CN114958653A - Bacillus subtilis and application thereof - Google Patents
Bacillus subtilis and application thereof Download PDFInfo
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- CN114958653A CN114958653A CN202210462267.7A CN202210462267A CN114958653A CN 114958653 A CN114958653 A CN 114958653A CN 202210462267 A CN202210462267 A CN 202210462267A CN 114958653 A CN114958653 A CN 114958653A
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Images
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
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 center for type culture Collection (CGMCC) of Beijing microorganisms, and the preservation number is as follows: CGMCC N0.24391, the preservation time is as follows: 11/02/2022; preparing culture, fermentation liquor, bacterial suspension and fermentation supernatant by using the bacillus subtilis; and the application of the bacillus subtilis in promoting the germination of plant seeds, the growth of plants and inhibiting the growth and reproduction of pathogenic fungi and other fungi. The bacillus subtilis can promote the germination of plant seeds and the growth of plants, and can inhibit the growth and the propagation 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 influencing crop harvest include development and growth conditions of crops and growth environment of crops. Wherein, the development and growth of the crops comprise the seed germination condition of the crops and the growth condition of plants; the growing environment of crops includes nutrients in the soil and pathogenic bacteria in the soil.
Generally, the method for promoting seed germination is mainly to pretreat the seeds: soaking the seeds in ozone, accelerating germination of the soaked seeds, then putting the seeds into a coating agent, stirring, taking out and airing. However, the time required for the pretreatment of the seeds is long and the operation is complicated. The method for promoting the growth of crop plants mainly comprises fertilization, however, when the addition amount of the high-concentration compound fertilizer in the soil exceeds the amount which can be kept by the soil, the fertilizer flows into the surrounding water along with rainwater, so that the eutrophication of water bodies is caused, algae is bred, the water environment is further damaged, and agricultural pollution is formed. In addition, the method for inhibiting the pathogenic bacteria in the plant growth environment is to apply a medicament or carry out high-temperature treatment, however, the medicament can remain on the plant and even indirectly enter the human body to cause great harm to the human body, and the high-temperature treatment is complex to operate and brings great inconvenience to operators.
Based on the above-mentioned drawbacks of the related art, it is necessary to provide a new method for improving the development and growth conditions of crops themselves and the growing environment of crops.
Disclosure of Invention
The application provides a bacillus subtilis and an application thereof, and the bacillus subtilis can promote the germination of plant seeds and the growth of plants, and can inhibit the growth and the propagation of pathogenic fungi and other fungi.
In a first aspect, the present application provides a bacillus subtilis, which adopts the following technical scheme:
the bacillus subtilis is preserved in China center for type culture Collection (CGMCC) of Beijing microorganisms, and the preservation number is as follows: CGMCC N0.24391, the preservation time is as follows: 11/02/2022.
The bacillus subtilis is obtained by screening a continuous cropping obstacle-free area (hereinafter referred to as continuous cropping obstacle-free soil) on soil of a plurality of years of continuous cropping potatoes. The bacillus subtilis can still keep the normal growth of crops in the field with continuous cropping obstacles, which indicates that the bacillus subtilis can improve the cultivation of the crops in the field with the continuous cropping obstacles. Through researches, the bacillus subtilis provided by the application can promote seed germination and plant growth, and can inhibit the growth and propagation of pathogenic bacteria. The growth and development conditions of the plants and the growth environment of the plants are improved by using the bacillus subtilis, so that the pollution of fertilization and pesticides to the environment can be avoided, and the operation is simple and convenient. Meanwhile, the bacillus subtilis is released into the soil, so that the structure of natural flora can be enriched, and the diversity of the natural flora is improved.
The bacillus subtilis is rod-shaped and round-ended, is mostly arranged in a single, a few pairs or a chain shape, and shows positive through gram staining; during plate culture, the surface of a colony is rough and opaque, is dirty white or yellow, has skin marks at the edge and is picked to be sticky or frozen.
Preferably, the culture conditions of the bacillus subtilis are as follows: the temperature is 28-32 ℃, the pH is 6.5-7.5, and the rotation speed of the shaking table is 130-190 r/min.
In a specific embodiment, the Bacillus subtilis may be cultured at 28 deg.C, 30 deg.C, 32 deg.C.
In some specific embodiments, the Bacillus subtilis may be cultured at 28-30 deg.C, 28-32 deg.C, and 30-32 deg.C.
In a particular embodiment, the bacillus subtilis may have a pH of 6.5, 7 or 7.5.
In some specific embodiments, the Bacillus subtilis may also be cultured at a pH of 6.5 to 7, 6.5 to 7.5, 7 to 7.5.
In a specific embodiment, the culture rotation speed of the bacillus subtilis can be 130r/min, 160r/min or 190 r/min.
In some specific embodiments, the culture rotation speed of Bacillus subtilis can also be 160r/min, 190r/min, 160-190 r/min.
By studying the proliferation of the bacillus subtilis provided by the application under different culture conditions, it can be known that the culture conditions of the bacillus subtilis are controlled as follows: the temperature is 28-32 ℃, the pH is 6.5-7.5, the rotation speed of the shaking table is 130-.
In a second aspect, the present application provides a culture comprising the bacillus subtilis above.
In a third aspect, the present application provides a fermentation broth comprising the bacillus subtilis above.
In a fourth aspect, the present application provides a bacterial suspension comprising the bacillus subtilis described above.
In a fifth aspect, the present application provides a fermentation supernatant comprising the bacillus subtilis above.
In a sixth aspect, the application of the bacillus subtilis in promoting seed germination, promoting plant growth and inhibiting the growth and reproduction of various pathogenic bacteria is provided.
Further, the bacterial content of Bacillus subtilis is (1-4). times.10 8 cfu/g. The seed germination rate is improved by 17-25%, the growth rate of the root length of the plant is improved by 59-126%, and the growth rate of the fresh weight of the plant is 66-166%.
Furthermore, 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 fusarium graminearum can reach 54.33%.
In summary, the present application has the following beneficial effects:
1. the application provides bacillus subtilis which is preserved in China center for type culture Collection (CGMCC) of Beijing microorganisms, and the preservation number is as follows: CGMCC N0.24391, preservation time is as follows: 11/02/2022.
2. The application provides a Bacillus subtilis in soilThe content of the bacteria in the culture medium is (1-4) multiplied by 10 8 And when cfu/g is adopted, 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 growth rate of the root length of the plants is improved by 59-126%, and the growth rate of the fresh weight 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 application provides a bacillus subtilis adds in soil, can not produce harm to human body, animal body and plant, also can not cause the pollution to the environment.
5. The bacillus subtilis is released into soil, so that the structure of natural flora can be enriched, and the diversity of the natural flora can be improved.
Drawings
FIG. 1 shows the results of culturing Bacillus subtilis in nutrient agar as provided in example 1.
FIG. 2 is a photograph of Bacillus subtilis under an optical microscope × 1000 (oil lens) as provided in example 1.
FIG. 3 shows a phylogenetic tree constructed from Bacillus subtilis based on the 16S rDNA sequence provided in example 2.
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 carbonarius, C is the inhibitory effect of Bacillus subtilis on Aspergillus flavus, and D is the inhibitory effect of Bacillus subtilis on Aspergillus niger).
Detailed Description
The application provides bacillus subtilis and provides a method for separating, purifying and culturing the bacillus subtilis. The strain shows positive through gram staining, the strain is rod-shaped and round-ended, most of the strain is arranged in a single, a few pairs or chain shape, the surface of a colony is rough and opaque, is dirty white or yellow when cultured on a plate, and the edge of the colony forms wrinkle films which are picked to be sticky or frozen. The application also provides a culture, fermentation liquor, fermentation supernatant and bacterial suspension prepared by using the bacillus subtilis.
The separation and purification of the bacillus subtilis adopt the following method:
(1) sampling: collecting a soil sample in the continuous cropping obstacle-free soil;
(2) primary separation of target bacteria: adding normal saline into the soil sample obtained in the step (1), oscillating, taking supernatant fluid to dilute to different concentrations, and performing plate coating culture until bacterial colonies grow on the plate;
(3) and (3) carrying out streak culture on the bacterial colony grown in the step (2), repeatedly carrying out purification for multiple times, finally obtaining a single bacterial strain, and storing.
Sequencing the 16S rDNA sequence of the strain, wherein the sequence of the 16S rDNA is shown in SEQ ID NO. 1; the strain is judged to be bacillus subtilis through identification. In addition, the application also provides the application of the bacillus subtilis in promoting seed germination, promoting plant growth and inhibiting the growth and propagation of various pathogenic bacteria.
The Bacillus subtilis provided by the application is collected from Hongyangxiang (east longitude 105 degrees 30 '37', north latitude 36 degrees 02 '20') of Xiji county of Ningxia Guyuan city without continuous cropping obstacle soil, is identified as Bacillus subtilis by adopting 16S rDNA, is named as Bacillus subtilis BGB-99R, is preserved in China center for type culture Collection of microorganisms (CGMCC) in Beijing, and has the preservation number: CGMCC N0.24391, preservation time is as follows: 11/02/2022.
The culture medium used in the present application has the following formulation:
1. the formula of the nutrient liquid culture medium is as follows: 10g/L of peptone, 5g/L, NaCl10g/L of beef extract and distilled water as a solvent;
2. the nutrient solid culture medium comprises the following components in percentage by weight: 10g/L of peptone, 5g/L, NaCl10g/L of beef extract and 20g/L of agar, wherein the solvent is distilled water;
3. the nutrient agar culture medium comprises the following components: 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 of agar and 25g/L of agar, and the solvent is distilled water;
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 121 deg.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 and 1000mL of water, and the pH value is natural.
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 the small pieces on a heater until the potatoes are boiled, maintaining the boiling state for 20-30min, filtering the small pieces on a beaker with 6 layers of gauze while the potatoes are hot, taking supernate, adding 20g of glucose and 18g of agar, fully dissolving the supernate, fixing the volume to 1L by using distilled water, sterilizing the supernate at 121 ℃ for 30min, pouring the mixture onto a plate, and standing the mixture for later use.
The preparation method of the nutrient soil used in the application comprises the following steps: mixing the soil conditioner, the perlite, the vermiculite and the coconut chaff according to the weight ratio of 3:1:2:4 to prepare the nutrient soil, and sterilizing. Wherein the nutritional indexes (dry basis) in the sterilized nutrient soil are as follows: 65% of organic matter, 1.45% of nitrogen, 0.37% of phosphorus and 0.16% of potassium.
The soil conditioner used in the application is a Walmeck soil conditioner of Beijing Jia Bo Wen Biotech limited company.
Perlite was purchased from: guangdong Turon agriculture, Inc.
Vermiculite was purchased from: the Ming Hui county of vermiculite production and marketing Ming.
Coconut coir was purchased from: the Yangde district in Fushan City is happy and quick trade business.
The present application is described in further detail below with reference to FIGS. 1-4, examples 1-18, and the test assays.
Examples
Example 1
Separating and purifying strain
(1) Collecting a soil sample: collected from Hongyouxiang (105 degrees 30 '37 degrees in east longitude and 36 degrees 02' 20 degrees in northern latitude) in Xiji county of Ningxia Guyuan.
(2) Primary separation of target bacteria: storing the collected sample at low temperature, sending to a laboratory, weighing 10g of the collected sample, adding into a pre-sterilized triangular flask containing 100ml of physiological saline under aseptic condition, fully oscillating for 30min at 28 ℃, and standing for 15 min. Taking the supernatant fluid to dilute the supernatant fluid,the dilution concentration is 10 in sequence -3 、10 -4 、10 -5 Respectively sucking 0.2ml of each diluted solution, adding the solution to a nutrient agar culture medium plate, coating, and placing the nutrient agar culture medium plate in a constant-temperature incubator at 28 ℃ for inverted culture for 20 hours after the solution is completely sucked. The observation shows that the colonies grow on the nutrient agar medium plate.
(3) And (3) purification: picking single colony from the nutrient agar culture medium plate by using a sterilized toothpick, transferring the single colony to a new nutrient agar culture medium plate for streaking, and culturing in an incubator at 28 ℃ for 20 h. Repeating the purification steps until a strain is on the nutrient agar culture medium plate, and obtaining the purified strain. The strain is preserved in nutrient agar slant culture medium at 4 deg.C for use.
Example 2
Identification of strains
Carrying out PCR specific amplification on the bacterial liquid of the single colony of the strain, wherein primers are 27F and 1492R respectively, and enzyme is 2 XStar Mix. The primer sequences are as follows:
forward primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3';
reverse primer 1492R: 5'-TACGGCTACCTTGTTACGACTT-3' are provided.
The PCR reaction system is shown in Table 1:
TABLE 116 SrDNA 2 × starMix enzyme reaction System
Sequencing the purified and amplified product to obtain a forward and reverse sequencing result, then obtaining a plurality of reference strain sequences from an NCBI (GenBank) database, analyzing and comparing the 16S rDNA sequences of the strain and the reference strain in full length by using software BioEdit and MEGA11, constructing a phylogenetic tree of the strain and the reference strain, and determining the phylogenetic related genera of the strain.
Detection result 1
Morphological characteristics
The isolated and purified bacterial species of example 1 were observed under a microscope and gram stained.
As can be seen by observation, the colony surface of the strain is rough and opaque, is white or yellow, forms wrinkles on the edge, and is sticky or frozen. Gram staining was positive. The morphology of the above strain in nutrient agar medium is shown in FIG. 1.
The strain is observed under an optical microscope multiplied by 1000 (oil lens), the vegetative cells of a single colony of the strain are rod-shaped and round-ended, and most vegetative cells are arranged in a single, a few pairs or a chain, as shown in FIG. 2.
Through the observation, the characteristics of the strain are close to those of the bacillus strain, and the strain is preliminarily judged to be the bacillus.
Identification
Example 2 provides a phylogenetic tree as shown in figure 3.
According to a phylogenetic tree, the genetic relationship between the strain provided by the application and the Bacillus subtilis strain is the closest, and the reliability is 99%. Based on the 16S rDNA phylogenetic analysis of the strain, the strain obtained by screening is determined to be Bacillus subtilis and named as BGB-99R. The 16S rDNA sequencing result is shown in 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 as follows: 11/02/2022.
Examples 3 to 7
Examples 3-7 provide methods for culturing Bacillus subtilis BGB-99R, respectively. The above embodiments differ in that: and (3) the culture temperature of the bacillus subtilis BGB-99R. Specifically, as shown in table 2.
The method for culturing the bacillus subtilis BGB-99R comprises the following steps:
the strain obtained in example 1 was activated, and the activated strain was inoculated into 50ml of a nutrient liquid medium, which was then cultured at 28 ℃ for 24 hours in a shaker at 160r/min, and the pH of the nutrient liquid medium was 7, to obtain an inoculum.
Adding the inoculum to new nutrient liquid culture medium according to 5%And placing the nutrient liquid culture medium in a constant-temperature shaking table at a certain temperature for culturing for 24 hours, wherein the pH value of the nutrient liquid culture medium is 7. After the culture is finished, detecting the strain concentration OD of the cultured bacillus subtilis BGB-99R 600 。
TABLE 2 cultivation temperature for examples 3-7
| Examples | Temperature of culture |
| 3 | 18 |
| 4 | 28 |
| 5 | 30 |
| 6 | 32 |
| 7 | 35 |
Examples 8 to 11
Examples 8 to 11 provide methods for culturing Bacillus subtilis BGB-99R, respectively.
The above embodiments are different 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 and 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 to 15 provide methods for culturing Bacillus subtilis BGB-99R, respectively.
The above embodiments are different from embodiment 4 in that: the rotational speed of the rocking platforms used in the cultivation method is specifically shown in Table 4.
TABLE 4 rotation speed of rocking platforms in the methods provided in example 4 and examples 12 to 15
| Examples | Rotational speed r/min |
| 4 | 160 |
| 12 | 100 |
| 13 | 130 |
| 14 | 190 |
| 15 | 220 |
Detection result two
The bacterial species concentration OD of Bacillus subtilis BGB-99R cultured by the culture methods provided in examples 3-15 respectively 600 The results of the tests are shown in Table 5.
TABLE 5 bacterial species concentrations of Bacillus subtilis BGB-99R cultured in examples 3-15
In the comparative examples 3 to 7, it is understood by combining Table 2 and Table 5 that the seed concentration of Bacillus subtilis BGB-99R is higher at a temperature of 28 to 32 ℃ than 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, namely the fermentation rate of the Bacillus subtilis BGB-99R under the condition that the temperature is 28-32 ℃.
In Table 3 and Table 5, it is understood by comparing example 4 and examples 8 to 11 that when the pH of Bacillus subtilis BGB-99R is controlled within the range of 6.5 to 7.5, the cell concentration of Bacillus subtilis BGB-99R is higher than that when the pH is 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.
With reference to table 4 and table 5, it can be seen from comparison between examples 4 and 12-15 that when the rotation speed of the shaking table is controlled within the range of 190R/min-.
In conclusion, the optimum growth temperature of the Bacillus subtilis BGB-99R provided by the application is 28-32 ℃, the optimum growth pH is 6.5-7.5, and the optimum rotation speed of the shaking table is 130-. Under the growth condition, the Bacillus subtilis BGB-99R grows and breeds fastest.
Example 16
Example 16 provides a fermentation broth of bacillus subtilis BGB-99R.
The preparation method of the fermentation liquor comprises the following steps: the strain provided in example 1 was activated and inoculated into a nutrient liquid medium for 20 hours at a temperature of 28 ℃ and a pH of the medium of 7 to obtain a fermentation broth.
Example 17
Example 17 provides a fermentation supernatant of bacillus subtilis BGB-99R.
The preparation method of the fermentation supernatant comprises the following steps: the fermentation liquid obtained in example 16 was centrifuged to obtain a supernatant and a precipitate, and the supernatant was the fermentation supernatant.
Example 18
Example 18 provides a bacterial suspension of bacillus subtilis BGB-99R.
The preparation method of the bacterial suspension comprises the following steps: the fermentation liquid obtained in example 16 was centrifuged to obtain a supernatant and a precipitate, which was the Bacillus subtilis BGB-99R, and the Bacillus subtilis BGB-99R was diluted with sterile water to obtain a suspension.
Detection test
Bacillus subtilis BGB-99R antagonism multiple fungus test
The types of fungi used in the detection tests include Fusarium graminearum, Aspergillus carbonarius, Aspergillus flavus, Aspergillus niger.
(1) Preparing fungus cake and Bacillus subtilis BGB-99R cake.
Activating fungi, inoculating into PDA culture medium, culturing at 30 deg.C for 96 hr to obtain fungi plate, and perforating with 5mm sterile perforator to obtain 5mm fungus cake.
Activating the Bacillus subtilis BGB-99R, inoculating into a nutrient solid culture medium, culturing for 24h at the culture 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 cake.
(2) And (3) placing the bacillus subtilis BGB-99R fungus cake at a position 2.5cm away from the center of the PDA culture medium, and taking the fungus cake at a position 2.5cm away from the center of the PDA culture medium and 5cm away from the other fungus cake as a treatment group.
In addition, 5mm fungal cake was placed on new PDA medium as a control. Both the treated and control groups were incubated at 32 ℃ for 72 h.
(3) Observing the bacteriostatic effect, and calculating the bacteriostatic rate;
the calculation formula is as follows:
the bacteriostatic rate (%) is (Rp-Rt)/Rp multiplied by 100%
Wherein Rp represents the growth radius of the fungal hyphae of the control group; rt represents the radius of hyphal growth of the fungi of the treated group.
The results of the culture of the treated group and the control group are shown in FIG. 4. The inhibitory effect of the obtained Bacillus subtilis BGB-99R on fungi was analyzed and shown in Table 6.
TABLE 6 inhibitory Effect of Bacillus subtilis BGB-99R on fungi
With reference to fig. 4 and table 6, it can be seen that bacillus subtilis BGB-99R has significant inhibitory effects on fusarium graminearum, carbon black mold, aspergillus flavus and aspergillus niger, the inhibitory rate is 40.61-54.33%, and the strongest inhibitory effect on pathogenic bacteria fusarium graminearum is 54.33%.
Seed germination test
The Bacillus subtilis BGB-99R bacterial suspensions obtained in example 18 were respectively diluted to 2X 10 10 、4×10 10 、6×10 10 、8×10 10 cfu/ml, seed germination test was performed.
The method of seed germination testing is as follows:
(1) placing 2kg of sterilized nutrient soil into a seedling raising tray, and thoroughly pouring the nutrient soil with 10ml of bacillus subtilis BGB-99R bacterial suspension with the concentration, wherein the bacterial content of the nutrient soil is 1 × 10 8 、2×10 8 、3×10 8 、4×10 8 cfu/g as test group; in addition, the same volume of water was used to thoroughly irrigate the nutrient soil as a control group.
(2) And (2) placing the seedling tray treated in the step (1) in a greenhouse at 28 ℃ for 12h, treating the cucumber seeds for 2min by using 0.2% sodium hypochlorite, washing with sterile water for 5 times, planting the cucumber seeds in the seedling tray in the step (1) in a hole sowing manner, and observing the germination condition of the cucumber seeds after 10 d.
(3) Detection of germination rates
And calculating the germination rate by a counting mode. The results are shown in Table 7.
TABLE 7 test results for seed Germination
By comparing the germination rates of the test group and the control group based on the results of the above tests, it is possible to compare the germination rates with those of the test group and the control group, in combination with Table 7The bacillus subtilis BGB-99R bacterial suspension is added into the nutrient soil, so that the germination of seeds can be promoted, 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) multiplied by 10 8 In the cfu/ml range, the germination rate of the seeds can be effectively improved, and the germination of the seeds is promoted.
Further, by comparing the seed germination rates when using different concentrations of Bacillus subtilis BGB-99R bacterial suspensions, it can be seen that when the bacterial content of Bacillus subtilis BGB-99R is (1-3). times.10 8 The germination rate of the seeds is above 93.45% when cfu/ml; especially when the bacterial content of the bacillus subtilis BGB-99R is 2 multiplied by 10 8 When cfu/ml is adopted, the germination rate of the seeds is 98.27 percent, which is 25 percent higher than that of the control group. When the bacterial content of the bacillus subtilis BGB-99R is 4 multiplied by 10 8 The germination rate of the seeds is 90.23% at cfu/ml. Therefore, the bacterial content of the Bacillus subtilis BGB-99R is controlled to be (1-3) multiplied by 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.
Third, plant growth test
The Bacillus subtilis BGB-99R bacterial suspensions obtained in example 18 were respectively diluted to 2.5X 10 10 、5×10 10 、7.5×10 10 、10×10 10 cfu/ml, plant growth test was performed.
The method of the plant growth test is as follows:
(1) placing 2.5kg sterilized nutrient soil in seedling tray, and respectively diluting with 10ml of bacterial suspension with Bacillus subtilis BGB-99R of above concentration to thoroughly pour the nutrient soil, wherein the bacterial content of the nutrient soil is 1 × 10 8 、2×10 8 、3×10 8 、4×10 8 cfu/g as test group; in addition, the nutrient soil was drenched with the same volume of water as a control group.
(2) And (2) placing the seedling tray treated in the step (1) in a greenhouse at 28 ℃ for 12h, transplanting cucumber seedlings cultured by a hollow white control group in a seed germination test into the seedling tray in the step (1), placing the transplanted seedlings in an artificial growth chamber (the photoperiod is 16h/8h at day/night, and the temperature is 30 ℃/24 ℃) for culturing for 30d, and detecting the root length (cm) and the fresh weight (g) of the plants.
(3) Detection of plant root length (cm) and plant fresh weight (g): the results are shown in Table 8.
Wherein, the root length growth rate (%) (experimental group root length-control group root length)/control group root length is multiplied by 100%; the fresh weight increase rate (%) was (fresh weight of experimental group-fresh weight of control group)/fresh weight of control group × 100%.
TABLE 8 test results for plant growth
By comparing the plant growth conditions of the test group and the control group through analyzing the test results in combination with table 8, it can be seen that the growth of the plants can be promoted by adding the bacillus subtilis BGB-99R bacterial suspension into the nutrient soil, 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) multiplied by 10 8 In the cfu/ml range, the growth of plants can be effectively promoted.
Further, by comparing the growth of plants using different concentrations of Bacillus subtilis BGB-99R suspensions, it can be seen that the bacterial content of Bacillus subtilis BGB-99R is (2-4). times.10 8 The root length of the plant is more than 13.26cm and the fresh weight is more than 3.28g when cfu/ml is used. The bacterial content of the bacillus subtilis BGB-99R is more than 1 multiplied by 10 8 At cfu/ml, the root length and fresh weight of the plants are. Especially when the bacterial content of the bacillus subtilis BGB-99R is 2 multiplied by 10 8 When cfu/ml is used, 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 improved by more than one time compared with those of the plants of the control group. Therefore, the bacterial content of the bacillus subtilis BGB-99R is controlled to be (2-4) multiplied by 10 8 In the cfu/ml range, the growth of the plant can be further promoted.
It will be understood that the above embodiments are merely exemplary embodiments taken to illustrate the principles of the present invention, which is not limited thereto. It will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the spirit and substance of the invention, and these modifications and improvements are also considered to be within the scope of the invention.
Sequence listing
<110> Beijing Jia Bo Wen Biotechnology Co., Ltd
<120> bacillus subtilis and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1451
<212> DNA
<213> Bacillus subtilis
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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 (10)
1. The bacillus subtilis is preserved in China center for type culture Collection (CGMCC) of Beijing microorganisms, and the preservation number is as follows: CGMCC N0.24391, the preservation time is as follows: 11/02/2022.
2. The Bacillus subtilis according to claim 1, wherein the Bacillus subtilis shows positive by gram staining, the strains are rod-shaped and round-ended, most strains are arranged in a single, a few pairs or chain form, the surfaces of colonies are rough and opaque during plate culture, the colonies are white or yellow, wrinkle films are formed on the edges, and the colonies are 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 using the Bacillus subtilis of claim 1.
5. A bacterial suspension prepared using the Bacillus subtilis of claim 1.
6. A fermentation supernatant prepared using the bacillus subtilis of claim 1.
7. The use of bacillus subtilis according to claim 1 for promoting seed germination, promoting plant growth and inhibiting the growth and reproduction of pathogenic bacteria.
8. The use of Bacillus subtilis according to claim 7, wherein the Bacillus subtilis has a bacterial content of (1-4). times.10 8 cfu/g。
9. The use of Bacillus subtilis according to claim 7, wherein the Bacillus subtilis has an inhibition ratio of 40.61-54.33% against fungi.
10. The use of Bacillus subtilis according to claim 7, wherein the Bacillus subtilis has an inhibition of Fusarium graminearum of up to 54.33%.
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