CN112680365B - Liquid culture medium for beauveria bassiana and preparation method of beauveria bassiana microbial inoculum - Google Patents
Liquid culture medium for beauveria bassiana and preparation method of beauveria bassiana microbial inoculum Download PDFInfo
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Abstract
The invention provides a liquid culture medium for beauveria bassiana and a preparation method of a beauveria bassiana microbial inoculum, which are used for realizing the rapid production of beauveria bassiana and are obtained by uniformly mixing fungus chaff, water, manganese sulfate and ammonium nitrate and then sterilizing the mixture by high-pressure steam, wherein 4 to 5ml of water is added into each gram of fungus chaff, 0.03 to 0.05g of manganese sulfate is added into each gram of fungus chaff, and 0.05 to 0.1g of ammonium nitrate is added into each gram of fungus chaff. To a concentration of 10 7 The method comprises the steps of inoculating a beauveria bassiana inoculation solution with spores/ml into a beauveria bassiana liquid culture medium, adding 0.05-0.10 ml of the inoculation solution into each gram of dry fungus chaff, sealing, and culturing in a shaker at 25-27 ℃ at 150rpm for 4-6 days to obtain the beauveria bassiana microbial inoculum.
Description
Technical Field
The invention belongs to the technical field of fermentation culture, and particularly relates to a liquid culture medium for beauveria bassiana and a preparation method of a beauveria bassiana microbial inoculum.
Background
Beauveria bassiana is a fungus of ascomycetes, and can kill pests through body wall contact infection under natural conditions. It can parasitize more than 700 kinds of 15 order 149 families of insects, is safe to human and livestock and environment, is not easy to generate drug resistance of pests, and can be used together with certain chemical pesticides (insecticides, acaricides, bactericides). Up to now, there are 17 kinds of registered products of beauveria bassiana internationally, which are used for controlling pests such as grubs, houseflies, scale insects, whiteflies, aphids, thrips, potato leafbeetles, cockroaches, locusts, grasshoppers, crickets, bollworms, cotton plant bugs, corn borers, longicorn, sugarcane cockchafers and the like. The beauveria bassiana high-spore powder is one of high-efficiency biological insecticides popularized by the national forestry bureau, and can be widely applied to forest pests, vegetable pests, dry-land crop pests and the like. For years, the beauveria bassiana is successfully applied to control nearly 40 agricultural and forestry pests in China, and the beauveria bassiana is mainly used in production nowadays for controlling pine caterpillars, corn borers, grubs, locusts, potato beetles, monochamus alternatus, termites, tea lesser leafhoppers, peach fruit borers and the like. The most successful beauveria bassiana control method is the grub in nursery gardens, lawns, farmlands and the like, and the grub damages various crops such as peanuts, soybeans, lawns, vegetables, nursery gardens and the like. The method is widely applied to the production of organic agricultural products and tea.
The economic and rapid mass propagation production of beauveria bassiana is a precondition for the massive application of beauveria bassiana in biological control practice. At present, scholars at home and abroad try to culture the beauveria bassiana by using local agricultural wastes, and obtain the beauveria bassiana with considerable spore quantity. The mushroom bran is the remainder of a culture medium after edible mushroom cultivation and harvesting, is used as a waste in agricultural production, and mainly comprises edible mushroom mycelia, wherein the cellulose content is about 10% -30%, the crude protein content is about 6% -13%, and the crude fat content is about 1% -3%. If the waste water is not timely treated and utilized, resource waste and environmental pollution are caused. Researches find that the nutrient substances in the waste mushroom bran can be used as nutrients of crops and can also be used for culturing other edible mushrooms. If the waste is utilized, the effect of changing waste into valuable can be achieved.
Disclosure of Invention
The invention aims to provide a liquid culture medium for beauveria bassiana to realize the rapid production of the beauveria bassiana, which is obtained by uniformly mixing fungus chaff, water, manganese sulfate and ammonium nitrate and then sterilizing the mixture by high-pressure steam, wherein 4 to 5ml of water is added into each gram of fungus chaff, 0.03 to 0.05g of manganese sulfate is added into each gram of fungus chaff, and 0.05 to 0.1g of ammonium nitrate is added into each gram of fungus chaff.
As a more preferable technical scheme, the fungus chaff is one or more of fungus chaff, oyster mushroom chaff and pholiota nameko fungus chaff which take wood chips as substrates.
As a more preferable technical scheme, 4ml of water is added into each gram of fungus chaff, 0.03g of manganese sulfate is added into each gram of fungus chaff, and 0.05g of ammonium nitrate is added into each gram of fungus chaff.
As a more excellent technical scheme of the invention, the temperature of the high-pressure steam is 121 ℃, and the steam pressure of water is 0.11MPa.
As a more excellent technical scheme of the invention, the high-pressure steam sterilization time is 30min.
The invention also aims to provide a preparation method of the beauveria bassiana microbial inoculum, which has the advantages of low production cost and short period, realizes the mass culture of the beauveria bassiana and comprises the following specific steps: inoculating the beauveria bassiana inoculation liquid into a beauveria bassiana liquid culture medium, adding 0.05-0.10 ml of inoculation liquid according to per gram of dry fungus chaff, sealing, culturing in a shaker at 25-27 ℃ at 150rpm for 4-6 days, and then harvesting the beauveria bassiana microbial inoculum.
As a more preferable technical scheme of the invention, 0.10ml of inoculation liquid is added into each gram of dry fungus bran.
The preparation method of the beauveria bassiana flat plate comprises the steps of placing beauveria bassiana blocks in a flat plate culture dish filled with a sterilized PDA culture medium by using sterile operation, and culturing in an incubator at 29 ℃ for 7-10 days, and then harvesting.
The beneficial effects are as follows:
compared with the existing solid fermentation, the invention reduces the fermentation time and increases the spore yield, the liquid fermentation in the invention needs 6 days, and the optimal spore concentration is 1.99 multiplied by 10 10 While the number of the obtained bacterial agent spores is 0.2 multiplied by 10 in the common solid fermentation time of 8 to 10 days 10 Left and right.
According to the invention, the liquid culture medium prepared from the fungus chaff and the additive is used for culturing the beauveria bassiana, most of nutrient substances are provided for the growth of the beauveria bassiana, ammonium nitrate and manganese sulfate are used as supplementary substances of the fungus chaff culture medium, an N source and inorganic salt components required by the growth are provided for the proliferation of the beauveria bassiana, and the spore yield is high; meanwhile, the fungus chaff is used as an agricultural waste material, the fungus chaff is economical and easy to obtain, and the application value of the fungus chaff is improved while the biocontrol bacteria are obtained by culturing the paecilomyces lilacinus with the fungus chaff.
The preparation method of the beauveria bassiana microbial inoculum has the advantages of simple operation, clear flow, low requirements on production environment and production equipment, low production cost and short production period, uses a shaking bed culture medium for mixing in the culture process, increases the oxygen supply amount, reduces the pollution problem of mixed bacteria in production, and is very suitable for small enterprises and production units to rapidly produce the beauveria bassiana.
Detailed Description
In order to better understand the present invention, the following examples are further provided to illustrate the content of the present invention, but the content of the present invention is not limited to the following examples, and should not be construed as limiting the scope of the present invention.
The beauveria bassiana is purchased from Guangdong province microorganism strain preservation center and is numbered as 3.428.
Example 1:
the first step is as follows: preparing a fungus chaff culture medium of beauveria bassiana;
placing 2g of fungus bran, 0.06g of manganese sulfate, 0.10g of ammonium nitrate and 8ml of distilled water into a 50ml conical flask, fully mixing with high-pressure steam at 121 ℃, and sterilizing for 30min to obtain the microbial agent.
The second step is that: preparing a beauveria bassiana flat plate;
the beauveria bassiana blocks are aseptically placed in a flat plate culture dish filled with sterilized PDA culture medium, and are cultured in an incubator at 25 ℃ for 10 days and then harvested.
The third step: by washing the plates with Beauveria bassiana to obtain a concentration of 10 7 Inoculating white muscardine fungi with spores/ml;
the fourth step: inoculating the fungus chaff culture medium in the example 1, adding 0.20ml of inoculation liquid into a sterilized 50ml conical flask filled with the fungus chaff culture medium, sealing the bottle mouth with a sealing film, and culturing in a shaking table at 25 ℃ for 6 days for harvesting. (ii) a
The fifth step: the harvested product was measured and filtered using 4 layers of sterile gauze to obtain a filtrate. The number of spores in the filtrate was measured.
The preparation of beauveria bassiana liquid culture medium by using various fungus chaff and sporulation quantity are shown in Table 1
Example 2:
this example differs from example 1 only in that: the volume of distilled water added in the first step was 10ml. The preparation of beauveria bassiana liquid culture medium by using various fungus chaff and sporulation quantity are shown in Table 1
Example 3:
this example differs from example 1 only in that: the mass of ammonium nitrate added in the first step was 0.16g. The preparation of Beauveria bassiana liquid culture medium by using various fungus chaff and the sporulation amount are shown in Table 1
Example 4:
this example differs from example 1 only in that: the mass of manganese sulfate added in the first step was 0.08g. The preparation of Beauveria bassiana liquid culture medium by using various fungus chaff and the sporulation amount are shown in Table 1
Example 5:
this example only differs from example 1 in that: the temperature set in the fourth step was 29 ℃. The preparation of beauveria bassiana liquid culture medium by using various fungus chaff and sporulation quantity are shown in Table 1
Example 6:
this example differs from example 1 only in that: the volume of the inoculum added in the fourth step was 0.14ml. The preparation of beauveria bassiana liquid culture medium by using various fungus chaff and sporulation quantity are shown in Table 1
Example 7:
this example differs from example 1 only in that: the time of incubation in the fourth step was 5 days. The preparation of beauveria bassiana liquid culture medium by using various fungus chaff and sporulation quantity are shown in Table 1
The preparation of a beauveria bassiana liquid medium using various fungus chaff and the spore production amounts in examples 1 to 7 are shown in table 1.
TABLE 1
The concentration of the beauveria bassiana spore liquid fermented by the agaric fungus chaff serving as the culture medium in the embodiment 1 is the largest, is improved by 3.0-5.0 percent compared with other fungus chaffs under the same condition, and is improved by 45.2 percent compared with the oyster mushroom chaff of the embodiment 2 with the lowest concentration of the beauveria bassiana spore liquid.
Compared with the existing solid fermentation, the method reduces the fermentation time and increases the spore yield. The time required for liquid fermentation in the invention is 6 days, and the optimal spore concentration is 1.99 multiplied by 10 10 While the common solid fermentation time is about 8-10 days, the number of obtained bacterial agent spores is 0.2 multiplied by 10 10 Left and right.
The culture medium has the advantages of simple formula and preparation method, easily obtained materials, low cost and high yield of beauveria bassiana spores. The culture method of the beauveria bassiana is simple to operate, clear in production process flow, short in production period, high in spore yield, low in requirements on production environment and production equipment, strong in operability and low in production cost.
The inoculum concentration in the present invention is a constant in the examples, and a preferred inoculum concentration used in many of the related documents is 10 7 Theoretically, 10 in the above step 7 The method can be changed into other data, and is estimated from the result of the research on the dosage of the inoculation liquid, under the condition that the dosage of the inoculation liquid is not changed, the concentration of the inoculation liquid is changed, and the final spore amount of the obtained beauveria bassiana microbial inoculum can be reduced after the final spore amount rises to the highest point.
The above description is only a preferred example of the present invention and is not intended to limit the present invention, and various modifications and changes may be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement and the like of the present invention shall be included in the protection scope of the present invention.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
The above-described embodiments of the present invention should not be construed as limiting the scope of the present invention. Any other corresponding changes and modifications made according to the technical idea of the present invention should be included in the protection scope of the claims of the present invention.
Claims (7)
1. A liquid culture medium for beauveria bassiana is characterized in that: the sterilization agent is prepared by uniformly mixing fungus chaff, water, manganese sulfate and ammonium nitrate and then sterilizing the mixture by high-pressure steam, wherein 4-5 ml of water is added into each gram of fungus chaff, 0.03-0.05g of manganese sulfate is added into each gram of fungus chaff, and 0.05-0.1g of ammonium nitrate is added into each gram of fungus chaff; the fungus chaff is one or more of fungus chaff, oyster mushroom chaff and nameko mushroom chaff which take sawdust as a substrate.
2. The liquid medium for beauveria bassiana according to claim 1, characterized in that: 4ml of water is added into each gram of fungus chaff, 0.03g of manganese sulfate is added into each gram of fungus chaff, and 0.05g of ammonium nitrate is added into each gram of fungus chaff.
3. The liquid medium for beauveria bassiana according to claim 1, characterized in that: the temperature of the high-pressure steam is 121 ℃, and the pressure of the water steam is 0.11MPa.
4. The liquid culture medium for beauveria bassiana according to claim 1, wherein: the time of the high-pressure steam sterilization is 30min.
5. A preparation method of a beauveria bassiana microbial inoculum is characterized by comprising the following specific steps: inoculating the beauveria bassiana inoculation solution into the liquid culture medium for the beauveria bassiana as defined in claim 1, adding 0.05-0.10ml of the inoculation solution into each gram of dry fungus chaff, sealing, culturing in a shaking table at the temperature of 25-27 ℃ at the speed of 150rpm for 4-6 days, and then harvesting the beauveria bassiana fungicide.
6. The method for preparing beauveria bassiana microbial inoculum according to claim 5, wherein 0.10ml of inoculation liquid is added per gram of dry bran.
7. The method for preparing a beauveria bassiana inoculum according to claim 5, wherein the beauveria bassiana inoculum is prepared by placing beauveria bassiana blocks in a PDA (personal digital assistant) culture medium filled with sterilized medium, culturing in an incubator at 29 ℃ for 7-10 days, and washing the plate to obtain the beauveria bassiana inoculum.
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| CN112760234B (en) * | 2021-02-02 | 2023-01-31 | 吉林农业大学 | Trichoderma harzianum and trichoderma viride liquid culture medium and preparation method of trichoderma harzianum and trichoderma viride microbial inoculum |
| CN112680366B (en) * | 2021-02-02 | 2023-01-31 | 吉林农业大学 | Liquid culture medium for paecilomyces lilacinus and preparation method of paecilomyces lilacinus microbial inoculum |
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