CN114736834A - Bacillus pumilus TS1 and application thereof - Google Patents
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- A—HUMAN NECESSITIES
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Abstract
The application protects a bacillus pumilus TS1 and application thereof. The Bacillus pumilus TS1 is acid and alkali resistant and has antibacterial activity. The application of the Bacillus pumilus TS1 in preparing the feed additive and the application of the Bacillus pumilus TS1 in preparing the feed additive for relieving the damage caused by stress are protected. In the experiment, when the feed added with the bacillus pumilus TS1 is used for feeding chickens, the weight of the chickens is obviously heavier than that of the chickens fed with the common feed under the same environment.
Description
Technical Field
The invention relates to the technical field of biology, in particular to bacillus pumilus TS1 and application thereof.
Background
Stress or stress response refers to the non-specific systemic response of the body when stimulated by various strong factors (stressors). Stressors are environmental events or situations that place an adaptation on demand and can cause objective changes in response to imbalances in homeostasis, and may also be referred to as stimuli or stimuli. Oxidative stress refers to the condition that when the organism is subjected to various harmful stimuli, the body generates excessive high-activity molecules such as active oxygen and active nitrogen, and the oxide removal capacity of cells at the moment is far from meeting the oxidation resistance which is required to resist the existing oxidation degree, and the oxidation system and the oxidation resistance system are unbalanced, so that the imbalance can cause damage to cells and important biomolecules and has potential influence on the whole organism. In the field of poultry farming, after poultry is stimulated by stressors, the immunity and resistance of the poultry are reduced, and finally, the feed intake of the poultry is reduced, the growth and development of the poultry are slowed down or stopped, the immunity is reduced, even the poultry die, and great economic loss is caused to the breeding industry.
The large-scale application of intensive cultivation in poultry cultivation has the obvious stimulation problem of various stress sources to the growth of poultry while achieving good economic benefit. Such as: the crowded breeding environment catches and drives the poultry, so that the poultry is frightened; temperature stimuli such as heat and cold, and various pathogenic microorganism infections can be the stressors. Many studies show that under the stimulation of various stressors, poultry can cause the activity of superoxide dismutase, glutathione peroxidase and the like in poultry tissues and organs or serum to be reduced and the activity of Reactive Oxygen Species (ROS), NO, Malondialdehyde (MDA) to be increased, thereby causing oxidative stress injury.
Stress in the poultry breeding process is inevitable, so that the method for relieving the damage caused by the stress is significant. Since stress is a non-specific damage to the animal body, there is no targeted drug. The bacillus pumilus can be added into feed to reduce the damage of tissues and organs caused by the oxidative stress of poultry. Therefore, the finding of the bacillus pumilus suitable for being added into the feed has important significance for reducing the tissue and organ damage caused by the oxidative stress of the poultry.
Disclosure of Invention
The application provides bacillus pumilus which is added into poultry feed to reduce tissue and organ damage caused by poultry oxidative stress.
In a first aspect, the application provides a Bacillus pumilus, namely Bacillus pumilus (Bacillus pumilus) TS1, wherein the preservation number of the Bacillus pumilus (Bacillus pumilus) TS1 is CCTCC No. M2022538.
The genus Bacillus, a genus of bacteria, is capable of forming spores (endospores). They have strong resistance to external harmful factors and wide distribution, and exist in soil, water, air, animal intestinal tracts and the like. Bacillus bacteria are large (4-10 μm), gram-positive, and are either strictly aerobic or facultative anaerobic, podded bacilli. An important property of this genus of bacteria is the ability to produce spores that are particularly resistant to adverse conditions.
Bacillus pumilus (Bacillus pumilus) is a bacterium of the genus Bacillus with a fine rod-like shape, and is generally 0.6 to 0.7. mu. m.times.2.0 to 3.0. mu.m, gram-positive. Bacillus pumilus is commonly used to control wheat root rot and strawberry gray mold.
Optionally, the 16s rDNA sequence of bacillus pumilus TS1 is: SEQ ID NO:1, the bacillus pumilus TS1 is acid-resistant and bile salt-resistant and has a bacteriostatic effect.
The advantages of the bacillus pumilus are as follows: (1) easy preservation and good stress resistance: the conditions of separation, culture and preservation are simple, the technical requirement on industrial chemical production is low, and the stable effect and high activity can be maintained in intestines and stomach; spores generated by the spore can resist the external severe environment (such as heat, ultraviolet rays, ionizing radiation, low pH (2-3) and the like); (2) in the process of producing and breeding poultry, the feed additive can promote the growth and development of the poultry and the growth performance of the poultry, has simple requirements on nutrition and can quickly promote metabolism; (3) can also relieve the injury of tissues and organs caused by poultry oxidative stress or pathogenic bacteria infection. The Bacillus pumilus has the advantages of easy preservation, high active ingredients and the like, and is worthy of being popularized and applied in a large scale.
Optionally, the bacillus pumilus TS1 is isolated from yak dung.
In a second aspect, the application provides an application of bacillus pumilus TS1 in preparing a feed additive.
The poultry industry has mainly regulated intestinal flora by feed additives to improve intestinal health. Although progress has been made, the actual production effect is not the same considering the types of additives, the types of poultry, the breeding environment, and other problems. Bacillus pumilus (Bacillus pumilus) is one of the feed additives, has unique advantages compared with other feed additives in animal production, and is widely applied.
Optionally, the feed additive is in the form of freeze-dried powder.
The freeze-dried powder is prepared by freezing the water in the liquid medicine in advance by adopting a vacuum freeze-drying method of a freeze dryer, and then sublimating the frozen water in the liquid medicine in a vacuum sterile environment, thereby obtaining freeze drying.
Optionally, the content of Bacillus pumilus TS1 in the freeze-dried powder is more than or equal to 109CFU/g。
Optionally, the lyophilized powder is used in an amount of up to 1 g/day.
In a third aspect, the application provides an application of bacillus pumilus TS1 in preparing a feed additive for relieving stress-caused injury.
The emergency reaction is the allergic reaction of the sensitive body caused by the stimulation of the poultry and livestock by a stressor. For example, in crowded breeding environments, the poultry is caught and driven, so that frightened groups of the poultry are caused; temperature stimuli such as heat and cold, and various pathogenic microorganism infections can be stressors.
Oxidative stress refers to the condition that when the organism is subjected to various harmful stimuli, the body generates excessive high-activity molecules, such as active oxygen and active nitrogen, and the oxide removal capacity of cells at the moment is far from meeting the requirement of the oxidation resistance which is provided for resisting the existing oxidation degree, and the oxidation system and the oxidation resistance system are unbalanced, so that the cells and important biomolecules are damaged and have potential influence on the whole organism. Many studies at present show that under the stimulation of various stress sources, poultry can cause the activity reduction of superoxide dismutase, glutathione peroxidase and the like in poultry tissues and organs or serum and the increase of active oxygen (ROS), NO and Malondialdehyde (MDA), so that oxidative stress injury is caused, the feed intake of the poultry is reduced, the growth and development are slowed down or stopped, the immunity is reduced, even death is caused, and huge economic loss is caused to the breeding industry.
Because the stress is nonspecific injury to animal organisms, no targeted medicine is provided, and the stress injury can be alleviated by adding a proper feed additive to improve the health of the poultry organisms, enhance the immunity, or improve the feeding environment and the like. Wherein TS1 belongs to Bacillus pumilus and microorganisms allowed to be added in feed additives.
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In order to more clearly explain the technical solution of the present application, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious to those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows the BLAST results for the 16S rDNA sequence at NCBI;
FIG. 2 is an agarose electrophoresis picture of the PCR product of Bacillus pumilus TS 1;
FIG. 3 shows the results of the survival rate of Bacillus pumilus TS1 cultured in LB medium at pH 7.12, 6.04, 5.03, 4.06 and 3.02 for 24 hours (wherein the abscissa is the pH of the LB medium and the ordinate is the survival rate of Bacillus pumilus TS 1);
FIG. 4 shows the results of the survival rate of Bacillus pumilus TS1 cultured in LB medium with bile salt concentrations of 0.05%, 0.1%, 0.2% and 0.3% for 24 hours (wherein the abscissa is the bile salt concentration of LB medium and the ordinate is the survival rate of Bacillus pumilus TS 1);
FIG. 5 shows the inhibition zone of Bacillus pumilus TS1 against gram-negative bacteria (wherein the left picture is E.coli and the right picture is Salmonella);
FIG. 6 shows the inhibition zone of Bacillus pumilus TS1 against gram-positive bacteria (wherein the left panel shows Streptococcus and the right panel shows Staphylococcus aureus);
FIG. 7 shows the results of the effect of Bacillus pumilus TS1 on chicken weight (wherein the abscissa is the number of days of feeding and the ordinate is the weight of the chickens; the darker bar in the bar graph is the control group and the lighter bar is the experimental group; and P is < 0.05).
Detailed Description
Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings. When the following description refers to the accompanying drawings, like numbers in different drawings represent the same or similar elements unless otherwise indicated. The embodiments described in the following examples do not represent all embodiments consistent with the present application. But merely as exemplifications of systems and methods consistent with certain aspects of the application, as recited in the claims.
Example 1: separation and purification of bacillus pumilus TS1
Clamping a small healthy yak dung sample from a army and horse field of Shandan Gansu with forceps, putting the sample into a glass test tube filled with 5mL of distilled water, sealing the tube opening with a sealing film, and boiling in boiling water for 10 minutes; taking 100 mu L of boiled supernatant to an EP tube filled with 5mL of LB liquid culture medium, and putting the supernatant into a shaker at 37 ℃ and 180rpm for shaking bacteria for 16-18 h; and adding 100 mu L of the shaken bacterial liquid into 5mL of liquid culture medium, and shaking the bacterial liquid for 16h again.
Streaking the dipped and shaken bacterial liquid in an LB flat plate, carrying out inverted culture in a 37 ℃ incubator for 12-18h, selecting a single colony inoculation flat plate, carrying out culture in the 37 ℃ incubator for 12-18h for purification, selecting a single colony again, shaking the bacterial liquid in 5mL of LB liquid culture medium for 16-18h to obtain the bacterial liquid, mixing the bacterial liquid with the same volume of LB frozen stock solution, and carrying out cryopreservation at-80 ℃.
Example 2: identification of Bacillus pumilus TS1
Sequencing by using a 16S universal primer to determine the sequence of the separated probiotics, wherein the 16S sequencing result is shown as SEQ ID NO. 1. The 16S rDNA sequences were compared with the database using BLAST software. The BLAST results for the 16S rDNA sequence at NCBI are shown in FIG. 1 and show more than 99% homology with Bacillus pumilus.
During PCR amplification reaction, fresh bacterial liquid, 16S universal primer and PCR enzyme are mixed in a PCR tube according to 25. mu.L of 2xTaq Plus Master Mix, 2. mu.L of primer 27F and 2. mu.L of primer 1492R, and total 50. mu.L of 1-5. mu.L of fresh bacterial liquid and secondary distilled water. The PCR amplification conditions were: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 deg.C for 15s, annealing at 50.6 deg.C for 15s, extension at 72 deg.C for 100s, and circulation for 30-35 times; final extension at 72 ℃ for 5 min.
And adding the PCR product into 1% agarose nucleic acid gel, carrying out electrophoresis in 1xTAE electrophoresis solution, and then irradiating the gel to obtain a 16S result. As a result, as shown in FIG. 2, the band of interest appeared around 1670bp, and the negative control showed no band.
Example 3: acid resistance test
Preparing an acidic culture medium: 36.0-38.0% hydrochloric acid solution 6.4mL is added into 500mL LB liquid culture medium, and the pH value of the solution is adjusted to 7.0, 6.0, 5.0, 4.0 and 3.0 at room temperature.
Shaking the bacteria: taking 50 mu L of activated bacteria liquid by 1% inoculation amount, respectively adding 4950 mu L of LB acidic liquid culture medium with pH 7.0, 6.0, 5.0, 4.0 and 3.0, then adding 50 mu L of activated bacteria liquid into 4950 mu L of normal LB liquid culture medium as a control, and simultaneously putting into shaking table bacteria with temperature of 37 ℃ and rotation speed of 160-180rpm for 16-18 h.
Measuring the light absorption value: the absorbance values (OD600) of the bacterial liquid cultured in the acidic medium and the normal medium at 600nm were measured at 0h and 24h, respectively, with shaking.
Calculating the survival rate: according to beer-lambert law a-Kbc (a: absorbance; K: molar absorption coefficient; b: absorption layer thickness; c: concentration), the bacteria have a maximum absorbance at a wavelength of 600nm, and the bacteria concentration c-a/kb, which is proportional to the absorbance.
Survival rate of X1/X0×100％(X1: culturing in an acidic culture medium for 24 h; x0: viable count of 0h in acid medium culture); the viable count is expressed as the bacterial concentration, and the final survival rate can be expressed as: survival rate ═ a1/A0×100％(A1: culturing in an acidic culture medium for 24 h; a. the0: culturing in acid medium for 0h light absorption value)
In the acid resistance test, the pH values of the prepared LB culture solution were 7.12, 6.04, 5.03, 4.06, and 3.02, and the survival rates were as shown in fig. 3, and bacillus pumilus TS1 had good acid resistance, wherein the survival rate of bacillus pumilus TS1 was the highest among the five groups at pH 7.12.
Example 4: alkali resistance test
Preparing a bile salt culture medium: to 500mL of LB liquid medium was added 1.5g of bovine bile salt to make the content 0.3%, and then the mixture was diluted in a gradient of 0.2%, 0.1%, 0.05%.
Shaking the bacteria: taking 1% inoculation amount to measure 50 μ L of activated bacteria liquid, respectively adding to 4950 μ L of culture liquid with different cholate concentrations, adding 50 μ L of activated bacteria liquid to 4950 μ L of normal LB liquid culture medium as a reference, and simultaneously placing into shaking table for shaking bacteria at 37 ℃ and 180rpm at 160-18 h.
Measuring the light absorption value: respectively measuring the OD600 values of the culture solution in the bile salt culture solution with different concentrations in 0h and 24h of shaking the bacteria;
calculating the survival rate: survival rate of B1/B0×100％(B1: culturing in a bile salt culture medium for 24h to obtain a light absorption value; b is0: culture in bile salt medium 0h light absorption value)
The result of the alkali resistance test is shown in FIG. 4, and the Bacillus pumilus TS1 has good acid resistance. The survival rate in LB culture solution with 0.05%, 0.1%, 0.2% and 0.3% of bile salt concentration for 24h is higher than 60%, the survival rate in LB culture solution with 0.05% of bile salt concentration exceeds 90%, and the survival rate decreases with the increase of the concentration.
Example 5: in-vitro bacteriostatic test of bacillus pumilus TS1 on gram-negative bacteria
And (3) culturing pathogenic bacteria: the culture of the escherichia coli and the salmonella is similar to the recovery and activation process of TS1, and finally, the escherichia coli and the salmonella are shaken synchronously with TS1 to obtain activated bacterial liquid;
pathogen dilution: taking five 10mL centrifuge tubes, adding 4.5mL sterile PBS into each tube, adding 500 mu L bacterial liquid into the first tube, mixing uniformly, taking 500 mu L diluted bacterial liquid in the first tube, adding into the second tube, mixing uniformly … … in sequence, and performing gradient dilution to obtain 10-concentration bacterial liquid-1、10-2、10-3、10-4、10-5The pathogenic bacteria liquid.
Paving a board: and blowing the LB nutrient agar plate beside an alcohol lamp and the like on a super clean bench for 5-10 minutes, pouring the diluted escherichia coli and salmonella bacteria liquid to a plate, uniformly mixing, sucking out the redundant escherichia coli and salmonella bacteria liquid, opening a plate cover in the super clean bench, and blowing aside the alcohol lamp for about 20 minutes.
Punching: after the flat plate is dried, punching small holes with the diameter of 5-6mm and regular edges in the flat plate by using a puncher;
culturing: and (3) taking 50-80 mu L of TS1 activated bacterial liquid to the hole, and culturing for 18-20 h in a constant temperature box at 37 ℃ after the bacterial liquid is completely absorbed.
As shown in FIG. 5, the results show that the Bacillus pumilus TS1 has a good bacteriostatic effect on gram-negative bacteria.
Example 6: in-vitro bacteriostatic test of bacillus pumilus TS1 on gram-positive bacteria
And (3) culturing pathogenic bacteria: the culture of streptococcus and staphylococcus aureus is similar to the recovery and activation process of TS1, and finally, bacteria shaking is carried out synchronously with TS1 to obtain activated bacteria liquid;
pathogen dilution: taking five 10mL centrifuge tubes, adding 4.5mL sterile PBS into each tube, adding 500 mu L bacterial liquid into the first tube, mixing uniformly, taking 500 mu L diluted bacterial liquid in the first tube, adding into the second tube, mixing uniformly …… gradient dilution was performed by analogy to obtain a concentration of 10-1、10-2、10-3、10-4、10-5The pathogenic bacteria liquid.
Paving a board: and blowing the LB nutrient agar plate beside an alcohol lamp and the like in a super clean bench for 5-10 minutes, pouring the diluted streptococcus and staphylococcus aureus liquid to a plate, uniformly mixing, sucking out the redundant streptococcus and staphylococcus aureus liquid, opening a plate cover in the super clean bench, and blowing aside the alcohol lamp for about 20 minutes.
Punching: after the flat plate is dried, punching small holes with the diameter of 5-6mm and regular edges in the flat plate by using a puncher;
culturing: and (3) taking 50-80 mu L of TS1 activated bacterial liquid to the hole, and culturing for 18-20 h in a constant temperature box at 37 ℃ after the bacterial liquid is completely absorbed.
The results are shown in fig. 6, which shows that the bacillus pumilus TS1 has a good bacteriostatic effect on gram-positive bacteria.
Example 7: effect of feed feeding with TS1 on Chicken weight
20 chickens were randomly divided into two groups, a control group and an experimental group. TS1 is added into the feed of the experimental group, and the feed is continuously fed for 15 days, wherein the addition amount of the feed is 109 CFU/pig/day; the control group was fed the same way for 15 days without TS1 in the feed. The body weights of the two groups of chickens were measured on days 6, 8, 10, 12, 14, 16, 18 and 20 and recorded.
The weight of the chickens increased with the number of days, the group with TS1 was significantly heavier than the control group without feed, and there was a significant difference between the two groups (P <0.05)
The embodiments provided in the present application are only a few examples of the general concept of the present application, and do not limit the scope of the present application. Any other embodiments extended according to the scheme of the present application without inventive efforts will be within the scope of protection of the present application for a person skilled in the art.
Sequence listing
<110> Ningxia Anlison Biotechnology Ltd
Nanjing university of agriculture
<120> Bacillus pumilus TS1 and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1422
<212> DNA
<213> Bacillus pumilus (Bacillus pumilus)
<400> 1
ctcataaggt tacctcaccg acttcgggtg ttaaaactct cgtggtgtga cgggcggtgt 60
gtacaaggcc cgggaacgta ttcaccgcgg catgctgatc cgcgattact agcgattcca 120
gcttcacgca gtcgagttgc agactgcgat ccgaactgag aacagattta tgggattggc 180
taaaccttgc ggtctcgcag ccctttgttc tgtccattgt agcacgtgtg tagcccaggt 240
cataaggggc atgatgattt gacgtcatcc ccaccttcct ccggtttgtc accggcagtc 300
accttagagt gcccaactaa atgctggcaa ctaagatcaa gggttgcgct cgttgcggga 360
cttaacccaa catctcacga cacgagctga cgacaaccat gcaccacctg tcactctgtc 420
cccgaaggga aagccctatc tctagggttg tcagaggatg tcaagacctg gtaaggttct 480
tcgcgttgct tcgaattaaa ccacatgctc caccgcttgt gcgggccccc gtcaattcct 540
ttgagtttca gtcttgcgac cgtactcccc aggcggagtg cttaatgcgt tagctgcagc 600
actaaggggc ggaaaccccc taacacttag cactcatcgt ttacggcgtg gactaccagg 660
gtatctaatc ctgttcgctc cccacgcttt cgctcctcag cgtcagttac agaccagaga 720
gtcgccttcg ccactggtgt tcctccacat ctctacgcat ttcaccgcta cacgtggaat 780
tccactctcc tcttctgcac tcaagtttcc cagtttccaa tgaccctccc cggttgagcc 840
gggggctttc acatcagact taagaaaccg cctgcgagcc ctttacgccc aataattccg 900
gacaacgctt gccacctacg tattaccgcg gctgctggca cgtagttagc cgtggctttc 960
tggttaggta ccgtcaaggt gcgagcagtt actctcgcac ttgttcttcc ctaacaacag 1020
agctttacga tccgaaaacc ttcatcactc acgcggcgtt gctccgtcag actttcgtcc 1080
attgcggaag attccctact gctgcctccc gtaggagtct gggccgtgtc tcagtcccag 1140
tgtggccgat caccctctca ggtcggctac gcatcgtcgc cttggtgagc cattacccca 1200
ccaactagct aatgcgccgc gggtccatct gtaagtgaca gccgaaaccg tctttcatcc 1260
ttgaaccatg cggttcaagg aactatccgg tattagctcc ggtttcccgg agttatccca 1320
gtcttacagg caggttaccc acgtgttact cacccgtccg ccgctaacat ccgggagcaa 1380
gctcccttct gttcgctcga cttgcatgta ttaggcacgc cg 1422
Claims (10)
1. The Bacillus pumilus is characterized by being named as Bacillus pumilus (TS 1), and the preservation number of the Bacillus pumilus (TS 1) is CCTCC No. M2022538.
2. The Bacillus pumilus of claim 1, wherein the Bacillus pumilus TS1 has the 16srDNA sequence: SEQ ID NO:1, the bacillus pumilus TS1 is acid-resistant, bile salt-resistant and has bacteriostasis.
3. The Bacillus pumilus of claim 1, wherein the Bacillus pumilus TS1 is isolated from yak feces.
4. The Bacillus pumilus of claim 3, wherein the isolation conditions are: clamping small healthy yak dung samples from a ramus army and horse farm in Gansu province by using forceps, sealing the opening of a tube by using a sealing film, and boiling in boiling water; taking the boiled supernatant to an EP tube filled with an LB liquid culture medium, and putting into a shaking table for shaking bacteria; adding the shaken bacterial liquid into a liquid culture medium, and shaking the bacteria again.
5. The Bacillus pumilus of claim 4, wherein the isolation conditions are: clamping a small healthy yak dung sample from a army and horse field of Shandan Gansu with forceps, putting the sample into a glass test tube filled with 5mL of distilled water, sealing the tube opening with a sealing film, and boiling in boiling water for 10 minutes; taking 100 mu L of boiled supernatant to an EP tube filled with 5mL of LB liquid culture medium, and putting the supernatant into a shaker at 37 ℃ and 180rpm for shaking bacteria for 16-18 h; and adding 100 mu L of the shaken bacterial liquid into 5mL of liquid culture medium, and shaking the bacterial liquid for 16h again.
6. Application of Bacillus pumilus TS1 in preparing feed additive.
7. The use according to claim 6, wherein the feed additive is in the form of a lyophilized powder.
8. The use of claim 7, wherein the lyophilized powder has a Bacillus pumilus TS1 content of 10 or more9CFU/g。
9. Use according to claim 7, wherein the lyophilized powder is used in an amount of up to 1 g/day.
10. Application of bacillus pumilus TS1 in preparation of feed additives for relieving injuries caused by stress.
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| CN102586144A (en) * | 2012-02-09 | 2012-07-18 | 中国科学院南海海洋研究所 | Bacillus pumilus, probiotics preparation and preparation method and application thereof |
| CN102911901A (en) * | 2012-10-25 | 2013-02-06 | 青岛蔚蓝生物集团有限公司 | Bacillus pumilus strain and application thereof |
| JP2014096996A (en) * | 2012-11-13 | 2014-05-29 | Tokyo Univ Of Agriculture & Technology | Novel bacillus nitrogen fixation bacterium, plant growth accelerator, and plant cultivation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586144A (en) * | 2012-02-09 | 2012-07-18 | 中国科学院南海海洋研究所 | Bacillus pumilus, probiotics preparation and preparation method and application thereof |
| CN102911901A (en) * | 2012-10-25 | 2013-02-06 | 青岛蔚蓝生物集团有限公司 | Bacillus pumilus strain and application thereof |
| JP2014096996A (en) * | 2012-11-13 | 2014-05-29 | Tokyo Univ Of Agriculture & Technology | Novel bacillus nitrogen fixation bacterium, plant growth accelerator, and plant cultivation method |
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