CN111172066B - Bacillus megaterium and application thereof - Google Patents
Bacillus megaterium and application thereof Download PDFInfo
- Publication number
- CN111172066B CN111172066B CN202010010082.3A CN202010010082A CN111172066B CN 111172066 B CN111172066 B CN 111172066B CN 202010010082 A CN202010010082 A CN 202010010082A CN 111172066 B CN111172066 B CN 111172066B
- Authority
- CN
- China
- Prior art keywords
- bacillus megaterium
- fermentation
- strain
- ysj3
- application
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, 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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/11—Bacillus megaterium
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/45—Tobacco
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, 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
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biodiversity & Conservation Biology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Ecology (AREA)
- Forests & Forestry (AREA)
- Hydrology & Water Resources (AREA)
- Environmental & Geological Engineering (AREA)
- Water Supply & Treatment (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of biological fermentation engineering, and particularly relates to bacillus megaterium and an application patent application thereof. The bacillus megaterium YSJ3 is preserved in the China general microbiological culture Collection center in 2019, 12 months and 5 days, and the preservation addresses are as follows: the microbial research institute of China academy of sciences No. 3 of Xilu No. 1 of Beijing, Chaoyang, Beijing, and the collection number is CGMCC 19088. In the application, the inventor finally obtains the bacillus megaterium YSJ3 strain with better growth vigor by the combined application of ultraviolet mutagenesis and plasma beam mutagenesis technology and directional screening breeding on the basis of the existing bacillus megaterium YSJ1 strain. Preliminary experiment results show that the highest fermentation activity of the YSJ3 strain can reach more than 150 hundred million/mL, the growth effect is good, and a good technical basis can be laid for the preparation of bacillus megatherium microbial preparation products.
Description
Technical Field
The invention belongs to the technical field of biological fermentation engineering, and particularly relates to bacillus megaterium and an application patent application thereof.
Background
Bacillus megaterium (B.)Bacillus megaterium) The bacillus subtilis is aerobic gram-positive bacterium capable of producing spores, the diameter of the bacterium is more than 1.0 mu m, the bacterium is commonly called as 'megaspore' bacterium and is beneficial to soil bacteria, and the prior research shows that the bacterium has good use for promoting crop production and improving crop quality. Therefore, in recent years, some attention has been paid to the study and application of the bacterium. But is limited by the limitations of weak growth activity, slow growth speed, low fermentation yield and the like of the existing bacteria, and the production and application of microbial preparations mainly comprising the bacteria are limited. Therefore, it is necessary to screen a bacillus megaterium strain with better growth effect and further optimize the fermentation process of the strain, thereby laying a technical foundation for the production and application of related microbial preparation products.
Disclosure of Invention
Based on the existing bacillus megaterium, through mutagenesis and screening, the application aims to provide the bacillus megaterium with better growth activity, thereby laying a certain technical foundation for relevant biological organic fertilizer, microbial preparation, antibiotic synthesis and enzyme industrial production application.
The technical solution adopted in the present application is detailed as follows.
A bacillus megaterium strain is specifically named as: bacillus megaterium (B.) (B.megaterium)Bacillus megaterium) YSJ3, which has been preserved in Chinese microbial strain in 2019, 12 months and 5 daysThe general microbiological center of the Tibetan management committee has the preservation address as follows: the microbial research institute of China academy of sciences No. 3, Xilu No. 1, Beijing, Chaoyang, with the collection number of CGMCC 19088;
the strain is obtained by carrying out multiple ultraviolet mutagenesis and plasma beam composite mutagenesis breeding on an original starting strain Bacillus megaterium YSJ 1.
The fermentation product prepared by the bacillus megaterium is prepared by the following steps:
(1) strain activation and seed liquid preparation
Activating and culturing the preserved Bacillus megaterium YSJ3 strain in an LB plate culture medium at 37 ℃ for 24h, collecting lawn, transferring the lawn to an LB liquid culture medium for further amplification, transferring the lawn to a seed culture medium in a seed tank, and fermenting at 37 ℃ and 180rpm for about 15h to obtain seed liquid;
the seed culture medium comprises the following components in percentage by mass: 2% of glucose, 3% of corn flour, 5% of bean cake powder, 1% of corn steep liquor dry powder, 0.2% of monopotassium phosphate, 1.5% of yeast powder and pH 7.0;
(2) preparation of fermentation broth
Transferring the seed solution prepared in the step (1) into a fermentation culture medium according to the inoculation amount of 10% by volume ratio, and fermenting at 30-38 ℃ and 180-year 200rpm for 24-48 h in a fermentation tank to prepare a fermentation liquid (fermenting at 37 ℃ and 180-year 200rpm for about 30h until the spore formation rate is more than 80%, and simultaneously placing the fermentation liquid in the tank when the pH value is obviously increased);
in order to facilitate the subsequent preparation of a microbial preparation finished product, 2 mass percent of anhydrous sodium sulfate can be added into fermented mash and is fully and uniformly stirred, then the mixture is further transferred into an emulsification tank for spray drying treatment (the specific spray drying treatment process reference parameters are that the air inlet temperature is controlled at 140-170 ℃, the air exhaust temperature is controlled at 60-65 ℃, and the negative pressure is 200-500 pa), and the mixture is further prepared into powdery raw powder which is compounded with other auxiliary materials or effective components to be used as a bacillus megaterium finished product preparation;
the fermentation medium is in a liquid state, and comprises the following specific components in percentage by mass: 5-8% of corn flour, 9-12% of bean cake powder, 0.5-1.5% of bran, 0.03% of calcium chloride, 0.1-0.5% of monopotassium phosphate, 0.01-0.03% of manganese sulfate and 6.5-7.2% of pH.
The bacillus megaterium or the fermentation product thereof is applied to crop cultivation, can be used as a beneficial microbial fertilizer, can obviously improve the phenotype of crops or promote the yield increase, and is particularly used for tobacco cultivation.
In the application, an inventor obtains a bacillus megaterium YSJ3 strain with good growth activity finally through combined application of ultraviolet mutagenesis and plasma beam mutagenesis technology and directional screening breeding on the basis of an existing bacillus megaterium YSJ1 strain (obtained by separating and screening in the soil of a Tianchi tobacco base in Mianchi county in the early stage, the bacterial colony of the strain is oval, the surface is smooth, the edge is neat, a cyst of a spore is not expanded through microscopic examination, and the spore is oval). Preliminary experiment results show that the highest fermentation activity of the YSJ3 strain can reach more than 150 hundred million/mL, the growth effect is good, and a good technical basis can be laid for the preparation of bacillus megatherium microbial preparation products.
Drawings
FIG. 1 is a graph showing the growth of Bacillus megaterium.
Detailed Description
The present application is further illustrated by the following examples. Before describing the specific embodiments, a brief description of the background of some of the experiments involved in the following examples is provided below.
Culture medium:
nutrient agar medium (Nutrient agar. na): 10.00 g of peptone, 3.00 g of beef extract, 5.00 g of sodium chloride, 15.00 g of agar, 1.00L of distilled water and pH 7.0;
beef extract medium (bee4.3 extract Agar): taking 500.00 g of lean meat without fat, cutting, adding 1.00L of distilled water into a beaker, putting the beaker in a refrigerator at 4 ℃ overnight, filtering the mixture by using cotton, boiling the filtrate in a water bath for 1 h to solidify protein, filtering the filtrate, adding 10.00 g of peptone and 5.00 g of sodium chloride into the filtrate, adjusting the pH to 7.4, supplementing 1.00L of liquid and 15.00 g of agar, subpackaging and sterilizing the mixture for later use;
method for determining activity of bacillus megaterium
Preparing diluents with different concentrations by a dilution inversion plate method according to a 10-time dilution method, respectively sucking a certain volume from the diluents and placing the volume in a sterile culture dish, and repeating for 3 times for each dilution;
then pouring about 12 mL of nutrient agar culture medium which is melted in advance and cooled to about 50 ℃ into each culture dish, shaking up, carrying out inverted culture for 24h after solidification, and carrying out colony counting;
taking thalli of 5-10 bacterial colonies from each dilution after culture, staining a smear, and identifying by a microscope;
selecting plates with proper dilution and 30-300 bacterial colonies for counting;
and a result calculation method comprises the following steps:
example 1
This example provides Bacillus megaterium (B.megaterium) according to the present applicationBacillus megaterium) The mutagenesis screening of the strain YSJ3 was carried out as follows.
Bacillus megaterium (B) provided by the applicationBacillus megaterium) YSJ3 is mainly obtained by ultraviolet mutagenesis and ion beam mutagenesis screening of the existing original strain Bacillus megaterium YSJ1, and the specific mutagenesis and screening processes are briefly described as follows.
(1) Ultraviolet mutagenesis
Firstly, ultraviolet mutation breeding is carried out on a starting strain of bacillus megaterium YSJ1, and the main process comprises the following steps:
taking 4ml of YSJ1 bacterial suspension cultured at 37 ℃ to logarithmic phase, and irradiating at 28 cm distance by using a 15W ultraviolet lamp, wherein the survival rate of the bacterial strain is gradually reduced along with the prolonging of the ultraviolet irradiation time in the irradiation process. In the radiation mutagenesis, the irradiation time was 40s as the ultraviolet ray mutagenesis dose (the measurement results showed that when the irradiation time exceeded 40s, the survival rate was less than 10%, and thus this time was selected as the optimum time).
After mutagenic screening such as multi-round irradiation, preliminary screening, secondary screening and the like, a mutant strain with high activity is obtained and named as YSJ 1-507. The measurement result shows that the fermentation activity is 83 hundred million/mL.
(2) Ion beam mutagenesis
The YSJ1-507 strain obtained by ultraviolet mutagenesis is used as an initial strain for ion beam mutagenesis, and the specific mutagenesis and screening processes are as follows:
taking 5mL of bacterial liquid obtained by culturing original strain YSJ1-507 to logarithmic phase, filtering, resuspending with sterile normal saline, taking 200 uL of bacterial suspension in a sterile culture dish, uniformly coating the bacterial suspension on the middle position of the culture dish, naturally drying in a sterile operation platform, and respectively carrying out drying according to the proportion of 5 multiplied by 1014 ions·cm-2、1×1015 ions·cm-2、5×1015 ions·cm-2、1×1016 ions·cm-2、5×1016 ions·cm-2Irradiating five doses with ion beams for 18s, 36s, 179s, 368s and 1790s respectively;
eluting with normal saline after irradiation, coating the eluted mutagenized strain on a culture medium, and culturing at 37 deg.C for 2 days;
finally, 5 new strains with good colony morphology and high growth speed are obtained by screening, and are respectively named as: YSJ2-107, YSJ2-219, YSJ2-505, YSJ2-326, and YSJ 2-432.
The growth performance (i.e. growth activity as a main index) of the 5 strains is evaluated, and the specific process is as follows:
carrying out shake flask fermentation culture on the selected 5 strains for 48h at 37 ℃, wherein the formula of a culture medium is as follows: 6% of corn flour, 9% of bean cake powder, 1% of bran, 0.03% of calcium chloride, 0.1% of monopotassium phosphate, 0.01% of manganese sulfate and pH 6.5;
after the culture, the activity of the bacillus megaterium in the fermentation broth was measured, and the specific culture results are shown in the following table.
TABLE 1, 5 Bacillus megaterium shake flask fermentation results
Further carrying out primary screening and secondary screening and continuously transferring for 10 generations so as to evaluate the growth vigor of the strains. The strain viability results of YSJ2-505 strain after 10 serial passages are shown in Table 2 below.
TABLE 2 continuous passage productivity results of Bacillus megaterium strain YSJ2-505
The result shows that the growth activity of the YSJ2-505 strain is stable, the number of thalli is stable above 150 hundred million/mL after fermentation for 48 hours (original strain of Bacillus megaterium YSJ1, the fermentation activity under the same condition is only about 50 hundred million/mL), therefore, the inventor carries out biological preservation on the strain and renames the strain as follows: bacillus megaterium (B.)Bacillus megaterium) YSJ3, which has been deposited in China general microbiological culture Collection center on 12 months and 10 days in 2019 at the deposition address: the microbial research institute of China academy of sciences No. 3, Xilu No. 1, Beijing, Chaoyang, and the preservation number is CGMCC 19088.
Part of the phenotypic characteristics of this strain are as follows: the flat plate colony is oval, the surface is smooth, the yellow color is light, the edge is neat, the microscopic bud cysts do not expand, and the spores are oval.
Example 2
Based on the strain obtained by screening in example 1, the inventors further optimize the fermentation process adapted to the strain to further improve the growth and fermentation capacity of the strain. The specific process is briefly described as follows.
(1) Strain activation and seed liquid preparation
Activating and culturing the preserved Bacillus megaterium YSJ3 strain in LB plate culture medium at 37 deg.C for 2d, collecting thallus Porphyrae, transferring to LB liquid culture medium for further amplification, transferring to seed culture medium in seed tank, and fermenting at 37 deg.C and 280rpm for 16h to obtain seed liquid;
the seed culture medium is as follows: 2% of glucose, 3% of corn flour, 5% of bean cake powder, 1% of corn steep liquor dry powder, 0.2% of monopotassium phosphate, 1.5% of yeast powder and 7.0% of pH value.
(2) Preparation of fermentation broth
Transferring the seed liquid prepared in the step (1) into a fermentation culture medium according to the inoculation amount of 10% by volume ratio, fermenting in a fermentation tank at 37 ℃ and 180rpm until the spore formation rate is over 80% (meanwhile, the pH of the fermentation liquid can be obviously increased), and putting the fermentation tank to obtain the fermentation liquid;
in order to facilitate the subsequent preparation of a microbial preparation finished product, 2 percent of anhydrous sodium sulfate is added into the fermented mash, then the fermented mash is further transferred into an emulsifying tank for spray drying treatment, and the fermented mash is further prepared into powdery raw powder which is compounded with other auxiliary materials or active ingredients to be used as a bacillus megaterium finished product preparation;
the fermentation medium is liquid and comprises the following specific components in percentage by mass: corn flour 6%, bean cake powder 9%, bran 1%, calcium chloride 0.03%, monopotassium phosphate 0.1%, manganese sulfate 0.01%, and pH 6.5.
The statistical list of the results of the tests on 5 batches of fermentation production is shown in table 3 below.
TABLE 3 Bacillus megaterium 30m3Experimental results of 5-batch fermentation production in fermentation tank
As can be seen from the results in the table above, the fermentation activity is stable, and the average fermentation activity is stable to more than 150 hundred million/mL.
Further, in the fermentation process of a certain batch, the fermentation broth is taken every 4h, the absorbance, i.e., the OD value, is measured under the condition of 600nm, and the change curve of the OD value is drawn as shown in FIG. 1. As can be seen, the strain grows rapidly, and the fermentation period is shortened to about 28-32 h.
(3) Preparation of Bacillus megaterium product
On the basis of the above experiment, referring to the above operation, the inventor takes fermentation liquor fermented at 37 ℃ and 180rpm for 30h as an example, and carries out spray drying treatment by using a centrifugal spray drying tower to further prepare powdery raw powder, wherein the spray drying treatment parameters are as follows: the air inlet temperature is controlled at 160 ℃, the air exhaust temperature is controlled at 65 ℃, and the negative pressure is 200 pa.
As a control, using the same fermentation process, the inventors also prepared a powdered raw powder, exemplified by the original starting strain Bacillus megaterium YSJ 1.
In the preparation process, the inventor determines the comprehensive yield of the final products prepared from different batches, and the results are shown in the following tables 4 and 5.
TABLE 4 Bacillus megaterium strain YSJ1 at 30m3Fermentation and yield condition table of fermentation tank
TABLE 5 Bacillus megaterium strain YSJ3 at 30m3Fermentation and yield condition table of fermentation tank
In the table, the yield is calculated by the formula:
。
from the results in the table above, it can be seen that the overall yield of the original bacillus megaterium YSJ1 is 77.8%, and the overall yield of the YSJ3 strain after mutagenesis and screening in the application can reach 93%, i.e., the better growth and fermentation activity ensures higher yield, and a good technical basis can be established for the preparation of bacillus megaterium microbial preparation products.
Example 3
The bacillus megaterium YSJ3 has multiple uses as a potential functional bacterium, for example, in the field of livestock breeding, the bacillus megaterium YSJ3 can improve the breeding environment and reduce the occurrence of livestock diseases; in the aspect of environmental treatment, the water body can be purified; in the field of crop cultivation, the microbial fertilizer can be prepared into a microbial fertilizer for improving soil microbial environment and increasing soil fertility, and meanwhile, the microbial fertilizer has multiple purposes of increasing crop biomass, increasing yield, preventing and treating plant diseases and insect pests, improving crop quality and the like. In the application, the inventor only takes the cured tobacco in Henan as an example and uses the cured tobacco in the cultivation of the cured tobacco to investigate the specific application effect, and the specific experimental situation is briefly described as follows.
Time and place of experiment:
the test is arranged in a field of a cross country village in the lakeside region of the three gorges city at the beginning of 2019 in 2 months;
the tested soil is brown soil, the soil fertility is medium, and the content of basic nutrients of the plough layer soil is as follows: 13.25g/kg of organic matter, 0.97g/kg of total nitrogen and quick-acting phosphorus (P)2O5) 16.8mg/kg, quick acting potassium (K)2O）121.7mg/kg；
The crop to be tested is flue-cured tobacco (Honghuadajinyuan);
the test of 'bacillus megaterium agent' (after dried, the original powder is diluted by talcum powder, and the number of effective viable bacteria is adjusted to be about 200 hundred million/g);
experiment design:
the experiment is provided with three treatments, random block arrangement and three times of repetition, and the area of each experimental cell is 30m2；
Treatment 1: conventional fertilization and bottom fertilization of 1kg of fine sand mixed with 40kg of organic fertilizer (specifically cow dung, the same below) per mu;
and (3) treatment 2: conventional fertilization and bottom application of 1kg of the test inactivated bacillus megaterium fungicide mixed with 40kg of organic fertilizer per mu;
and (3) treatment: conventional fertilization and bottom application of 1kg of test bacillus megaterium fungicide mixed with 40kg of organic fertilizer per mu;
the conventional fertilization comprises the following steps: applying 40kg of compound fertilizer (azophoska = 20-15-5) on the bottom of each mu;
the experimental process comprises the following steps:
applying a fertilizer to be tested, an inactivated fertilizer, fine sand and an organic fertilizer to each treatment group before land preparation in the last 2 months according to the requirements of a test scheme, transplanting tobacco seedlings in the middle 3 months in the period of three leaves, and setting the row spacing to be 0.6m multiplied by 1.2 m;
in the last ten days of month 4 and the last ten days of month 5, respectively randomly extracting 20 plants in each processing cell for biological character investigation; harvesting in batches in the last 5 th, last 6 th, middle 6 th, last 6 th and last 7 th of month, and performing field investigation and seed test; recording the actual yield by a single-harvest single-term in a cell during harvesting; the test is carried out according to the requirements of the scheme, and other management measures are the same as the common production.
Results of the experiment
(1) Influence of bottom application of 'Bacillus megaterium' on biological phenotypic characters of flue-cured tobaccos in Henan province
Some of the biological trait statistics are shown in the following table:
TABLE 6 agronomic characteristics of tobacco plants in the clumping period (in late 4-month ten days) for different treatment groups
(Note: the data in the Table is the average of three treatment triplicates)
TABLE 7 agronomic traits of tobacco plants in the Dome period (5 Yuehai ten days) for different treatment groups
(Note: the data in the table is the average of three treatment replicates).
As can be seen from the results in the above table 6, in the treatment 3 in the clumping period, the plant height is increased by 0.7cm and 0.5 cm, the number of the leaves of each plant is increased by 0.2 and 0.1, the maximum leaf length is increased by 3.0cm and 2.1cm, and the maximum leaf width is increased by 1.7cm and 1.3cm, respectively, compared with the treatments 1 and 2; as can be seen from table 7: compared with treatments 1 and 2, the plant height of the treatment 3 in the dome period is increased by 4.9cm and 4.0cm, the diameter circumference is increased by 0.04cm and 0.02cm, the maximum leaf length is increased by 3.5cm and 3.0cm, and the maximum leaf width is increased by 2.0cm and 1.5 cm. That is, comprehensively, on the basis of conventional fertilization, phenotypic characters such as plant height, single-plant leaf number, diameter circumference, leaf length and width and the like can be obviously improved by applying the bacillus megaterium microbial inoculum at the bottom.
(2) Influence of bottom application of 'Bacillus megaterium' on yield of flue-cured tobacco in Henan province
The specific yield results are shown below in table 8.
TABLE 8 statistical Table of yield results
The data in the table above were further analyzed for variance and summarized in tables 9 and 10 below:
TABLE 9 ANOVA TABLE
TABLE 10 multiple comparison tables
As can be seen from table 8 above: compared with treatment 1, the treatment 3 increases the yield by 166.8kg on average per mu, and the yield is increased by 6.13%; compared with the treatment 2, the treatment 3 increases the yield by 146.7kg per mu averagely, and the yield is increased by 5.35 percent. Analysis of variance was performed on the results of each treatment (see table 9) and the yield differences between treatments were of a very significant level. When multiple comparisons were performed by the PLSD method (see Table 10), the differences in yield between treatment 3 and treatment 1 and treatment 2 were very significant, and the differences in yield between treatment 2 and treatment 1 were not significant.
The combination of the above results shows that: on the basis of local conventional fertilization, compared with the bacillus megaterium inoculant applied by mixing 1kg of the bacillus megaterium inoculant for test with 40kg of organic fertilizer at the bottom of each mu of organic fertilizer and applying the bacillus megaterium inoculant after the bacillus megaterium inoculant is inactivated in equal amount at the bottom of each mu of organic fertilizer, the plant height, the number of leaves per plant, the diameter circumference and the leaf length width of flue-cured tobacco in Henan can be obviously increased, the yield is increased by 173.96kg per mu on average, the yield increase rate is 6.40%, and the yield difference reaches an extremely obvious level.
Of course, it should be noted that the fermentation product of the microbial strain is also suitable for planting vegetable crops such as pepper, cucumber, cabbage and watermelon, field crops such as wheat, corn and rice, and fruit trees, and the preliminary experiment results of part of the crops also show better expected effects of improving the biological phenotype and increasing the yield.
In a word, the functional microbial inoculum provided by the application can be prepared and uniformly mixed with organic fertilizers or sandy soil for base application in soil preparation, and the mixture is mixed with fertilizers according to the dosage of 1 kg/mu and then applied as base fertilizers, or used as biological bacterial fertilizers singly and directly sprayed on leaf surfaces or poured into soil (once every week and 2-3 times) according to the dosage of 150 g/mu plus water (directly diluted by 500 times).
Claims (5)
1. The bacillus megaterium is characterized by being named as follows: bacillus megaterium (B.)Bacillus megatherium) YSJ3, which is preserved in China general microbiological culture Collection center on 12 th and 5 th month in 2019 at the preservation address of: the microbial research institute of China academy of sciences No. 3, Xilu No. 1, Beijing, Chaoyang, and the preservation number is CGMCC 19088.
2. A fermentation product produced by using the Bacillus megaterium of claim 1, which is obtained by the steps of:
(1) strain activation and seed liquid preparation
Activating and culturing the stored bacillus megaterium YSJ3 strain, and inoculating the bacillus megaterium YSJ3 strain into a seed culture medium to prepare a seed solution;
the seed culture medium comprises the following components in percentage by mass: 2% of glucose, 3% of corn flour, 5% of bean cake powder, 1% of corn steep liquor dry powder, 0.2% of monopotassium phosphate, 1.5% of yeast powder and pH 7.0;
(2) preparation of fermentation broth
Transferring the seed liquid prepared in the step (1) into a fermentation culture medium according to the inoculation amount of 10% by volume ratio, and fermenting the seed liquid in a fermentation tank at 30-38 ℃ and 180-year rotation speed for 24-48 h to prepare fermentation liquid;
the fermentation medium has a pH of 6.5-7.2 by mass percent, and comprises the following specific components: 5-8% of corn flour, 9-12% of bean cake powder, 0.5-1.5% of bran, 0.03% of calcium chloride, 0.1-0.5% of monopotassium phosphate and 0.01-0.03% of manganese sulfate.
3. The fermentation product of claim 2, which is prepared by using the bacillus megaterium of claim 1, wherein after 2% of anhydrous sodium sulfate by mass is added into the fermentation liquid of the step (2) and fully and uniformly stirred, the fermentation product is subjected to spray drying treatment, and the specific spray drying treatment process parameters are as follows: the inlet air temperature is controlled as follows: 140 minus 170 ℃, the air exhaust temperature is controlled at 60-65 ℃, and the negative pressure is 200 minus 500 Pa.
4. Use of bacillus megaterium in crop cultivation according to claim 1, as a beneficial microbial fertilizer for tobacco cultivation.
5. Use of the fermentation product according to claim 2 or 3 in crop cultivation, characterized in that it is used in tobacco cultivation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010010082.3A CN111172066B (en) | 2020-01-06 | 2020-01-06 | Bacillus megaterium and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010010082.3A CN111172066B (en) | 2020-01-06 | 2020-01-06 | Bacillus megaterium and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111172066A CN111172066A (en) | 2020-05-19 |
| CN111172066B true CN111172066B (en) | 2022-07-01 |
Family
ID=70624036
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010010082.3A Active CN111172066B (en) | 2020-01-06 | 2020-01-06 | Bacillus megaterium and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111172066B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112553119B (en) * | 2020-12-25 | 2022-07-29 | 宁夏启元药业有限公司 | Bacillus megaterium strain and application thereof |
| CN114702357B (en) * | 2022-01-13 | 2023-05-23 | 山东绿邦生物科技有限公司 | High-activity compound microbial fertilizer and preparation method thereof |
| CN116731916B (en) * | 2023-06-01 | 2024-04-12 | 中化农业（临沂）研发中心有限公司 | Salt-tolerant bacillus megaterium and application thereof |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005151887A (en) * | 2003-11-26 | 2005-06-16 | Idemitsu Kosan Co Ltd | Agent and method for controlling soil-borne disease |
| KR20090077528A (en) * | 2008-01-11 | 2009-07-15 | 경북대학교 산학협력단 | Bacillus megaterium knuc251 for controlling plant disease and accelerating plant growth, and plant disease controlling agent and plant growth accelerant using the same |
| US7977384B1 (en) * | 2007-12-17 | 2011-07-12 | The University Of Louisiana At Monroe | Anticancer tobacco cembranoids |
| CN102168036A (en) * | 2010-11-30 | 2011-08-31 | 浙江工业大学 | Preparation of R-4-chloro-3-hydroxybutyrate and S-3-hydroxy-butyrolactone by microbial transformation and strain |
| CN103122328A (en) * | 2012-12-07 | 2013-05-29 | 贵州省烟草公司毕节市公司 | Bacillus megaterium strain and application thereof |
| CN103320360A (en) * | 2013-06-25 | 2013-09-25 | 云南省烟草公司玉溪市公司 | Bacillus megatherium SJ-7 strain and application thereof |
| CN103468613A (en) * | 2013-09-12 | 2013-12-25 | 江苏省苏微微生物研究有限公司 | Bacillus megatherium, method for preparing microbial inoculum through solid fermentation of bacillus megatherium and application of microbial inoculum |
| CN105255785A (en) * | 2015-11-17 | 2016-01-20 | 山东省农业科学院农业资源与环境研究所 | Fermentation method of bacillus megatherium with high rate of sporation |
| CN106011035A (en) * | 2016-08-04 | 2016-10-12 | 大连知微生物科技有限公司 | Bacillus amyloliquefaciens for producing surfactin, and application thereof |
| CN106520630A (en) * | 2016-12-14 | 2017-03-22 | 南京秦邦吉品农业开发有限公司 | Bacillus megatherium QBJP-F6 and application thereof |
| CN109207392A (en) * | 2018-09-10 | 2019-01-15 | 青岛秾贝尔生物工程有限公司 | One plant of bacillus megaterium and its application in prevention and treatment American ginseng root maize ear rot |
| CN110358697A (en) * | 2018-04-11 | 2019-10-22 | 中国科学院上海生命科学研究院 | Bacillus megaterium and its application in promotion plant growth |
-
2020
- 2020-01-06 CN CN202010010082.3A patent/CN111172066B/en active Active
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005151887A (en) * | 2003-11-26 | 2005-06-16 | Idemitsu Kosan Co Ltd | Agent and method for controlling soil-borne disease |
| US7977384B1 (en) * | 2007-12-17 | 2011-07-12 | The University Of Louisiana At Monroe | Anticancer tobacco cembranoids |
| KR20090077528A (en) * | 2008-01-11 | 2009-07-15 | 경북대학교 산학협력단 | Bacillus megaterium knuc251 for controlling plant disease and accelerating plant growth, and plant disease controlling agent and plant growth accelerant using the same |
| CN102168036A (en) * | 2010-11-30 | 2011-08-31 | 浙江工业大学 | Preparation of R-4-chloro-3-hydroxybutyrate and S-3-hydroxy-butyrolactone by microbial transformation and strain |
| CN103122328A (en) * | 2012-12-07 | 2013-05-29 | 贵州省烟草公司毕节市公司 | Bacillus megaterium strain and application thereof |
| CN103320360A (en) * | 2013-06-25 | 2013-09-25 | 云南省烟草公司玉溪市公司 | Bacillus megatherium SJ-7 strain and application thereof |
| CN103468613A (en) * | 2013-09-12 | 2013-12-25 | 江苏省苏微微生物研究有限公司 | Bacillus megatherium, method for preparing microbial inoculum through solid fermentation of bacillus megatherium and application of microbial inoculum |
| CN105255785A (en) * | 2015-11-17 | 2016-01-20 | 山东省农业科学院农业资源与环境研究所 | Fermentation method of bacillus megatherium with high rate of sporation |
| CN106011035A (en) * | 2016-08-04 | 2016-10-12 | 大连知微生物科技有限公司 | Bacillus amyloliquefaciens for producing surfactin, and application thereof |
| CN106520630A (en) * | 2016-12-14 | 2017-03-22 | 南京秦邦吉品农业开发有限公司 | Bacillus megatherium QBJP-F6 and application thereof |
| CN110358697A (en) * | 2018-04-11 | 2019-10-22 | 中国科学院上海生命科学研究院 | Bacillus megaterium and its application in promotion plant growth |
| CN109207392A (en) * | 2018-09-10 | 2019-01-15 | 青岛秾贝尔生物工程有限公司 | One plant of bacillus megaterium and its application in prevention and treatment American ginseng root maize ear rot |
Non-Patent Citations (4)
| Title |
|---|
| Isolation, Identification and Characteristics of an Endophytic Quinclorac Degrading Bacterium Bacillus megaterium Q3;Min Liu et al.;《PLOS ONE》;20140922;第9卷(第9期);第1-8页 * |
| 一株紫茎泽兰根际细菌分离鉴定及对烟草促生作用研究;李祖红等;《云南农业大学学报》;20140618;第29卷(第4期);第602-605页 * |
| 巨大芽孢杆菌发酵培养基的优化;饶犇等;《湖北农业科学》;20140831;第53卷(第15期);第3539-3542页 * |
| 巨大芽孢杆菌在生物肥料上的研究现状与发展方向;鲍朋等;《农技服务》;20131231;第30卷(第6期);第601-602页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111172066A (en) | 2020-05-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP2479253B1 (en) | Antagonistic bacteria for preventing and treating panama wilt disease of continuously planted banana and microorganism organic fertilizer thereof | |
| KR101039176B1 (en) | Antagonistic bacteria for preventing and eliminating the bacterial wilt of continuous cropping tobacco and their microbial organic fertilizer | |
| CN111172066B (en) | Bacillus megaterium and application thereof | |
| CN111500489B (en) | Bacillus coagulans and application thereof in tea planting | |
| CN108277177B (en) | Streptomyces microflavus solid fermentation medium, preparation method and fermentation method thereof, fermentation product, biocontrol product and application | |
| CN111944716B (en) | Special compound microbial agent for tobacco seedling culture and preparation method and application thereof | |
| CN111073825B (en) | Bacterium with plant soil-borne disease resistance effect and application thereof | |
| CN103570448B (en) | Large-scale harmless treatment technique of tobacco stems | |
| CN106011022A (en) | Streptomyces roseoflavus solid fermentation medium and preparation and fermentation method thereof | |
| CN110981610A (en) | Microbial agent with disease prevention function and preparation method thereof | |
| CN109526986A (en) | A kind of tobacco diseases prevention biological prevention and control agent and preparation method thereof | |
| CN107338056B (en) | Green microbial soil conditioner and preparation method thereof | |
| CN113980871B (en) | Bacillus belgii strain HT-9 and application thereof | |
| CN117736944B (en) | Streptomyces griseus as well as microbial inoculum and application thereof | |
| CN111172065B (en) | High-yield paenibacillus polymyxa and application thereof | |
| CN110229757B (en) | Trichoderma citrinoviride JS84 capable of effectively promoting crop growth and bio-organic fertilizer developed by trichoderma citrinoviride JS84 | |
| CN111172064B (en) | Bacillus laterosporus and application thereof | |
| CN115747100B (en) | Siamese bacillus N2 and application thereof in preventing and treating ginseng root rot | |
| CN110791459A (en) | Bacillus subtilis for preventing and controlling continuous cropping lily soil-borne blight and application thereof | |
| CN109749938B (en) | Endophytic fungus for reducing incidence rate of panax notoginseng root rot and microbial inoculum thereof | |
| CN113999778B (en) | Trichoderma atroviride microsclerotia and preparation method and application of preparation thereof | |
| CN114058556B (en) | Preparation method of composite microbial agent | |
| CN110106115B (en) | Bacillus subtilis synergist and application thereof in preparation of bacillus subtilis microbial inoculum | |
| CN113773988A (en) | Bacillus subtilis and application thereof in disease prevention and yield increase of pepper | |
| CN113773126B (en) | Biological organic fertilizer for preventing and treating clubroot of Chinese cabbage and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |