CN1236049C - Method for preparing forest mycorrhizal fungi preparation from different area and raw material - Google Patents

Abstract

The invention relates to a method for preparing forest mycorrhizal fungus inoculants with different regions and sources of raw materials. After selecting and matching substrate sawdust, bran, perlite, peat, etc., they are mixed according to weight ratio, and inorganic salts and some nutritional elements are added to the preparation method. According to the water content of the substrate, the nutrient solution is prepared as a nutrient solution for mixing materials. After the material solution is evenly mixed, it is packaged and sealed, and sterilized by pressurized steam; after cooling, it will be produced by secondary biological fermentation in advance. Bacteria of 2-10% were inoculated in the matrix, and cultured at a constant temperature for 10-30 days. The invention is not limited by raw material sources and regions, has low cost and is suitable for popularization and application.

Description

A kind of preparation method of forest mycorrhizal fungus inoculum with different regions and raw material sources

technical field

The invention relates to fungal inoculum, in particular to a method for preparing forest mycorrhizal fungal inoculum with different regions and sources of raw materials.

Background technique

Mycorrhiza is a common phenomenon in the symbiosis of fungi and plants in nature. Mycorrhizae can promote the absorption of water and nutrients by plants, produce biologically active substances, and enhance the stress resistance of plants. For some plants or under specific environmental conditions, the existence of mycorrhizae will become a determinant factor affecting plant growth and development. Therefore, as a biotechnology, mycorrhizal is widely used in agricultural and forestry production. The nutrient mycelium of mycorrhizal fungi under the state of pure culture is used for artificial inoculation to accelerate the formation of seedling mycorrhizae to promote the growth of seedlings, thereby improving the quality of seedlings. Studying the nutritional conditions of mycorrhizal fungi and the production technology of bacterial agents to realize artificial mycorrhizalization of forest tree seedlings has broad application prospects in breeding and afforestation, especially in afforestation of barren hills, which has an irreplaceable role. Therefore, it has attracted great attention from mycorrhizal experts and scholars at home and abroad, and has done a lot of work in introduction, seedling cultivation, afforestation and environmental protection, and has achieved remarkable results. However, when mycorrhizal technology is applied in production, the preparation of bacterial agents is often completed in the laboratory, mainly using 60-70% of the substrate weight of bran as a nitrogen source or the following waste rice bran as a nitrogen source supplement, carbon source with miscellaneous Sawdust is the main method, and the bacterial agent needs to be carried or consigned to the actual site for application, resulting in high cost of the bacterial agent, which severely restricts the promotion and application of this practical new technology.

Contents of the invention

In order to overcome the above-mentioned problems, the purpose of the present invention is to provide a method for preparing forest mycorrhizal fungal agents with low cost, not limited by regions, and suitable for popularization and application.

In order to achieve the above object, the technical scheme of the present invention is: mix and prepare each component by weight ratio, can prepare according to the following process:

1) Matrix mix: the matrix consists of 3-4 parts of sawdust mixed with 1-2 parts of bran;

2) Preparation of nutrient solution: add 0.1-0.5g of diammonium hydrogen phosphate, 2-10g of glucose, 0.01-0.08g of calcium chloride, 0.01-0.2g of magnesium sulfate, and 0.001-0.005g of ferric chloride into 1 liter of water , sodium chloride 0.05-0.3g, vitamin B ₁ 0.01-0.05g and potassium dihydrogen phosphate 0.5-5g and/or dipotassium hydrogen phosphate 0.32-3.2g to make a nutrient solution;

3) Sterilization: Mix the above-mentioned nutrient solution with the matrix so that the water content of the mixed matrix is 50-70%, pack and seal, and steam sterilize under pressure at 105-125°C for 1-2 hours. cooling standby;

4) Bacteria preparation: take the strains and culture medium after the primary culture, mix the strains and the culture medium in a ratio of 2-5:100, and prepare the strains by the secondary biological fermentation method;

5) Bacteria agent preparation: inoculate the sterilized matrix with 2-10% of the bacteria body amount, and culture at a constant temperature of 23-30° C. for 10-30 days.

Also can add the filler that accounts for 10-70% of substrate gross weight in the described substrate, and filler is one or more in peat, wheat orange, rice straw, rice husk, corn stalk; Add 10-50% perlite accounting for the total weight of the substrate as a carrier; one or more of the following substances can also be selected to be added in the preparation of the nutrient solution: 0.05-0.3g of yeast extract, 1.0-3.0g of peptone, and the concentration of ferrous sulfate is 10-50ppm, zinc sulfate concentration is 10-50ppm, sodium molybdate concentration is 10-50ppm;

The sawdust is one or a mixture of birch sawdust, larch sawdust, maple sawdust, masson pine sawdust;

When the sawdust is coniferous sawdust, it needs to be pre-steamed with steam at 120 degrees Celsius for more than 30 minutes or at normal pressure for more than 2 hours before mixing the matrix, and the gas is discharged at the same time.

The strains are forest mycorrhizal fungi: one or more of the following types of milky mushrooms, burr sarcophagus, gray ring boletus, pine cone bovine liver, high ring peduncle, and chrysostomus.

The present invention has the following advantages

1. Wide range of material sources and low cost. The material that the present invention prepares bacterial agent is the waste material that produces in the wood processing process---sawdust, the leftovers of agricultural crops (as wheat orange, straw, rice husk, corn stalk) and building material perlite, the peat of swamp, these Compared with the prior art, which mainly adopts 60-70% of the substrate weight of bran as a nitrogen source or the following waste rice bran as a nitrogen source supplement, the material does not require high investment and is easy to obtain.

2. The operation of the present invention is not restricted by region. Due to the characteristics of symbiotic nutrition and saprophytic characteristics of forest mycorrhizal fungi, the wood chips of trees are naturally the preferred carrier substrate for their growth and development. However, due to differences in regions, the types of raw materials are not the same. The present invention adjusts the technology for preparing bacterial agents: pretreating unsuitable coniferous sawdust materials; selecting and matching local abundant materials to reduce costs, The practical new technology can be widely popularized and applied by adjusting the selection ratio and dosage of various nutritional components adapted to it.

3. The present invention can adapt measures to local conditions, obtain local materials, simplify due to leakage, and process and apply locally, which will further enhance the local adaptability of the bacterial agent, and can be widely used.

Detailed ways

Below in conjunction with embodiment the present invention is described in further detail:

Embodiment 1. adopts larch sawdust as the inoculant prepared by matrix (mainly used in Greater/Little Khingan Mountains)

Its preparation process is as follows:

(1) Substrate ratio: take 70% of larch sawdust for pre-steaming, and discharge the air at normal pressure for 2 hours to volatilize the aromatic substances in the substrate, and identify them by the degree of aroma, then take 30% of bran and larch sawdust Mix by weight;

(2) Nutrient solution mixed with substrate: add 0.2g of diammonium hydrogen phosphate, 5g of glucose, 0.05g of calcium chloride, 0.05g of sodium chloride, 3g of potassium dihydrogen phosphate, 1.0g of peptone, and 0.05g of magnesium sulfate in 1 liter of water , ferric chloride 0.004g, zinc sulfate concentration is 10ppm, sodium molybdate concentration is 10ppm and vitamin B ₁ 0.05g, makes matrix water content reach 65%;

(3) Sterilization: bottle the mixed matrix with a suitable solid-liquid ratio, seal it, and steam sterilize it at 110°C for 2 hours at a pressure slightly higher than one atmosphere, and cool it for later use;

(4) Bacteria production: Inoculate the strain after primary culture (incline culture) with the culture medium at a ratio of 2:100, and produce it by biological fermentation (shaking table shaking culture), specifically take Hebeloma mesophaeum , Suillus aeruginascens and Cortinarius russus, in 1 liter of water add: potato 100g, glucose 10g, potassium dihydrogen phosphate 1g, magnesium sulfate 0.5g, vitamin B ₁ 0.02 g, make culture medium, be that 100ml is subpackaged in the Erlenmeyer flask of 500ml with volume, shake constant temperature culture with shaking table for 3 days;

(5) Bacteria agent: Inoculate the prepared bacteria in the above-mentioned solid substrate in an amount of 7%, and culture at a constant temperature of 28° C. for 15 days.

Embodiment 2. adopt perlite, peat as the inoculant prepared by matrix (mainly used in cold temperate zone and north temperate zone)

(1) Substrate proportioning: mix 25% of birch sawdust, 40% of perlite, 20% of peat, and 15% of bran by weight;

(2) Nutrient solution mixed with substrate: Add 0.3g of diammonium hydrogen phosphate, 10.0g of glucose, 3.0g of peptone, 0.08g of calcium chloride, 0.2g of sodium chloride, 2.0g of potassium dihydrogen phosphate, and magnesium sulfate to 1 liter of water 0.01g, 0.005g of ferric chloride, and 0.04g of vitamin _B1 , so that the water content of the matrix reaches 70%;

(3) Sterilization: bag and seal the substrate with a suitable mixed solid-liquid ratio, steam sterilize at 115°C slightly higher than one atmospheric pressure for 1.5 hours, and cool it for later use;

(4) Bacterial production: mix the first-level cultured bacteria with the culture medium at a ratio of 5:100, and produce it by biological fermentation (shaking table shaking culture), specifically, Lactarius insulsus and burr slippery rust Hebeloma mesophaeum and Cortinarius russus; in 1 liter of water, add: 300g of potatoes, 20g of glucose, 3g of potassium dihydrogen phosphate, 1.5g of magnesium sulfate, 0.05g of vitamin _B1 , and make a culture Base, with a volume of 130ml, divided into 500ml Erlenmeyer flasks, and cultured at a constant temperature on a shaker for 5 days;

(5) Bacteria agent: Inoculate the prepared bacterium in the above-mentioned solid substrate in an amount of 10%, and culture at a constant temperature of 23° C. for 30 days.

Embodiment 3. adopt masson pine sawdust as the inoculant prepared by matrix (mainly used in Guangxi Yulin area)

(1) Pretreatment of masson pine sawdust: take 60% of masson pine sawdust and pre-steam at 120 degrees Celsius for 30 minutes, then discharge the gas, so that the aromatic substances in the matrix can be volatilized, and the degree of aroma can be used to identify it, and then take 40% of the bran Mix with masson pine sawdust in weight ratio;

(2) Nutrient solution mixed with substrate: add 0.1 g of diammonium hydrogen phosphate, 4.0 g of glucose, 0.03 g of calcium chloride, 0.1 g of sodium chloride, 3.0 g of potassium dihydrogen phosphate, and 0.2 g of yeast extract in 1 liter of water, Magnesium sulfate 0.1g, iron trichloride 0.001g, vitamin B ₁ 0.04, the concentration of zinc sulfate is 20ppm, the concentration of sodium molybdate is 30ppm, so that the water content of the matrix reaches 60%;

(3) Sterilization: bottle and seal the substrate with a suitable mixed solid-liquid ratio, and steam sterilize it at 113°C slightly higher than one atmospheric pressure for 1.8 hours, and cool it for later use;

(4) Bacteria production: inoculate the strain after primary culture with the culture medium at a ratio of 4:100, and produce it by biological fermentation (shaking table shaking culture), specifically take Cortinarius russus, burr slippery rust Umbrella (Hebeloma mesophaeum), Lactarius insulsus and Lepiota procera; add medium to 1 liter of water: potatoes 200 g, glucose 15 g, wort 4 degrees Baume, potassium dihydrogen phosphate 4g, 2.5g of magnesium sulfate, and 0.04g of vitamin _B1 were used to make a culture medium, which was divided into 500ml Erlenmeyer flasks with a volume of 120ml, and cultured on a shaker at a constant temperature for 7 days;

(5) Bacteria agent: inoculate the prepared bacteria in the above-mentioned solid substrate in an amount of 5%, and culture at a constant temperature of 27° C. for 20 days.

Embodiment 4. adopt the inoculation agent (mainly used in non-forest area) of agricultural by-product leftovers as substrate preparation

(1) Substrate proportioning: get maple sawdust 30%, rice husk 45%, straw 15%, bran 10% and mix by weight;

(2) Nutrient solution mixed with substrate: Add 0.4g of diammonium hydrogen phosphate, 8g of glucose, 0.01g of calcium chloride, 0.3g of sodium chloride, 2.0g of peptone, 0.3g of yeast extract, and 1g of potassium dihydrogenphosphate to 1 liter of water , dipotassium hydrogen phosphate 2g, magnesium sulfate 0.2g, ferrous sulfate concentration is 50ppm, ferric chloride 0.002g and vitamin B ₁ 0.02g, make the matrix water content reach 50%;

(3) Sterilization: Pack the substrate with a suitable mixed solid-liquid ratio into bags or seal the bottle, perform steam sterilization at 121°C and 1.5 atmospheres for 1.0 hour, and cool for later use;

(4) Bacteria production: inoculate the strain after primary culture with the culture medium at a ratio of 3:100, and produce by biological fermentation (shaking table shaking culture). Specifically get Hebelomamesophaeum (Hebelomamesophaeum), bad taste milk mushroom (Lactarius insulsus) and pine cone beef liver (Boletus floccopus); add in 1 liter of water: potato 100g, glucose 18g, potassium dihydrogen phosphate 5g, magnesium sulfate 2g, Vitamin B ₁ 0.03g, made into a culture medium, divided into 500ml Erlenmeyer flasks with a volume of 140ml, and cultured on a shaker for 5 days;

(5) Bacteria agent: inoculate the prepared bacteria in the above-mentioned solid substrate in an amount of 4%, and culture at 25° C. for 25 days.

Utilize the bacterial agent of the present invention, apply in the restoration and renewal of the forest resources of Daxing'an Mountains burnt land, the cultivated mycorrhized seedlings are planted on the burned land, the survival rate of the year reaches more than 95%; the high growth of the year, the growth rate of Xing'an larix is higher than that of the control 80%, Pinus sylvestris increased by 128% compared with the control. Utilize the bacterium agent of the present invention, make the masson pine seedling raising of state-owned 60,000 forest farms in Yulin, Guangxi, change the stubble once every 2-3 years from the original annual stubble change, and save 400-500 yuan/mu of expenditure for the forest farm.

Claims (7)

1. the preparation method of the area forest mycorrhizal fungi microbial inoculum different with raw material sources is characterized in that by weight each component of mixed preparing, according to the following procedure preparation:

1) the matrix collocation mixes: matrix comprises that 3-4 part wood chip and 1-2 part wheat bran mix;

2) nutritive medium preparation: by adding Secondary ammonium phosphate 0.1-0.5g, glucose 2-10g, calcium chloride 0.01-0.08g, sal epsom 0.01-0.2g, iron trichloride 0.001-0.005g, sodium-chlor 0.05-0.3g, vitamins B in the 1 liter water ₁0.01-0.05g be made into nutritive medium with potassium primary phosphate 0.5-5g and/or dipotassium hydrogen phosphate 0.32-3.2g;

3) sterilization: above-mentioned nutritive medium and matrix are puddled, and making the water content of substrate after puddling is 50-70%, and packing is sealed, and carries out under 105-125 ℃ steam sterilizing 1-2 hour, cools off standby;

4) thalline preparation: bacterial classification and substratum after the one-level of learning from else's experience is cultivated, be 2-5 with bacterial classification with the blending ratio of substratum: 100 mix, and produce thalline with the two stage biological fermentation method;

5) fungicide preparation: the matrix after will sterilizing was inoculated with the biomass of its 2-10%, at 23-30 ℃ of following constant temperature culture 10-30 days.

2. according to the preparation method of the described forest mycorrhizal fungi of claim 1 microbial inoculum, it is characterized in that: also can add the adding of 10-70% that accounts for the matrix gross weight in the described matrix and fill agent, add fill agent be in the peat composed of rotten mosses, wheat tangerine, straw, rice husk, the corn stalk one or more.

3. according to the preparation method of the described forest mycorrhizal fungi of claim 1 microbial inoculum, it is characterized in that: also can add in the described matrix and account for matrix gross weight 10-50% perlite as carrier.

4. according to the preparation method of the described forest mycorrhizal fungi of claim 1 microbial inoculum, it is characterized in that: also can select to add one or more following materials in the described nutritive medium preparation: yeast extract paste 0.05-0.3g, peptone 1.0-3.0g, ferrous sulfate concentration is 10-50ppm, zinc sulfate concentration is 10-50ppm, and Sodium orthomolybdate concentration is 10-50ppm.

5. according to the preparation method of the described forest mycorrhizal fungi of claim 1 microbial inoculum, it is characterized in that: described wood chip is one or more mixing in birch bits, tamarack wood chip, maple bits, the Pinus massoniana Lamb wood chip.

6. according to the preparation method of claim 1 or 5 described forest mycorrhizal fungi microbial inoculums, it is characterized in that: need when described wood chip is the needle wood chip before mixed substrate, to steam 2 hours in advance, carry out gaseous emission simultaneously with 120 degrees centigrade of pre-steamings of steam 30 minutes or normal pressure.

7. according to the preparation method of the described forest mycorrhizal fungi of claim 1 microbial inoculum, it is characterized in that: described bacterial classification is the forest mycorrhizal fungi: one or more mixing in kakgeusia breast mushroom, the sliding rust of burr umbrella, ash ring Suillus bovines, pinecone beef liver, high handle mushroom, the reddish brown cortina bacterium.