CN114958653B - Bacillus subtilis and application thereof - Google Patents
Bacillus subtilis and application thereof Download PDFInfo
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- CN114958653B CN114958653B CN202210462267.7A CN202210462267A CN114958653B CN 114958653 B CN114958653 B CN 114958653B CN 202210462267 A CN202210462267 A CN 202210462267A CN 114958653 B CN114958653 B CN 114958653B
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- PGNXLDQQCINNPZ-BURFUSLBSA-N n-methyl-n-[(2s,3r,4r,5r)-2,3,4,5,6-pentahydroxyhexyl]undecanamide Chemical compound CCCCCCCCCCC(=O)N(C)C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO PGNXLDQQCINNPZ-BURFUSLBSA-N 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Virology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The application relates to the technical field of crop cultivation, and particularly discloses bacillus subtilis and application thereof. The bacillus subtilis is preserved in China general microbiological culture Collection center (CGMCC), and the preservation number is as follows: CGMCC N0.24391, the preservation time is: 2022, 02, 11; cultures, fermentation liquor, bacterial suspension and fermentation supernatant prepared by the bacillus subtilis; and the application of the bacillus subtilis in the aspects of promoting the germination of plant seeds, the growth of plants and inhibiting the growth and propagation of pathogenic fungi and other fungi. The bacillus subtilis provided by the application can promote germination of plant seeds and growth of plants, and can inhibit growth and reproduction of various fungi.
Description
Technical Field
The application relates to the technical field of crop cultivation, in particular to bacillus subtilis and application thereof.
Background
Factors affecting crop harvest include the development of the crop itself, the growth conditions, and the growth environment of the crop. The development and growth of the crops comprise seed germination conditions of the crops and growth conditions of plants; the growing environment of crops includes nutrients in the soil and pathogenic bacteria in the soil.
In general, methods for promoting seed germination mainly pretreat seeds: soaking seeds in ozone, accelerating germination of the soaked seeds, then placing the seeds into a coating agent for stirring, taking out and airing. But the seed pretreatment takes a long time and is complicated to operate. The method for promoting the growth of crop plants mainly comprises fertilization, but when the addition amount of the high-concentration compound fertilizer in soil exceeds the amount which can be maintained by the soil, the fertilizer flows into surrounding water along with rainwater, so that water eutrophication is caused, algae are bred, and then the water environment is damaged, so that agricultural pollution is formed. In addition, the method for inhibiting pathogenic bacteria in the plant growth environment is to apply a medicament or high-temperature treatment, however, the medicament can remain on the plant and even indirectly enter the human body, so that the human body is greatly harmed, the high-temperature treatment is relatively complex in operation, and great inconvenience is brought to operators.
Based on the drawbacks of the related art, it is necessary to provide a new method for improving the development and growth conditions of the crops themselves and the growth environment of the crops.
Disclosure of Invention
The application provides bacillus subtilis and application thereof, and the bacillus subtilis can promote germination of plant seeds and growth of plants, and can inhibit growth and reproduction of pathogenic fungi and other fungi.
In a first aspect, the present application provides a bacillus subtilis, which adopts the following technical scheme:
bacillus subtilis is preserved in China general microbiological culture Collection center (CGMCC), and the preservation number is: CGMCC N0.24391, the preservation time is: 2022, 02, 11.
The application obtains the bacillus subtilis by screening from a region without continuous cropping obstacle (hereinafter referred to as 'continuous cropping obstacle-free soil') on the soil of the potato for many years. The bacillus subtilis can ensure that crops can still keep normal growth in the field with continuous cropping obstacle, which indicates that the bacillus subtilis can improve cultivation of crops in the field with continuous cropping obstacle. Through researches, the bacillus subtilis provided by the application can promote seed germination and plant growth, and can inhibit growth and reproduction of pathogenic bacteria. The bacillus subtilis is used for improving the growth and development conditions of plants and the growth environment of the plants, so that the pollution of fertilization and pesticides to the environment can be avoided, and the operation is simple and convenient. Meanwhile, bacillus subtilis is released into soil, so that the natural flora structure can be enriched, and the natural flora diversity is improved.
The bacillus subtilis is in a rod shape, round ends, most of the bacillus subtilis is single, few bacillus subtilis are arranged in pairs or chains, and the bacillus subtilis is positive after gram staining; the colony surface is rough and opaque, dirty white or yellow, and the edge forms a wrinkle surface, and the colony is picked into a sticky or frozen shape during plate culture.
Preferably, the culturing conditions of the bacillus subtilis are as follows: the temperature is 28-32 ℃, the pH is 6.5-7.5, and the rotating speed of the shaking table is 130-190r/min.
In a specific embodiment, the culturing temperature of the bacillus subtilis may be 28 ℃,30 ℃, 32 ℃.
In some specific embodiments, the culturing temperature of the bacillus subtilis may also be 28-30 ℃, 28-32 ℃, 30-32 ℃.
In a specific embodiment, the bacillus subtilis may have a pH of 6.5, 7 or 7.5.
In some specific embodiments, the bacillus subtilis may also have a culture pH of 6.5-7, 6.5-7.5, 7-7.5.
In a specific embodiment, the culturing speed of the bacillus subtilis can be 130r/min, 160r/min or 190r/min.
In some specific embodiments, the culturing speed of the bacillus subtilis can be 130-160r/min, 130-190r/min and 160-190r/min.
By exploring the proliferation conditions of the bacillus subtilis provided by the application under different culture conditions, the culture conditions of the bacillus subtilis can be known as follows: the temperature is 28-32 ℃, the pH is 6.5-7.5, the rotating speed of the shaking table is 130-190r/min, and under the culture condition, the rapid propagation of bacillus subtilis can be promoted.
In a second aspect, the present application provides a culture comprising the bacillus subtilis described above.
In a third aspect, the present application provides a fermentation broth comprising the bacillus subtilis described above.
In a fourth aspect, the present application provides a bacterial suspension comprising bacillus subtilis as described above.
In a fifth aspect, the present application provides a fermentation supernatant comprising the bacillus subtilis described above.
In a sixth aspect, the application of the bacillus subtilis in promoting seed germination, promoting plant growth and inhibiting growth and reproduction of various pathogenic bacteria is provided.
Further, the bacillus subtilis has a bacterial content of (1-4). Times.10 8 cfu/g. The germination rate of the seeds is improved by 17% -25%, the growth rate of plant roots is improved by 59% -126%, and the fresh weight growth rate of the plants is 66% -166%.
Further, the bacillus subtilis can inhibit the reproduction of pathogenic fungi and other fungi, and the inhibition rate is 40.61-54.33%. Wherein, the inhibition effect on pathogenic bacteria Fusarium graminearum can reach 54.33 percent.
In summary, the present application has the following beneficial effects:
1. the application provides a bacillus subtilis which is preserved in China general microbiological culture collection center (CGMCC), and the preservation number is as follows: CGMCC N0.24391, the preservation time is: 2022, 02, 11.
2. The bacillus subtilis has the bacterial content of (1-4) multiplied by 10 in soil 8 When cfu/g is carried out, the germination rate of seeds can be improved, the growth of plants is promoted, the germination rate of the seeds is improved by 17-25%, the root growth rate of the plants is improved by 59-126%, and the fresh weight growth rate of the plants is 66-166%.
3. The inhibition rate of the bacillus subtilis to various pathogenic bacteria is 40.61-54.33%.
4. The bacillus subtilis provided by the application is added into the soil, so that the harm to human bodies, animal bodies and plants is avoided, and the pollution to the environment is avoided.
5. The bacillus subtilis thalli are released into soil, so that the natural flora structure can be enriched, and the natural flora diversity is improved.
Drawings
FIG. 1 shows the result of culturing Bacillus subtilis according to example 1 in nutrient agar.
Fig. 2 is a photograph of bacillus subtilis provided in example 1 under an optical microscope×1000 (oil mirror).
FIG. 3 is a phylogenetic tree of Bacillus subtilis provided in example 2 constructed based on a 16S rDNA sequence.
FIG. 4 shows the inhibitory effect of Bacillus subtilis on various fungi (A is the inhibitory effect of Bacillus subtilis on Fusarium graminearum; B is the inhibitory effect of Bacillus subtilis on Aspergillus carbon black; C is the inhibitory effect of Bacillus subtilis on Aspergillus flavus; D is the inhibitory effect of Bacillus subtilis on Aspergillus niger).
Detailed Description
The application provides bacillus subtilis and provides methods for separating, purifying and culturing the strain. The strain is positive through gram staining, is in a rod shape, round ends, is in a single shape, is small in a pair or chain shape, is rough and opaque in colony surface, is dirty white or yellow when in flat culture, forms a wrinkle at the edge, and is picked into a sticky shape or a frozen shape. The application also provides a culture, a fermentation broth, a fermentation supernatant and a bacterial suspension prepared by using the bacillus subtilis.
The bacillus subtilis is separated and purified by the following method:
(1) Sampling: collecting soil samples from soil without continuous cropping obstacle;
(2) Primary separation of target bacteria: adding normal saline into the soil sample obtained in the step (1), oscillating, taking supernatant, diluting to different concentrations, and performing plate coating culture until colonies grow on the plate;
(3) And (3) carrying out streak culture on the colony grown in the step (2), repeatedly purifying for a plurality of times, and finally obtaining a single strain and preserving.
Sequencing the sequence of the 16S rDNA of the strain, wherein the sequence of the 16S rDNA is shown as SEQ ID NO. 1; the strain is identified and judged to be bacillus subtilis. In addition, the application also provides application of the bacillus subtilis in promoting seed germination, plant growth and various pathogenic bacteria growth and propagation.
The bacillus subtilis is collected from red Yao village (105 degrees 30 '37' of east longitude and 36 degrees 02 '20' of north latitude) in the Ningxia solid city of Wenji county, and is identified as bacillus subtilis (Bacillus subtilis) by adopting 16S rDNA, and is named as bacillus subtilis BGB-99R and is preserved in China general microbiological culture Collection center (CGMCC), and the preservation number is as follows: CGMCC N0.24391, the preservation time is: 2022, 02, 11, the deposit address is: beijing, chaoyang area, north Chen Xili No.1, 3, china academy of sciences, microbiological institute.
The medium formulation used in this application is as follows:
1. the formula of the nutrient liquid culture medium is as follows: 10g/L peptone, 5g/L, naCl g/L beef extract and distilled water as solvent;
2. the formula of the nutrient solid culture medium is as follows: 10g/L peptone, 5g/L, naCl g/L beef extract and 20g/L agar, wherein the solvent is distilled water;
3. the formula of the nutrient agar medium is as follows: mannitol 10g/L, mgSO 4 ·7H 2 O0.2g/L, naCl0.1g/L, yeast powder 3g/L, K 2 HPO 4 0.25g/L、CaCO 3 3g/L, 25g/L of agar, and distilled water as solvent;
the preparation method of each culture medium comprises the following steps: mixing the above components, dissolving in 1L distilled water, adjusting pH to 7, and sterilizing at 121deg.C for 30min to obtain the desired culture medium.
The formula of the PDA culture medium is as follows: 200g of potato, 20g of glucose, 18g of agar, 1000mL of water and natural pH value.
The preparation method of the PDA culture medium comprises the following steps: weighing 200g of peeled potatoes, cutting the potatoes into small pieces, putting the small pieces into a pot, adding 1000ml of distilled water, heating to boil on a heater, maintaining for 20-30min, filtering on a beaker with 6 layers of gauze while the potato is hot, taking supernatant, adding 20g of glucose and 18g of agar, fully dissolving, fixing the volume with distilled water to 1L,121 ℃ for 30min, sterilizing, pouring the plates, and standing for later use.
The preparation method of the nutrient soil used in the application comprises the following steps: mixing soil conditioner, perlite, vermiculite and coconut husk according to a weight ratio of 3:1:2:4 to prepare nutrient soil, and sterilizing. Wherein, the nutrition index (dry basis) in the sterilized nutrient soil is as follows: 65% of organic matters, 1.45% of nitrogen, 0.37% of phosphorus and 0.16% of potassium.
The soil conditioner used in the application is Wo Meike soil conditioner of Beijing Jiabo biotechnology Co.
Perlite purchasing is from: guangdong agricultural Co., ltd.
Vermiculite purchasing: the vermiculite production and marketing company in the Ming of life and county.
Purchasing coco coir: the forward and backward district of the bergamot market is Xinxing Jie trade business.
The present application is described in further detail below in conjunction with FIGS. 1-4, examples 1-18, and detection assays.
Examples
Example 1
Isolation and purification of strains
(1) Soil sample collection: collected from red Yao rural areas (105 degrees 30 '37' east longitude, 36 degrees 02 '20' north latitude) in the West Ji county of Ningxia solid original city without continuous cropping obstacle soil.
(2) Primary separation of target bacteria: the collected sample is preserved in a low-temperature environment and sent to a laboratory, 10g of the collected sample is weighed, the sample is added into a presterilized triangular flask containing 100ml of physiological saline under the aseptic condition, and the sample is fully oscillated for 30min and is kept stand for 15min at the temperature of 28 ℃. Diluting the supernatant with the concentration of 10 -3 、10 -4 、10 -5 0.2ml of each diluted concentration solution is respectively sucked and added to a nutrient agar culture medium plate for coating, and after the liquid is sucked out, the mixture is placed in a constant temperature incubator at 28 ℃ for inversion culture for 20 hours. As can be seen, colonies grew on the nutrient agar plates.
(3) Purifying: single colonies were picked from the nutrient agar plates with sterilized toothpicks and transferred to new nutrient agar plates for streaking and placed in a constant temperature incubator at 28℃for 20h. Repeating the above purification steps until a strain is present on the nutrient agar medium plate to obtain the purified strain. The strain is preserved in nutrient agar slant culture medium at 4deg.C for use.
Example 2
Identification of strains
And (3) carrying out PCR specific amplification on single bacterial colony liquid of the strain, wherein the primers are 27F and 1492R respectively, and 2 xstar Mix is adopted as enzyme. The primer sequences were as follows:
forward primer 27F:5'-AGAGTTTGATCCTGGCTCAG-3';
reverse primer 1492R:5'-TACGGCTACCTTGTTACGACTT-3'.
The PCR reaction system is shown in Table 1:
TABLE 1 16SrDNA2×Starmix enzyme reaction System
Sequencing the purified and amplified product to obtain forward and reverse sequencing results, obtaining a plurality of reference strain sequences from NCBI (GenBank) database, comparing the 16S rDNA sequences of the strain and the reference strain by using software BioEdit and MEGA11 in full-length analysis, constructing a phylogenetic tree of the strain and the reference strain, and determining the genus related to the phylogenetic of the strain.
Detection result I
Morphological features
The isolated and purified strain of example 1 was observed by a microscope and subjected to gram staining.
As can be seen by observation, the colony surface of the strain is rough and opaque, dirty white or yellow, the edge forms a wrinkle and is picked into a sticky or frozen shape. Gram staining was positive. The morphology of the strain in nutrient agar medium is shown in FIG. 1.
The strain was observed under an optical microscope x 1000 (oil microscope) and the vegetative cells of the individual colonies of the strain were rod-shaped, round-ended, mostly single, few pairs or chain-shaped, as shown in fig. 2.
Through the observation, the characteristics of the strain are close to those of bacillus strains, and the strain is primarily judged to be bacillus.
Authentication
The phylogenetic tree provided in example 2 is shown in fig. 3.
As can be seen from phylogenetic tree, the strain provided by the application and Bacillus subtilis strain show the closest relationship, and the reliability is 99%. Based on the 16S rDNA phylogenetic analysis of the strain, the strain obtained by screening was determined to be Bacillus subtilis (Bacillus subtilis), and designated as BGB-99R. The 16S rDNA sequencing result is shown as SEQ ID NO. 1. The bacillus subtilis BGB-99R is preserved in China general microbiological culture collection center (CGMCC), and the preservation number is as follows: CGMCC N0.24391, the preservation time is: 2022, 02, 11.
Examples 3 to 7
Examples 3-7 provide a method for culturing BGB-99R of Bacillus subtilis, respectively. The above-described embodiments differ in that: culturing temperature of bacillus subtilis BGB-99R. As shown in table 2.
The culturing method of the bacillus subtilis BGB-99R comprises the following steps:
the strain obtained in example 1 was used for activation, and the activated strain was inoculated into 50ml of a nutrient broth, and cultured in a shaker at 28℃and 160r/min for 24 hours, the pH of the nutrient broth being 7, to obtain an inoculum.
The inoculation liquid is added into a new nutrient liquid culture medium according to the inoculation amount of 5 percent, the nutrient liquid culture medium is placed into a constant temperature shaking table with a certain temperature for culture, the culture time is 24 hours, and the pH value of the nutrient liquid culture medium is 7. After the culture is completed, detecting the strain concentration OD of the cultured bacillus subtilis BGB-99R 600 。
TABLE 2 culture temperatures for examples 3-7
| Examples | Culture temperature |
| 3 | 18 |
| 4 | 28 |
| 5 | 30 |
| 6 | 32 |
| 7 | 35 |
Examples 8 to 11
Examples 8-11 provide a method for culturing BGB-99R of Bacillus subtilis, respectively.
The above embodiments differ from embodiment 4 in that: the pH of the nutrient broth is shown in Table 3.
TABLE 3 pH values of the media of example 4, examples 8-11
| Examples | pH value of culture |
| 4 | 7 |
| 8 | 6 |
| 9 | 6.5 |
| 10 | 7.5 |
| 11 | 8 |
Examples 12 to 15
Examples 12-15 provide a method for culturing BGB-99R of Bacillus subtilis, respectively.
The above embodiments differ from the embodiment 4 in that: the rotational speed of the shaker used in the cultivation method is shown in Table 4.
Table 4 rotational speed of the shaker in the method provided in example 4, examples 12-15
| Examples | Rotational speed r/min |
| 4 | 160 |
| 12 | 100 |
| 13 | 130 |
| 14 | 190 |
| 15 | 220 |
Second detection result
Bacterial species concentration OD of bacillus subtilis BGB-99R obtained by culturing the culture methods provided in examples 3-15 respectively 600 The results of the detection are shown in Table 5.
TABLE 5 concentration of species of Bacillus subtilis BGB-99R cultured in examples 3-15
| Examples | OD 600 Value of |
| 3 | 0.22 |
| 4 | 1.42 |
| 5 | 1.35 |
| 6 | 1.05 |
| 7 | 0.31 |
| 8 | 0.35 |
| 9 | 0.72 |
| 10 | 0.68 |
| 11 | 0.56 |
| 12 | 0.38 |
| 13 | 0.49 |
| 14 | 0.58 |
| 15 | 0.35 |
As can be seen from the combination of Table 2 and Table 5, comparative examples 3 to 7 have a higher strain concentration of Bacillus subtilis BGB-99R at a temperature of 28 to 32℃than that of Bacillus subtilis BGB-99R at a temperature of 18℃or 35 ℃. Therefore, the bacillus subtilis BGB-99R provided by the application can improve the propagation speed of the bacillus subtilis BGB-99R under the condition that the temperature is 28-32 ℃, namely, the fermentation rate of the bacillus subtilis BGB-99R can be improved.
As is clear from the results of comparative examples 4 and 8 to 11, when the pH of B.subtilis BGB-99R was controlled in the range of 6.5 to 7.5, the concentration of B.subtilis BGB-99R was high, and the concentration of B.subtilis BGB-99R was higher than when the pH was less than 6.5 or greater than 7.5. Therefore, the bacillus subtilis BGB-99R provided by the application can improve the propagation speed of the bacillus subtilis BGB-99R within the pH range of 6.5-7.5, namely the fermentation rate of the bacillus subtilis BGB-99R.
By combining the table 4 and the table 5, the comparative examples 4 and 12-15 show that when the rotation speed of the shaking table is controlled within the range of 130-190R/min, the bacterial concentration of the bacillus subtilis BGB-99R is higher and is greater than that of the bacillus subtilis BGB-99R when the rotation speed of the shaking table is less than 100R/min or greater than 220R/min, so that the propagation speed of the bacillus subtilis BGB-99R can be improved when the rotation speed of the shaking table is within the range of 130-190R/min.
In conclusion, the optimal growth temperature of the bacillus subtilis BGB-99R provided by the application is 28-32 ℃, the optimal growth pH is 6.5-7.5, and the optimal rotation speed of the shaking table is 130-190R/min. Under the growth condition, the bacillus subtilis BGB-99R grows and breeds fastest.
Example 16
Example 16 provides a fermentation broth of B.subtilis BGB-99R.
The preparation method of the fermentation broth comprises the following steps: the strain provided in example 1 was activated and transferred to a nutrient broth for 20h at 28℃and pH 7 to obtain a broth.
Example 17
Example 17 provides a fermentation supernatant of B.subtilis BGB-99R.
The preparation method of the fermentation supernatant comprises the following steps: centrifuging the fermentation broth obtained in the example 16, and obtaining supernatant and precipitate after centrifugation, wherein the supernatant is fermentation supernatant.
Example 18
Example 18 provides a bacterial suspension of B.subtilis BGB-99R.
The preparation method of the bacterial suspension comprises the following steps: centrifuging the fermentation broth obtained in the example 16, and obtaining supernatant and precipitate after centrifugation, wherein the precipitate is bacillus subtilis BGB-99R thallus, and diluting the bacillus subtilis BGB-99R thallus with sterile water to obtain bacterial suspension.
Detection test
Bacillus subtilis BGB-99R antagonism test for various fungi
The fungal species used in the detection assay include Fusarium graminearum, aspergillus carbon black, aspergillus flavus, and Aspergillus niger.
(1) Preparing fungus cakes and bacillus subtilis BGB-99R cakes.
Activating fungi, transferring to PDA culture medium, culturing for 96 hr at 30deg.C to obtain fungi plate, and punching with 5mm aseptic puncher to obtain 5mm fungi cake.
Activating the bacillus subtilis BGB-99R, transferring the bacillus subtilis BGB-99R into a nutrient solid culture medium, culturing for 24 hours at the temperature of 30 ℃ to obtain a bacillus subtilis BGB-99R flat plate, and punching by using a 5mm sterile puncher to obtain a 5mm bacillus subtilis BGB-99R bacterial cake.
(2) The bacillus subtilis BGB-99R bacterial cake is placed at a position which is 2.5cm away from the center of the PDA culture medium, and the fungal bacterial cake is placed at a position which is 2.5cm away from the center of the PDA culture medium and 5cm away from the other bacterial cake, so as to be used as a treatment group.
In addition, a 5mm fungal cake was placed on the new PDA medium as a control. Both the treated group and the control group were incubated at 32℃for 72h.
(3) Observing the antibacterial effect and calculating the antibacterial rate;
the calculation formula is as follows:
antibacterial ratio (%) = (Rp-Rt)/rp×100%
Wherein Rp represents the fungal hypha growth radius of the control group; rt represents the fungal hypha growth radius of the treatment group.
The results of the culture of the above-mentioned treatment group and control group are shown in FIG. 4. The inhibition effect of BGB-99R of bacillus subtilis on fungi obtained by analysis is shown in Table 6.
TABLE 6 inhibition of fungi by Bacillus subtilis BGB-99R
Referring to FIG. 4 and Table 6, it can be seen that Bacillus subtilis BGB-99R has significant inhibition effect on Fusarium graminearum, carbon black mold, aspergillus flavus and Aspergillus niger, the inhibition rate is 40.61-54.33%, and the inhibition effect on pathogenic bacteria Fusarium graminearum is strongest at 54.33%.
Seed germination test
Obtaining example 18The obtained bacillus subtilis BGB-99R bacterial suspension is respectively diluted to 2 multiplied by 10 10 、4×10 10 、6×10 10 、8×10 10 cfu/ml, seed germination experiments were performed.
The method of the seed germination test is as follows:
(1) 2kg of sterilized nutrient soil is placed in a seedling tray, 10ml of bacillus subtilis BGB-99R bacterial suspension with the concentration is respectively used for thoroughly watering the nutrient soil, namely the bacterial contents of the nutrient soil are respectively 1 multiplied by 10 at the moment 8 、2×10 8 、3×10 8 、4×10 8 cfu/g as test group; in addition, nutrient soil was thoroughly poured with the same volume of water as a control group.
(2) Placing the seedling raising tray treated in the step (1) in a greenhouse at 28 ℃ for 12 hours, treating cucumber seeds with 0.2% sodium hypochlorite for 2 minutes, washing with sterile water for 5 times, planting in the seedling raising tray in the step (1) in a hill-drop manner, and observing germination conditions of the cucumber seeds after 10 days.
(3) Detection of germination Rate
Germination rate was calculated by means of counting. The test results are shown in Table 7.
TABLE 7 test results of seed germination
By analyzing the test results and comparing the germination rates of the test group and the control group, according to the table 7, it can be seen that the germination rate of seeds can be promoted by adding the bacillus subtilis BGB-99R bacterial suspension into the nutrient soil, and the germination rate is improved by at least 17%. Therefore, the bacterial content of the bacillus subtilis BGB-99R is controlled to be (1-4). Times.10 8 In the cfu/ml range, the germination rate of seeds can be effectively improved, and the germination of the seeds is promoted.
Further, it is known that when seed germination rates are compared with those of the suspensions of Bacillus subtilis BGB-99R having different concentrations, the bacterial content of Bacillus subtilis BGB-99R is (1-3). Times.10 8 When cfu/ml, the germination rate of the seeds is more than 93.45 percent; especially when the bacterial content of the bacillus subtilis BGB-99R is 2×10 8 When cfu/ml, the germination rate of the seeds is 98.27%, which is improved by 25% compared with the germination rate of the control group. While when the bacillus subtilis BGB-99R has a bacterial content of 4 multiplied by 10 8 At cfu/ml, the germination rate of the seeds was 90.23%. Therefore, the bacterial content of the bacillus subtilis BGB-99R is controlled to be (1-3). Times.10 8 In the cfu/ml range, the germination rate of the seeds can be further improved, and the germination of the seeds is further promoted.
Thirdly, plant growth test
The Bacillus subtilis BGB-99R bacterial suspensions obtained in example 18 were diluted to 2.5X10 respectively 10 、5×10 10 、7.5×10 10 、10×10 10 cfu/ml, plant growth experiments were performed.
The method for plant growth test is as follows:
(1) 2.5kg of sterilized nutrient soil is placed in a seedling raising tray, 10ml of bacillus subtilis BGB-99R diluted bacterial suspension with the concentration is respectively used for thoroughly watering the nutrient soil, namely the bacterial contents of the nutrient soil are respectively 1 multiplied by 10 at the moment 8 、2×10 8 、3×10 8 、4×10 8 cfu/g as test group; in addition, nutrient soil was thoroughly poured with the same volume of water as a control group.
(2) Placing the seedling raising tray treated in the step (1) in a greenhouse at 28 ℃ for 12 hours, transplanting cucumber seedlings cultured in a hollow white control group in a second seed germination test into the seedling raising tray in the step (1), placing the transplanted seedlings in an artificial growth chamber (photoperiod: day/night=16 h/8h; temperature: day/night=30 ℃/24 ℃) for culturing, and detecting plant root length (cm) and plant fresh weight (g) after culturing for 30 days.
(3) Plant root length (cm) and plant fresh weight (g): the test results are shown in Table 8.
Wherein root length increase rate (%) = (experimental group root length-control group root length)/control group root length×100%; fresh weight increase (%) = (experimental fresh weight-control fresh weight)/control fresh weight x 100%.
TABLE 8 test results of plant growth
By analyzing the above test results and comparing the plant growth conditions of the test group and the control group, it can be seen that the addition of the Bacillus subtilis BGB-99R suspension to the nutrient soil can promote the growth of plants, wherein the root length is increased by at least 59%, and the fresh weight is increased by at least 66%. Therefore, the bacterial content of the bacillus subtilis BGB-99R is controlled to be (1-4). Times.10 8 In the cfu/ml range, the growth of plants can be effectively promoted.
Further, comparing plant growth conditions when using different concentrations of the Bacillus subtilis BGB-99R bacterial suspension, it is known that when the bacterial content of the Bacillus subtilis BGB-99R is (2-4). Times.10 8 When cfu/ml, the root length of the plant is above 13.26cm, and the fresh weight is above 3.28 g. The bacterial content of BGB-99R is 1×10 8 At cfu/ml, the root length of the plant is equal to the fresh weight. Especially when the bacillus subtilis BGB-99R has a bacterial content of 2×10 8 When cfu/ml, the root length of the plant is 18.23cm, the fresh weight is 4.78g, and the root length and the fresh weight of the plant are increased by more than one time compared with those of the plant of the control group. Therefore, the bacterial content of the bacillus subtilis BGB-99R is controlled to be (2-4). Times.10 8 In the cfu/ml range, the growth of plants can be further promoted.
It is to be understood that the above embodiments are merely illustrative of the application of the principles of the present invention, but not in limitation thereof. Various modifications and improvements may be made by those skilled in the art without departing from the spirit and substance of the invention, and are also considered to be within the scope of the invention.
Sequence listing
<110> Beijing Jiabo Biotech Co., ltd
<120> a Bacillus subtilis and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1451
<212> DNA
<213> Bacillus subtilis (Bacillus subtilis)
<400> 1
ggcggggtgc tataatgcaa gtcgagcgga cagatgggag cttgctccct gatgttagcg 60
gcggacgggt gagtaacacg tgggtaacct gcctgtaaga ctgggataac tccgggaaac 120
cggggctaat accggatgct tgtttgaacc gcatggttca aacataaaag gtggcttcgg 180
ctaccactta cagatggacc cgcggcgcat tagctagttg gtgaggtaat ggctcaccaa 240
ggcaacgatg cgtagccgac ctgagagggt gatcggccac actgggactg agacacggcc 300
cagactccta cgggaggcag cagtagggaa tcttccgcaa tggacgaaag tctgacggag 360
caacgccgcg tgagtgatga aggttttcgg atcgtaaagc tctgttgtta gggaagaaca 420
agtgccgttc aaatagggcg gcaccttgac ggtacctaac cagaaagcca cggctaacta 480
cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg tccggaatta ttgggcgtaa 540
agggctcgca ggcggtttct taagtctgat gtgaaagccc ccggctcaac cggggagggt 600
cattggaaac tggggaactt gagtgcagaa gaggagagtg gaattccacg tgtagcggtg 660
aaatgcgtag agatgtggag gaacaccagt ggcgaaggcg actctctggt ctgtaactga 720
cgctgaggag cgaaagcgtg gggagcgaac aggattagat accctggtag tccacgccgt 780
aaacgatgag tgctaagtgt tagggggttt ccgcccctta gtgctgcagc taacgcatta 840
agcactccgc ctggggagta cggtcgcaag actgaaactc aaaggaattg acgggggccc 900
gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accaggtctt 960
gacatcctct gacaacccta gagatagggc ttccccttcg ggggcagagt gacaggtggt 1020
gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac 1080
ccttgatctt agttgccagc attcagttgg gcactctaag gtgactgccg gtgacaaacc 1140
ggaggaaggt ggggatgacg tcaaatcatc atgcccctta tgacctgggc tacacacgtg 1200
ctacaatgga cagaacaaag ggcagcgaga ccgcgaggtt aagccaatcc cacaaatctg 1260
ttctcagttc ggatcgcagt ctgcaactcg actgcgtgaa gctggaatcg ctagtaatcg 1320
cggatcagca tgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca 1380
cgagagtttg taacacccga agtcggtgag gtaaccttta tggagccagc cgccgaaggt 1440
cacagagttg t 1451
Claims (9)
1. The bacillus subtilis is characterized in that the bacillus subtilis is preserved in China general microbiological culture collection center (CGMCC), and the preservation number is as follows: CGMCC N0.24391, the preservation time is: 2022, 02, 11.
2. The bacillus subtilis according to claim 1, wherein the bacillus subtilis is gram-positive, the strain is in a rod shape, round end, a plurality of single strains, a few pairs or chain-shaped strains, the colony surface is rough and opaque, dirty white or yellow, a wrinkle is formed at the edge during plate culture, and the colony is picked to be sticky or frozen.
3. A culture comprising the bacillus subtilis of any one of claims 1-2.
4. A fermentation broth prepared by using the Bacillus subtilis of claim 1.
5. A bacterial suspension prepared using the bacillus subtilis of claim 1.
6. The use of the bacillus subtilis according to claim 1, wherein the bacillus subtilis is used for promoting germination of cucumber seeds, promoting growth of cucumber seedlings and inhibiting growth and reproduction of fusarium graminearum, carbon black mold, aspergillus flavus and aspergillus niger.
7. The use of the bacillus subtilis according to claim 6, wherein the bacillus subtilis has a bacterial content of (1-4) ×10 8 cfu/g。
8. The use of bacillus subtilis according to claim 6, characterized in that the inhibition rate of the bacillus subtilis against fungi is 40.61-54.33%.
9. The use of bacillus subtilis according to claim 6, characterized in that the inhibition rate of bacillus subtilis against fusarium graminearum is up to 54.33%.
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