for the experimental group: The biocontrol agent containing formulation 2 in Example 1 was used.

[0056] 2.2.2 Preparation of the control group:

[0057] Blank group: Sterile water was used instead of the protective solution, and the rest of the operation was the same as the above experimental group.

[0058] Control group 1: Betaine was used instead of the protective agent solution, and the rest of the operation was the same as the above experimental group.

[0059] Control group 2: Sorbitol was used instead of the protective agent solution, and the rest of the operation was the same as the above experimental group.

[0060] Control group 3: Glycerin was used instead of the protective solution, and the rest of the operation was the same as the above experimental group.

[0061] Control group 4: Sucrose was used instead of the protective agent solution, and the rest of the operation was the same as the above experimental group.

[0062] 2.2.3 Viability determination

[0063] The number of viable bacteria in the bacterial agent was determined periodically using the dilution plating method.

[0064] Viable bacteria rate (%) = (number of viable bacteria after storage / number of viable bacteria before storage) × 100%.

[0065] 2.2.4 Test Results

[0066] Table 2 Effects of different treatments on the storage stability of biocontrol bacteria

[0067]

[0068] The results showed that after adding the protective agent of the present invention, the viability of biocontrol bacteria *Talaromyces kabodanensis* increased from 52.3% to 86.7% after 90 days of refrigeration at 4°C; and increased from 28.6% to 71.2% after 60 days at room temperature (25°C). This demonstrates that the protective agent of the present invention can significantly improve the stability of biocontrol bacteria *Talaromyces kabodanensis*.

[0069] Example 3: Effect of composite protectant on shelf life of biocontrol bacterium *Talaromyces kabodanensis*

[0070] 3.1 Test Methods

[0071] The biocontrol agent prepared in Example 1 and the control group were stored at a constant temperature of 25°C. The viability rate was measured every 10 days, and the shelf life was terminated when the viability rate dropped to below 70%.

[0072] 3.2 Test Results

[0073] Table 3. Changes in viable cell rate (%) during storage at 25℃ under different treatments

[0074]

[0075] The results showed that the shelf life (viable bacterial rate ≥70%) of the blank group was approximately 10 days; the shelf lives of the single protectant control groups (based on viable bacterial rate ≥70%) were approximately 25 days for control group 1, 20 days for control group 2, 20 days for control group 3, and 30 days for control group 4; while the shelf life of the experimental group was as long as approximately 60 days (viable bacterial rate was 71.2% at 60 days). If calculated based on a viable bacterial rate ≥50%, the shelf life of the experimental group could be extended to over 100 days. This indicates that the compound protectant of the present invention significantly extends the shelf life of the biocontrol agent.

[0076] Example 4: Effect of compound protectant on the field colonization ability of biocontrol fungus Talaromyces kabodanensis

[0077] 1. Test location: Strawberry greenhouse in Jiangsu Agricultural Expo Park, Baitu Town, Jurong City, Zhenjiang City, Jiangsu Province;

[0078] 2. Strawberry variety tested: Hongyan, two-year-old seedling.

[0079] 3. Test treatment

[0080] (1) Blank group: Only biocontrol agent of Talamoyces kabodanensis (1 dose of live bacteria (118 live bacteria, for two years) was applied by root irrigation, 100 mL per plant.

[0081] (2) Control group: The control group 1 to control group 4 described in Example 2 were applied by root irrigation, 100 mL per plant.

[0082] (3) Experimental group: The experimental group in Example 2 was applied by root irrigation, 100 mL per plant.

[0083] 4. Field survival rate determination

[0084] Seven days after application, rhizosphere soil samples were collected, and the viable count of biocontrol bacteria Talaromyces kabodanensis was isolated and counted using selective medium (PDA medium containing 50 mg / L ampicillin and 50 mg / L rifampin).

[0085] Field survival rate (%) = (Number of viable bacteria in the rhizosphere on day 7 / Number of viable bacteria initially applied) × Number of viable bacteria initially applied.

[0086] 5. Test Results

[0087] Table 4. Effects of different treatments on the field colonization ability of biocontrol bacteria.

[0088]

[0089] The results showed that the survival rate of the control group was 41.5% 7 days after application in the field, while that of the experimental group was 78.3%. This indicates that the compound protectant significantly enhanced the colonization and survival ability of Talaromyces kabodanensis in the field rhizosphere environment.

[0090] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A type of enhanced biocontrol bacteria Talaromyces kabodanensis A stability protectant, characterized in that, It is mainly composed of the following ingredients in parts by weight: 0.5-1.0 parts betaine, 2-3 parts sorbitol, 1-2 parts glycerol, and 2-4 parts sucrose.

2. The enhanced biocontrol bacteria according to claim 1 Talaromyces kabodanensis A stability protectant, characterized in that, It is mainly composed of the following ingredients in parts by weight: 1.0 part betaine, 2 parts sorbitol, 1.5 parts glycerin, and 2.5 parts sucrose.

3. A biocontrol agent, characterized in that, Contains the enhanced biocontrol bacteria as described in claim 1 or 2 Talaromyces kabodanensis Stability protectants and biocontrol bacteria Talaromyces kabodanensis The weight ratio of the protective agent to the biocontrol bacteria is 7:

93.

4. The biocontrol agent according to claim 3, characterized in that, In the biocontrol agent, biocontrol bacteria Talaromyces kabodanensis The number of live bacteria is not less than 1 and the number of live bacteria is not less than CFU / mL.

5. The biocontrol agent according to claim 4, characterized in that, The biocontrol agent can be formulated into a form acceptable to microbial pesticides.

6. The biocontrol agent according to claim 4, characterized in that, The dosage forms include liquid bacterial agents, wettable powders, water-dispersible granules, or solid granules.

7. A method for preparing the biocontrol agent according to any one of claims 3-6, characterized in that, Includes the following steps: (1) Activation of bacterial strains: Biocontrol bacteria Talaromyces kabodanensis Inoculate into PDA medium and culture 25 cells in the dark for 3-5 days; (2) Seed culture preparation: Take mycelial blocks of activated strains, inoculate them into PDB liquid medium, and culture at 25°C and 160 rpm for 5-7 days to obtain seed culture; (3) Fermentation culture: Inoculate the seed liquid into the fermentation medium at an inoculation rate of 5-10%, and culture at 160 rpm for 7-10 days to obtain the fermentation broth; (4) Collection of bacterial culture: After centrifuging and filtering the fermentation broth, the filtrate is collected to obtain bacterial culture. (5) Preparation of protective agent: Weigh betaine, sorbitol, glycerol and sucrose according to the weight ratio of claim 1 or 2, dissolve them in sterile water to obtain protective agent solution; (6) Forming of bacterial agent: Mix the bacterial liquid and the protective agent solution evenly to obtain the liquid bacterial agent.

8. The method for preparing the biocontrol agent according to claim 7, characterized in that, In step (3), the fermentation medium consists of: 200 g / L potato starch, 20 g / L maltose, 5 g / L peptone, 3 g / L yeast extract, 1 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, and pH 5.5~6.0.

Images