CN111676142A - Preparation and application method of beauveria bassiana powder - Google Patents

Preparation and application method of beauveria bassiana powder Download PDF

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Publication number
CN111676142A
CN111676142A CN202010291805.1A CN202010291805A CN111676142A CN 111676142 A CN111676142 A CN 111676142A CN 202010291805 A CN202010291805 A CN 202010291805A CN 111676142 A CN111676142 A CN 111676142A
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beauveria bassiana
seed culture
powder
temperature
culture
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Inventor
张家韬
崔传斌
陈超
董绘阳
王安然
王平平
任广伟
韩猛
安德荣
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Shaanxi Co Of China Tobacco General Corp
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Shaanxi Co Of China Tobacco General Corp
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14Fungi; Culture media therefor

Abstract

The invention discloses a preparation method and an application method of beauveria bassiana powder, and relates to the technical field of microorganisms. The invention comprises the following steps: the method comprises the steps of performing first-stage seed culture on beauveria bassiana, performing second-stage seed culture on the beauveria bassiana, performing third-stage seed culture on the beauveria bassiana, inoculating seed liquid obtained by the third-stage seed culture to a solid culture medium, wherein the inoculation amount is 20%, performing solid fermentation, stopping fermentation when the number of viable spores of the beauveria bassiana in the solid culture medium is more than or equal to 20 hundred million/ml, obtaining a bacterium powder intermediate, naturally air-drying the bacterium powder intermediate at 25 ℃, crushing the bacterium powder intermediate after air-drying, and crushing the bacterium powder to 70 meshes.

Description

Preparation and application method of beauveria bassiana powder
Technical Field
The invention relates to the technical field of microorganisms, in particular to a preparation method and an application method of beauveria bassiana powder.
Background
Tobacco belongs to plants in the genus of Nicotiana in the family of Solanaceae, an important commercial crop, and tobacco leaves, a product of the tobacco leaves, are a main production raw material of cigarette commodities; the tobacco is sensitive to the temperature reaction, and different temperature conditions have large influence on the quality and yield of the tobacco; the black cutworms in the tobacco planting field gnaw tobacco plants, and the increase of the number of the black cutworms inevitably affects the yield of tobacco leaves, so that an insecticidal means is needed for preventing and treating the tobacco planting field and inhibiting the number of the black cutworms.
The chemical insecticide on the market is not developed aiming at the agrotis ypsilon, so that the insecticidal effect of the insecticide is not obvious, and the chemical insecticide is easy to damage the soil structure to influence the growth of tobacco, so that the development of a preparation and application method of beauveria bassiana powder specially aiming at the agrotis ypsilon is a technical problem to be solved by technical personnel in the field.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a preparation method and an application method of beauveria bassiana powder, and solves the problems in the background art.
In order to achieve the purpose, the invention is realized by the following technical scheme: a preparation method of beauveria bassiana powder comprises the following steps:
SS01 performing first-stage seed culture on the beauveria bassiana;
SS02 performing secondary seed culture on the beauveria bassiana;
SS03 performing three-stage seed culture on the beauveria bassiana;
SS04 inoculating the seed liquid obtained by the third-stage seed culture to a solid culture medium, wherein the inoculation amount is 20%, performing solid fermentation, and stopping fermentation when the number of live spores of beauveria bassiana in the solid culture medium is more than or equal to 20 hundred million/ml to obtain a bacterial powder intermediate;
SS05 air drying the intermediate at 25 deg.C, pulverizing, and pulverizing to 70 mesh to obtain Beauveria bassiana powder.
Further, the primary seed culture, the secondary seed culture and the tertiary seed culture all adopt fermentation culture media, and the fermentation culture media are composed of the following raw materials in percentage by weight:
0.8-1.0% of rice flour, 1.2-1.5% of glucose, 1.6-2% of cane sugar, 2.7-3.0% of soybean cake powder, 0.6-1.2% of silkworm chrysalis powder, 0.9-1.3% of yeast powder, 0.015-0.03% of monopotassium phosphate, 0.007-0.01% of magnesium sulfate and 0.016-0.02% of calcium chloride.
Further, the first-stage seed culture, the second-stage seed culture and the third-stage seed culture all adopt stirrers, and the rotating speed of the stirrers is 160 rpm/min.
Further, the aeration and temperature of the primary seed culture, the secondary seed culture and the tertiary seed culture are controlled as follows:
in the time range of 0-24 h: the gas-liquid ratio is 0.5:1, and the temperature is 27 ℃;
within the time range of 24 h-48 h: the gas-liquid ratio is 1:1, and the temperature is 26 ℃;
within the time range of 48 h-60 h: the gas-liquid ratio was 1.1:1 and the temperature was 26 ℃.
Further, the solid culture medium is prepared from the following raw materials in percentage by weight:
4 to 5 percent of rice, 2.5 to 3 percent of corn flour, 0.5 to 1 percent of glucose, 1.6 to 2 percent of chaff, 2.7 to 3 percent of wheat bran, 0.8 to 1 percent of silkworm chrysalis meal, 1.7 to 2 percent of bean cake meal and the balance of water.
Further, the ventilation, temperature and humidity in the solid state fermentation are controlled as follows:
within the time range of 0-24 h: ventilation volume is 0.3:1, temperature is 28 ℃, and humidity is 55%;
in the time range of 24-48 h: ventilation volume is 0.5:1, temperature is 28 ℃, and humidity is 60%;
after 48 h: the ventilation volume is 0.6:1, the temperature is 27 ℃, and the humidity is 45%.
An application method of beauveria bassiana powder comprises the following steps:
mixing beauveria bassiana powder and water in a ratio of 1: 150, then fully shaking up, using the tobacco land soil, and carrying out statistics investigation on the number of the black cutworms in the soil and the number of wormholes on tobacco plants every 3 days.
The invention has the following beneficial effects:
the preparation method and the application method of the beauveria bassiana powder have obvious lethal effect on the black cutworms, can improve the prevention and treatment rate, and meanwhile, the beauveria bassiana powder is a green pollution-free biological insecticide, does not damage the soil structure of a tobacco planting field, and can ensure that the tobacco planting field can be recycled for a long time.
Of course, it is not necessary for any product in which the invention is practiced to achieve all of the above-described advantages at the same time.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic view of the preparation process of a Beauveria bassiana (balsamo) powder of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example one
Referring to fig. 1, the present invention provides a technical solution: a preparation method of beauveria bassiana powder comprises the following steps:
SS01 performing first-stage seed culture on beauveria bassiana;
SS02 performing secondary seed culture on beauveria bassiana;
SS03 performing three-stage seed culture on beauveria bassiana; the purity of the flora can be improved through three-stage culture, and the interference of mixed bacteria is avoided;
SS04 inoculating the seed liquid obtained by the third-stage seed culture to a solid culture medium, wherein the inoculation amount is 20%, performing solid fermentation, and stopping fermentation when the number of live spores of beauveria bassiana in the solid culture medium is more than or equal to 20 hundred million/ml to obtain a bacterial powder intermediate;
SS05 air drying the intermediate at 25 deg.C, pulverizing, and pulverizing to 70 mesh to obtain Beauveria bassiana powder.
Wherein, the first-stage seed culture, the second-stage seed culture and the third-stage seed culture all adopt fermentation culture media, and the fermentation culture media are composed of the following raw materials by weight percent:
1.0% of rice flour, 1.5% of glucose, 2% of sucrose, 3.0% of soybean cake powder, 1.2% of silkworm chrysalis powder, 1.3% of yeast powder, 0.03% of monopotassium phosphate, 0.01% of magnesium sulfate and 0.02% of calcium chloride.
Wherein, the first-stage seed culture, the second-stage seed culture and the third-stage seed culture all adopt stirrers, and the rotating speed of the stirrers is 160 rpm/min.
Wherein, the aeration and temperature of the first-level seed culture, the second-level seed culture and the third-level seed culture are controlled as follows:
in the time range of 0-24 h: the gas-liquid ratio is 0.5:1, and the temperature is 27 ℃; within the time range of 24 h-48 h: the gas-liquid ratio is 1:1, and the temperature is 26 ℃; within the time range of 48 h-60 h: the gas-liquid ratio was 1.1:1 and the temperature was 26 ℃.
Wherein the solid culture medium is prepared from the following raw materials in percentage by weight:
5% of rice, 3% of corn flour, 1% of glucose, 2% of chaff, 3% of wheat bran, 1% of silkworm chrysalis meal, 2% of bean cake meal and the balance of water.
Wherein, the ventilation volume, the temperature and the humidity in the solid state fermentation are controlled as follows:
within the time range of 0-24 h: ventilation volume is 0.3:1, temperature is 28 ℃, and humidity is 55%; in the time range of 24-48 h: ventilation volume is 0.5:1, temperature is 28 ℃, and humidity is 60%; after 48 h: the ventilation volume is 0.6:1, the temperature is 27 ℃, and the humidity is 45%.
An application method of beauveria bassiana powder comprises the following steps:
mixing beauveria bassiana powder and water in a ratio of 1: 150, then fully shaking up, using the No. 1 soil of the tobacco planting field, carrying out statistical investigation on the number of the cutworms in the soil and the number of the wormholes on the tobacco plants every 3 days, and bringing the statistical data into a first table and a second table.
Example two
The invention also provides another technical scheme: a preparation method of beauveria bassiana powder comprises the following steps:
SS01 performing first-stage seed culture on beauveria bassiana;
SS02 performing secondary seed culture on beauveria bassiana;
SS03 performing three-stage seed culture on beauveria bassiana;
SS04 inoculating the seed liquid obtained by the third-stage seed culture to a solid culture medium, wherein the inoculation amount is 20%, performing solid fermentation, and stopping fermentation when the number of live spores of beauveria bassiana in the solid culture medium is more than or equal to 20 hundred million/ml to obtain a bacterial powder intermediate;
SS05 air drying the intermediate at 25 deg.C, pulverizing, and pulverizing to 70 mesh to obtain Beauveria bassiana powder.
Wherein, the first-stage seed culture, the second-stage seed culture and the third-stage seed culture all adopt fermentation culture media, and the fermentation culture media are composed of the following raw materials by weight percent:
0.8% of rice flour, 1.2% of glucose, 1.6% of cane sugar, 2.7% of soybean cake powder, 0.6% of silkworm chrysalis powder, 0.9% of yeast powder, 0.015% of monopotassium phosphate, 0.007% of magnesium sulfate and 0.016% of calcium chloride.
Wherein, the first-stage seed culture, the second-stage seed culture and the third-stage seed culture all adopt stirrers, and the rotating speed of the stirrers is 160 rpm/min.
Wherein, the aeration and temperature of the first-level seed culture, the second-level seed culture and the third-level seed culture are controlled as follows:
in the time range of 0-24 h: the gas-liquid ratio is 0.5:1, and the temperature is 27 ℃; within the time range of 24 h-48 h: the gas-liquid ratio is 1:1, and the temperature is 26 ℃; within the time range of 48 h-60 h: the gas-liquid ratio was 1.1:1 and the temperature was 26 ℃.
Wherein the solid culture medium is prepared from the following raw materials in percentage by weight:
4% of rice, 2.5% of corn flour, 0.5% of glucose, 1.6% of chaff, 2.7% of wheat bran, 0.8% of silkworm chrysalis meal, 1.7% of bean cake meal and the balance of water.
Wherein, the ventilation volume, the temperature and the humidity in the solid state fermentation are controlled as follows:
within the time range of 0-24 h: ventilation volume is 0.3:1, temperature is 28 ℃, and humidity is 55%; in the time range of 24-48 h: ventilation volume is 0.5:1, temperature is 28 ℃, and humidity is 60%; after 48 h: the ventilation volume is 0.6:1, the temperature is 27 ℃, and the humidity is 45%.
An application method of beauveria bassiana powder comprises the following steps:
mixing beauveria bassiana powder and water in a ratio of 1: 150, then fully shaking up, using the No. 2 soil of the tobacco planting field, carrying out statistical investigation on the number of the cutworm in the soil and the number of the wormholes on the tobacco plants every 3 days, and bringing the statistical data into a first table and a second table, wherein the rest contents are consistent with the embodiment.
EXAMPLE III
The invention also provides a comparison technical scheme: in the pesticide insecticidal control group, the Laienlawn AnRuiyuan granular pesticide (pesticide registration number: PD20181336) is used for No. 3 fields of the tobacco planting fields, the number of the cutworms in the soil and the number of wormholes on tobacco plants are subjected to statistical investigation once every 3 days, and statistical data are included in a first table and a second table.
Example four
The invention also provides another comparison technical scheme: and (3) irrigating the No. 4 field of the tobacco planting field by using clear water in the CK clear water control group, carrying out statistical investigation on the number of the black cutworms in the soil and the number of wormholes on tobacco plants every 3 days, and bringing statistical data into a table I and a table II.
The data statistics of the above examples 1 to 4 are drawn into a first table and a second table, the first table is a survey information table of the number of harmful insects in the tobacco fields, the second table is a survey information table of the number of wormholes on tobacco plants, and the specific table information is as follows:
table-survey information table of pest number in tobacco grassland
Figure RE-GDA0002621799600000071
Figure RE-GDA0002621799600000081
Table two tobacco plant wormhole quantity investigation information table
Figure RE-GDA0002621799600000082
The tobacco plant variety is the commercial common flue-cured tobacco, and the No. 1 land, the No. 2 land, the No. 3 land and the No. 4 land of the test land are tobacco planting lands with common soil.
From the table information, the death rate of the black cutworm in the number 1 land per square meter is 92.4%, and the wormhole reduction rate in 100 square meters is 88.57%, so that the first embodiment in the embodiments 1 to 4 is the best embodiment, has obvious lethal effect on the black cutworm, can improve the prevention and treatment rate, and meanwhile, the beauveria bassiana powder is a green pollution-free biological insecticide, does not damage the soil structure of the tobacco planting land, and can ensure that the tobacco planting land can be recycled for a long time.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Claims (7)

1. A preparation method of beauveria bassiana powder is characterized by comprising the following steps:
SS01 performing first-stage seed culture on the beauveria bassiana;
SS02 performing secondary seed culture on the beauveria bassiana;
SS03 performing three-stage seed culture on the beauveria bassiana;
SS04 inoculating the seed liquid obtained by the third-stage seed culture to a solid culture medium, wherein the inoculation amount is 20%, performing solid fermentation, and stopping fermentation when the number of live spores of beauveria bassiana in the solid culture medium is more than or equal to 20 hundred million/ml to obtain a bacterial powder intermediate;
SS05 air drying the intermediate at 25 deg.C, pulverizing, and pulverizing to 70 mesh to obtain Beauveria bassiana powder.
2. The method for preparing beauveria bassiana fungus powder as claimed in claim 1, wherein the first seed culture, the second seed culture and the third seed culture all adopt fermentation culture media, and the fermentation culture media are composed of the following raw materials by weight percentage:
0.8-1.0% of rice flour, 1.2-1.5% of glucose, 1.6-2% of cane sugar, 2.7-3.0% of soybean cake powder, 0.6-1.2% of silkworm chrysalis powder, 0.9-1.3% of yeast powder, 0.015-0.03% of monopotassium phosphate, 0.007-0.01% of magnesium sulfate and 0.016-0.02% of calcium chloride.
3. The method for preparing beauveria bassiana fungus powder as claimed in claim 1, wherein the first-stage seed culture, the second-stage seed culture and the third-stage seed culture all adopt a stirrer, and the rotation speed of the stirrer is 160 rpm/min.
4. The method for preparing beauveria bassiana fungus powder as claimed in claim 1, wherein the aeration and temperature of the first seed culture, the second seed culture and the third seed culture are controlled as follows:
in the time range of 0-24 h: the gas-liquid ratio is 0.5:1, and the temperature is 27 ℃;
within the time range of 24 h-48 h: the gas-liquid ratio is 1:1, and the temperature is 26 ℃;
within the time range of 48 h-60 h: the gas-liquid ratio was 1.1:1 and the temperature was 26 ℃.
5. The method for preparing beauveria bassiana fungus powder as claimed in claim 1, wherein the solid culture medium is prepared from the following raw materials by weight percentage:
4 to 5 percent of rice, 2.5 to 3 percent of corn flour, 0.5 to 1 percent of glucose, 1.6 to 2 percent of chaff, 2.7 to 3 percent of wheat bran, 0.8 to 1 percent of silkworm chrysalis meal, 1.7 to 2 percent of bean cake meal and the balance of water.
6. The method for preparing beauveria bassiana powder according to claim 1, wherein the ventilation, temperature and humidity in the solid state fermentation are controlled as follows:
within the time range of 0-24 h: ventilation volume is 0.3:1, temperature is 28 ℃, and humidity is 55%;
in the time range of 24-48 h: ventilation volume is 0.5:1, temperature is 28 ℃, and humidity is 60%;
after 48 h: the ventilation volume is 0.6:1, the temperature is 27 ℃, and the humidity is 45%.
7. The method for applying beauveria bassiana powder as claimed in any one of claims 1 to 6, which comprises the following steps:
mixing beauveria bassiana powder and water in a ratio of 1: 150, then fully shaking up, using the tobacco land soil, and carrying out statistics investigation on the number of the black cutworms in the soil and the number of wormholes on tobacco plants every 3 days.
CN202010291805.1A 2020-04-14 2020-04-14 Preparation and application method of beauveria bassiana powder Pending CN111676142A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114058518A (en) * 2021-11-26 2022-02-18 盐城市神微微生物菌种科技有限公司 Culture method of beauveria bassiana
CN114438011A (en) * 2022-03-07 2022-05-06 广西大学 Fermentation production method and application of beauveria bassiana PfBb spore powder

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CN106614423A (en) * 2016-12-30 2017-05-10 四川省农业科学院蚕业研究所 Artificial preparation method of white muscardine silkworms
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CN106614423A (en) * 2016-12-30 2017-05-10 四川省农业科学院蚕业研究所 Artificial preparation method of white muscardine silkworms
CN107347922A (en) * 2017-08-18 2017-11-17 临沂市农业科学院 A kind of application of Strain of Beauveria bassiana
CN110093283A (en) * 2019-05-15 2019-08-06 云南星耀生物制品有限公司 Strain of Beauveria bassiana and its cultural method

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Cited By (3)

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Publication number Priority date Publication date Assignee Title
CN114058518A (en) * 2021-11-26 2022-02-18 盐城市神微微生物菌种科技有限公司 Culture method of beauveria bassiana
CN114438011A (en) * 2022-03-07 2022-05-06 广西大学 Fermentation production method and application of beauveria bassiana PfBb spore powder
CN114438011B (en) * 2022-03-07 2024-03-22 广西大学 Fermentation production method and application of beauveria bassiana PfBb spore powder

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