Optimization and fermentation method of dominant beauveria bassiana liquid fermentation medium for preventing and treating dioryctria abietella

Abstract

The invention relates to an optimization and culture method of a dominant beauveria bassiana liquid fermentation culture medium for preventing and treating Dioryctria abietella. The liquid fermentation culture medium comprises the following components: 40g of soluble starch, 10g of yeast extract, 2g of anhydrous calcium chloride, 5g of yeast powder and 1000ml of distilled water. Compared with an original culture medium, the optimized liquid fermentation culture medium has the advantage that the hypha growth amount of beauveria bassiana CFCC81428 is obviously increased.

Description

Technical Field

[0001] This invention relates to the field of microbial insecticides, and in particular to an optimization of the liquid fermentation culture medium and fermentation method for controlling the fir shoot borer Beauveria bassiana. Background Technology

[0002] Beauveria bassiana, with its wide host range, strong pathogenicity, ease of cultivation, good control effect, and safety and non-toxicity to humans, livestock, and crops, has broad application prospects in the field of biological control. Using Beauveria bassiana spores as the active ingredient in insecticides, and adding carriers and various adjuvants to prepare Beauveria bassiana preparations, facilitates application and enhances the insecticidal activity of the infectants. A large quantity of Beauveria bassiana spores is produced through liquid-solid mixed fermentation. The focus of this invention is to improve the mycelium produced by the liquid fermentation of Beauveria bassiana by screening the carbon source, nitrogen source, and inorganic salts in the culture medium, thereby increasing the production of more Beauveria bassiana conidia in the subsequent solid-state fermentation. Summary of the Invention

[0003] The purpose of this invention is to provide a liquid culture medium that improves the mycelium production of Beauveria bassiana during liquid fermentation.

[0004] Another objective of this invention is to provide a fermentation method for improving the mycelium produced by the liquid fermentation of Beauveria bassiana.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] A culture medium for improving the mycelium and budding spores produced by liquid fermentation of Beauveria bassiana is mainly composed of the following weight components: 40 g / L soluble starch, 10 g / L yeast extract, 2 g / L anhydrous calcium chloride, and 5 g / L yeast powder.

[0007] Carbon sources, nitrogen sources, and trace elements are essential nutrients for the normal growth of Beauveria bassiana. The culture medium containing these components used in this invention is readily available and inexpensive.

[0008] The optimization of the fermentation medium and fermentation method for Beauveria bassiana includes the following steps:

[0009] 1) Activate the slant culture in liquid Sacchari medium, and culture it on a shaker at 28°C and 160 r / min for three days. Take out the culture and spread it on PDA medium, and then place it in an incubator at 25°C for 7 days.

[0010] 2) Preparation of Beauveria bassiana spore suspension: Rinse the spores on the PDA medium with 0.1% Tween 80 sterile water in a clean bench, filter out the mycelia through a 300-mesh sieve, and dilute the concentration to 1×10⁻⁶. 7 Spores / ml, store at 4℃ for later use.

[0011] 3) Culture medium preparation and sterilization: Prepare liquid culture medium by placing 50 ml into a 150 ml Erlenmeyer flask. Sterilize in an autoclave at 121℃ for 20 minutes, and then cool to room temperature.

[0012] 4) Fermentation method: Inoculate the sterilized culture medium from step 3) with 3% spore suspension and culture it on a shaker at 28°C and 160 r / min for three days.

[0013] 5) Method for weighing the dry weight of mycelium: Dry the filter paper in an oven at 80℃ and measure its weight W1. Filter the fermented liquid using a Buchner funnel connected to a vacuum pump, dry it in an oven at 80℃, and weigh it to obtain its dry weight W2. The dry weight of the mycelium is then determined.

[0014] =(W 2- W1) / 0.05g / L.

[0015] 6) Screening of basal culture media: The spore suspension from step 2) was inoculated at a ratio of 3% into different basal culture media for fermentation. The dry weight of the mycelium was measured, and the optimal basal culture medium was selected.

[0016] 7) Screening of carbon source, nitrogen source and inorganic salt: Replace the carbon source, nitrogen source and inorganic salt in the screened basic culture medium respectively, measure the dry weight of mycelium after fermentation, and screen out the best carbon source, nitrogen source and inorganic salt.

[0017] The technical advantages and positive effects of this invention are as follows:

[0018] 1) Carbon source, nitrogen source and trace elements are essential nutrients for the normal growth of Beauveria bassiana. The culture medium raw materials of the above components used in this invention are abundant and inexpensive.

[0019] 2) The liquid fermentation medium described above can significantly improve the mycelium produced by liquid fermentation. Attached Figure Description

[0020] The invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0021] Figure 1 This is a flowchart of the experimental process of the present invention. Detailed Implementation

[0022] like Figure 1 As shown, the invention mainly includes seven processes, starting with activating the bacterial strain, followed by preparing 1×10 7Spores / ml spore suspension. The spore suspension was inoculated at a ratio of 3% into different basal media for fermentation, and the optimal basal media was screened. Subsequently, the carbon source, nitrogen source, and inorganic salts in the screened basal media were replaced, and the optimal carbon source, nitrogen source, and inorganic salts were selected based on the measured mycelial dry weight after fermentation. Finally, the optimized culture medium formula most suitable for the liquid fermentation of *Beauveria bassiana* was obtained.

Claims

1. An optimized liquid fermentation culture medium and fermentation method for controlling the fir shoot borer using Beauveria bassiana, characterized in that: It contains, by weight percentage: 40 g / L soluble starch, 10 g / L yeast extract, 2 g / L anhydrous calcium chloride, and 5 g / L yeast powder.

2. The Beauveria bassiana liquid fermentation medium according to claim 1, characterized in that: The Beauveria bassiana strain mentioned is Beauveria bassiana strain CFCC81428.

3. A method for liquid fermentation of Beauveria bassiana, characterized by comprising the following steps: 1) Culture of Beauveria bassiana: The slant culture was activated in liquid Saxony medium and cultured on a shaker at 28°C and 160 r / min for three days. The culture was then spread on PDA medium and incubated in a 25°C incubator for 7 days. 2) Preparation of Beauveria bassiana spore suspension: Rinse the spores on the PDA medium with 0.1% Tween 80 sterile water in a clean bench, filter out the mycelia through a 300-mesh sieve, and dilute the concentration to 1×10⁻⁶. 7 Spores / ml. 3) Liquid fermentation: After sterilizing the liquid fermentation medium of claim 1, inoculate the spore suspension in 2) under aseptic conditions.

4. The liquid fermentation method for Beauveria bassiana according to claim 3, characterized in that, The conical flask used in step 3) is 150ml, and 50ml of liquid fermentation culture medium is placed inside.

5. The liquid fermentation method for Beauveria bassiana according to claim 3, characterized in that, In step 3), the concentration of the spore suspension is 1×10⁻⁶. 7 Spores / ml.

6. The liquid fermentation method for Beauveria bassiana according to claim 3, characterized in that, In step 3), the inoculation amount is 3% of the culture medium, i.e., 1.5 ml.

7. The liquid fermentation method for Beauveria bassiana according to claim 3, characterized in that, In step 3), the shaking speed is 160 r / min.

8. The liquid fermentation method for Beauveria bassiana according to claim 3, characterized in that, The fermentation temperature in step 3) is 28℃.

9. A liquid fermentation method for Beauveria bassiana according to claims 3-8, characterized in that, The Beauveria bassiana strain was CFCC81428.

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