Preparation method of microbial inoculum of mycorrhizal fungi wax mushroom bicolor

Abstract

The invention discloses a preparation method of a microbial inoculum of mycorrhizal fungi wax mushroom bicolor. The preparation method of the microbial inoculum mainly comprises preparation of a culture medium and preparation of the microbial inoculum. The formula of the liquid culture medium is as follows: 10g of glucose, 2g of maltose, 1g of yeast extract (yeast extract), 0.5 g of monopotassium phosphate, 0.25 g of diammonium hydrogen phosphate, 0.15 g of magnesium sulfate, 5mL of calcium chloride dihydrate with the concentration of 1%, 2.5 mL of sodium chloride with the concentration of 1% and 1.2 mL of ferric chloride with the concentration of 1% are dissolved in each liter of distilled water. The formula for preparing the solid culture medium is consistent with that of the liquid culture medium, and 15 g of agar needs to be added per liter. The culture medium needs high-temperature and high-pressure sterilization. The method comprises the following steps: cutting a solid microbial inoculum into small blocks of about 1cm < 3 >, putting one small block in the center of a culture dish, pasting one side with hyphae on a culture medium, sealing with a sealing film, and culturing in a dark place to obtain the solid microbial inoculum. In the culture of the liquid fungicide, small solid fungicide blocks with hyphae need to be put into a sterilized liquid culture medium, 15-20 small blocks are put into each conical flask (250mL conical flask, and 200mL liquid culture medium is put into each conical flask), and the conical flasks in which the fungicide blocks are put are put into a shaking table to be cultured in a dark place at the temperature of 25 DEG C and the rotating speed of 160 revolutions. The method provided by the invention can be used for rapidly and massively preparing the bicolor wax mushroom microbial inoculum, and is convenient for experiment and production practice of the bicolor wax mushroom microbial inoculum. The culture medium is low in cost, and the microbial inoculum is stable in quality.

Description

Technical Field

[0001] The invention relates to a method for preparing a bacterial agent of mycorrhizal fungus wax mushroom. Background Art

[0002] At present, people are more and more aware of environmental protection, but the requirements for production development are getting higher and higher. The application of a large amount of chemical fertilizers in production will bring many adverse effects on the environment and human health, and microbial fertilizer is an environmentally friendly and safe fertilizer. Therefore, the demand for microbial fertilizer is increasing. Laccaria bicolor is an ectomycorrhizal fungus belonging to the Basidiomycotina, Tricholomataceae, and Laccaria. This fungus is distributed all over the world and is very common in forest nurseries and mature forest stands. It can colonize the roots of plants, promote the formation of plant lateral roots, increase the absorption of nutrients in the soil by plants, and has a significant growth-promoting effect on host plants. It can also improve the resistance of plants to abiotic adversities such as heavy metals and salt stress. It is an excellent ectomycorrhizal fungus. The fungus can be cultured in pure culture and used to make biological fertilizers, but it is still less used in production and has a low yield. In order to facilitate the research and promotion of this excellent strain, it is of great significance to screen a method suitable for mass cultivation of bicolor wax mushroom agents. Summary of the invention

[0003] In order to facilitate the research and promotion of the excellent strain of wax mushroom, and to overcome the shortcomings of the existing culture medium formula and culture method, the purpose of the present invention is to provide a method for efficiently preparing a wax mushroom agent, so that the culture cycle of the wax mushroom agent is shorter and the quality is higher, which helps the promotion, research and application of the strain and provides technical guarantee for the production of biological fertilizer.

[0004] The technical solution adopted by the present invention is described as follows.

[0005] The formula of the liquid culture medium is: 10g glucose, 2g maltose, 1g yeast extract (yeast essence), 0.5g potassium dihydrogen phosphate, 0.25g diammonium hydrogen phosphate, 0.15g magnesium sulfate, 5mL 1% calcium chloride dihydrate, 2.5mL 1% sodium chloride, and 1.2mL 1% ferric chloride dissolved in each liter of distilled water.

[0006] The preparation of solid culture medium is consistent with the above liquid culture medium formula, and 15g of agar is added per liter.

[0007] Solid inoculum culture: cut the inoculum into 1 cm 3 Take a small piece and place it in the center of the culture dish with the culture medium, with the side with mycelium attached to the culture medium. Seal it with a sealing film and culture it away from light.

[0008] For the cultivation of liquid inoculants, small pieces of solid inoculants with mycelium are placed into sterilized liquid culture medium, with 15-20 small pieces placed in each bottle (250 mL conical flask, each conical flask contains 200 mL of liquid culture medium). The conical flasks with the inoculum pieces are placed in a shaker for light-proof cultivation at 25°C and 160 rpm. DETAILED DESCRIPTION

[0009] The implementation method of the present invention is described in detail below.

[0010] 1. Preparation of liquid culture medium: Dissolve 10g glucose, 2g maltose, 1g yeast extract (yeast essence), 0.5g potassium dihydrogen phosphate, 0.25g diammonium hydrogen phosphate, 0.15g magnesium sulfate, 5mL 1% calcium chloride dihydrate, 2.5mL 1% sodium chloride, and 1.2mL 1% ferric chloride in each liter of distilled water.

[0011] 2. Preparation of solid culture medium: Dissolve 10g glucose, 2g maltose, 1g yeast extract (yeast essence), 0.5g potassium dihydrogen phosphate, 0.25g diammonium hydrogen phosphate, 0.15g magnesium sulfate, 5mL 1% calcium chloride dihydrate, 2.5mL 1% sodium chloride, 1.2mL 1% ferric chloride, and 15g agar in each liter of distilled water.

[0012] The prepared solid and liquid culture media need to be divided into 250mL conical flasks, with 200mL of liquid culture medium in each bottle and 3g of agar added to each bottle of solid culture medium. After sealing with sealing film, place in an autoclave and sterilize with high-pressure steam at 121℃ for 20 minutes.

[0013] The sterilized solid culture medium needs to be dispensed into sterilized culture dishes while hot, and the culture dishes and liquid culture medium need to be placed on a sterile operating table to room temperature for inoculation.

[0014] 3. Cultivation method of solid bacterial agent: Cut the cultivated solid bacterial agent into small pieces of about 1cm3, take a small piece and place it in the center of the culture dish with the culture medium, with the side with hyphae attached to the culture medium, seal it with sealing film and culture it in the dark. Cultivate for 7-10 days. When the hyphae are about to cover the surface of the culture medium, it can be used for the preparation of the next batch of bacterial agents. The solid bacterial agents that are not used temporarily should be stored in a 4℃ refrigerator in the dark.

[0015] 4. Cultivation method of liquid bacterial agent: put small pieces of solid bacterial agent with mycelium into the sterilized liquid culture medium, put 15-20 small pieces in each bottle (250mL conical flask, each conical flask puts 200mL liquid culture medium), seal the conical flask with bacterial blocks with sealing film, and place it in a shaker for light-proof cultivation at 25℃ and 160 rpm. Cultivate for 7-10 days, when the agar pieces in the liquid culture medium grow into spherical colonies, and the volume of all bacterial balls in the bottle accounts for more than 2 / 3 of the volume of the liquid culture medium, the liquid bacterial agent is mature and can be used for experimental inoculation or production practice. Liquid bacterial agents that are not used temporarily should be stored in a 4℃ refrigerator away from light.

Claims

1. A method for preparing a bacterial agent for the mycorrhizal fungus wax mushroom. The formula of the liquid culture medium is: 10g glucose, 2g maltose, 1g yeast extract (yeast essence), 0.5g potassium dihydrogen phosphate, 0.25g diammonium hydrogen phosphate, 0.15g magnesium sulfate, 5mL 1% calcium chloride dihydrate, 2.5mL 1% sodium chloride, 1.2mL 1% ferric chloride. The solid culture medium is made in accordance with the formula of the liquid culture medium, and 15g agar is added per liter. The culture medium needs to be sterilized at high temperature and high pressure. The solid bacterial agent is cut into small pieces of about 1cm3, a small piece is taken and placed in the center of the culture dish with the culture medium poured, and the side with mycelium is attached to the culture medium, and the sealing film is sealed and cultured in the dark to cultivate the solid bacterial agent. For the cultivation of liquid inoculants, small pieces of solid inoculants with mycelium need to be placed into sterilized liquid culture medium. 15-20 small pieces should be placed in each bottle (250mL conical flask, each conical flask contains 200mL liquid culture medium). The conical flasks with the inoculum pieces should be placed in a shaker for light-proof cultivation at 25°C and 160 rpm.

2. The method for preparing a bacterial agent of the mycorrhizal fungus Lachnos nucifera according to claim 1, characterized in that: When preparing liquid culture medium, the solution needs to be heated to 60-70°C to accelerate dissolution.

3. The method for preparing a bacterial agent of the mycorrhizal fungus Lachnos bicolor according to claim 1, characterized in that: When making solid culture medium, the 15 g agar required for 1 liter of culture medium should be divided into 5 250 mL conical flasks, 3 g in each flask, and then 200 mL of liquid culture medium should be poured in.

4. The method for preparing a bacterial agent of the mycorrhizal fungus Lachnos bicolor according to claim 1, characterized in that: All culture media must be placed in conical flasks, sealed with sealing film, and placed in an autoclave for high-pressure steam sterilization at 121°C for 20 minutes.

5. The method for preparing a bacterial agent of the mycorrhizal fungus Lachnos bicolor according to claim 1, characterized in that: The preparation process of solid and liquid inoculants must be carried out on a sterile operating table, and the operation process must be strictly sterilized. The solid and liquid inoculants should be sealed in time after inoculation to prevent the incorporation of foreign bacteria into the cultured bicolor wax mushroom inoculants.

6. The method for preparing a bacterial agent of the mycorrhizal fungus Lachnos nucifera according to claim 1, characterized in that: When cultivating solid bacterial agents, the solid bacterial agents cut into small pieces need to be attached with the mycelium side to the surface of the new culture medium to ensure that the mycelium can smoothly contact the culture medium and grow and reproduce on the new culture medium as soon as possible.

7. The method for preparing a bacterial agent of the mycorrhizal fungus Lachnos bicolor according to claim 1, characterized in that: Solid culture medium (culture dish) should be cultured at room temperature away from light for 7-10 days. When the mycelium is almost growing all over the surface of the culture medium, it can be used to prepare the next batch of bacterial agents. Solid bacterial agents that are not used temporarily should be stored away from light in a 4°C refrigerator.

8. The method for preparing a bacterial agent of the mycorrhizal fungus Lachnos bicolor according to claim 1, characterized in that: The liquid culture medium should be placed in a shaker and cultured in the dark at 25°C and 160 rpm for 7-10 days. When the agar pieces in the liquid culture medium grow into spherical colonies and the volume of all bacterial balls in the bottle accounts for more than 2 / 3 of the volume of the liquid culture medium, the liquid culture agent is mature and can be used for experimental inoculation or production practice. Liquid culture agents that are not used temporarily should be stored in a 4°C refrigerator away from light.