CN112608873A - Paenibacillus bacteria with growth promoting activity and preparation and application thereof - Google Patents
Paenibacillus bacteria with growth promoting activity and preparation and application thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title abstract description 8
- 230000001580 bacterial effect Effects 0.000 claims abstract description 25
- 241000193397 Paenibacillus pabuli Species 0.000 claims abstract description 9
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- 239000001963 growth medium Substances 0.000 claims description 32
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- 241000894006 Bacteria Species 0.000 claims description 26
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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
- A01N63/25—Paenibacillus
-
- 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 invention relates to a paenibacillus bacterium with growth promoting activity, and the strain is paenibacillus (Bacillus subtilis)Paenibacilluspabuli) B03 with the preservation number of CGMCC No. 21386. Meanwhile, the invention also discloses a preparation method and application of the strain. The invention separates the bacterial strain from the root of the plant, and the metabolite of the bacterial strain acts on the pasture to improve the biochemical fertility status of the grassland soil, improve the functional stability of the ecological system of the grassland soil, recover and improve the productivity of the grassland, increase the yield and improve the quality, thereby promoting the development of the animal husbandry.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a Paenibacillus bacterium with growth promoting activity and preparation and application thereof.
Background
Plant growth-promoting rhizobacteria (PGPR) are a type of bacteria that colonize the rhizosphere, phyllosphere and body of plants and promote the growth, development and stress tolerance of plants under biotic and abiotic stress. In recent years, based on the important value of PGPR in the fields of agriculture, environmental protection, etc., it has become a hot spot for researchers at home and abroad. Pseudomonas bacteria (A), (B)Pseudomonas) Bacillus bacteria (b)Bacillus) Bacillus like bacteria (B)Paenibacillus) Agrobacterium, a bacterium (A) or (B)Agrobacterium) Flavobacterium (II)Flavobacterium) More than 20 rhizosphere organisms have the potential of preventing diseases and promoting growth.
In recent years, researches show that the functions of the grassland ecosystem are seriously threatened by the acidification of grassland soil, nutrition imbalance, reduction of biodiversity, productivity reduction, grassland degradation and the like caused by excessive application of chemical fertilizers. Under the background, the inoculation of PGPR can be used for improving the biochemical fertility condition of the grassland soil, improving the functional stability of the ecological system of the grassland soil, recovering and improving the productivity of the grassland, increasing the yield and improving the quality, thereby promoting the development of animal husbandry. Therefore, the microbial agent becomes a new research hotspot, and is used for improving the growth and disease resistance of pasture by breeding endophyte and rhizosphere microorganism with disease resistance and growth promotion characteristics, thereby treating the degenerated pasture.
At present, the development of a safe and effective biogenic growth promoter is receiving more and more attention, and the secondary metabolites of microorganisms become one of the important sources for the research and development of new growth promoters.
Disclosure of Invention
The invention aims to provide a Paenibacillus bacterium with growth promoting activity.
Another technical problem to be solved by the present invention is to provide a method for producing the bacterium belonging to the genus Bacillus.
The third technical problem to be solved by the invention is to provide the application of the bacillus.
In order to solve the above problems, the present invention provides a Paenibacillus bacterium having growth promoting activity, wherein: the strain is paenibacillus (Paenibacilluspabuli) B03. The strain B03 has been deposited in China general microbiological culture Collection center on 17.12.2020, and the addresses of the deposit centers are as follows: beijing, Ind. region of the republic of Yangyang, institute of microbiology, academy of China, zip code 100101, accession number CGMCC No. 21386.
The method for producing a Paenibacillus bacterium having growth promoting activity as described above, comprising: selecting healthy stipa purpurea with roots, cutting fresh roots of the stipa purpurea into small sections with the length of 0.2-0.5 cm, washing the small sections with sterilized distilled water for three times after disinfection, and placing the small sections on sterile filter paper to absorb water; then placing on an NA plate culture medium, and culturing at 28 +/-2 ℃ in a dark place; after the macroscopic bacteria grow on the plate, picking a few of bacteria with a sterile toothpick and transferring the bacteria to a new NA plate culture medium for purification, and if the bacteria are not polluted, transferring the bacteria to an NA slant culture medium for scribing and storing for a short time at 4 ℃ or transferring the bacteria to a mixed solution of an NA liquid culture medium and glycerol for storing for a long time at-70 ℃.
The disinfection means that fresh roots of the cut Philippine fescue are firstly disinfected for 30 s by ethanol with the volume concentration of 75 percent and then disinfected for 5 min by sodium hypochlorite with the mass concentration of 3 percent.
The NA plate culture medium is prepared by adding 10g of peptone, 3g of beef powder, 5g of sodium chloride and 15g of agar into 1000mL of water, uniformly mixing, adjusting the pH value to 7.2-7.4, and sterilizing at 121 ℃ for 30 min.
The NA liquid culture medium is prepared by adding 10g of peptone, 3g of beef powder and 5g of sodium chloride into 1000mL of water, uniformly mixing, adjusting the pH value to 7.2-7.4, and sterilizing at 121 ℃ for 30 min.
The macroscopic bacterial colonies are colony-forming units, and CFU is the colony with the average diameter of more than or equal to 1 mm.
The counting method of the macroscopic bacterial colonies refers to a concentration plate method, namely the macroscopic single scattered bacterial colonies in a culture dish with the diameter of 90 mm have the number of 100-150 CFU.
The mixed solution of the NA liquid culture medium and the glycerol is a solution obtained by uniformly mixing the NA liquid culture medium and the glycerol according to the volume ratio of 3: 1.
The use of a Paenibacillus bacterium having growth promoting activity as described above, wherein: sterilizing Paenibacillus (A), (B) and (C)Paenibacilluspabuli) B03 diluting the bacterial liquid by 100 times to obtain the bacterial liquid as the plant growth promoter of the stipa capillata.
Compared with the prior art, the invention has the following advantages:
1. the invention is prepared from Philippine fescue (Stipa purpurea) The strain is isolated from the roots of (1) and a metabolite of the strain is allowed to act on the plant. Strain B03 belongs to the genus Paenibacillus (B)Paenibacillus) Its bacterial colony is yellow, transparent, smooth and moist, and has luster.
2. The present invention relates to Paenibacillus (Paenibacilluspabuli) B03 is used as plant growth regulator, and its metabolite has obvious growth promoting activity to grass growth, so that its root system is developed, the biomass of aerial parts is increased, and the anti-adversity activity of grass is enhanced. Treatment with metabolites at different dilution concentrations and control H2The O treatment has obvious difference, and the growth promoting effect of the diluted fermentation liquor is very obvious.
3. The raw materials of the invention are from the roots of plants, have no adverse effect on the environment and all organisms such as plants, human beings, animals and the like, and have the advantages of low production cost, simple production process, no environmental pollution and suitability for popularization and application.
4. The biological growth regulator is a pure biological preparation, can regulate the growth of the tall fescue grass in northwest alpine grassland, is used for improving the biochemical fertility condition of the grassland soil, improving the functional stability of the grassland soil ecosystem, recovering and improving the productivity of the grassland, increasing the yield and improving the quality, thereby promoting the development of animal husbandry.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 shows the preliminary experiment of the growth of Philippine fescue grass by using the bacillus paenibacillus B03 bacterial liquid, wherein the upper grass is a control group (distilled water) and the lower grass is the bacterial liquid group.
FIG. 2 shows the effect of metabolites of Paenibacillus B03 at different concentrations on the weight of Achillea alpina grass.
FIG. 3 shows the effect of various concentrations of metabolites of Paenibacillus B03 of the present invention on Phlebia purpurea grass (root length).
FIG. 4 shows the effect of various concentrations of metabolites of Paenibacillus B03 of the present invention on Phlebia purpurea grass (stalk length).
Detailed Description
A Paenibacillus bacterium with growth promoting activity, which is Paenibacillus foeniculi (B.feeniculus) (B.feeniculus)Paenibacilluspabuli) B03. The strain B03 has been deposited in China general microbiological culture Collection center on 17.12.2020, and the addresses of the deposit centers are as follows: beijing, Ind. region of the republic of Yangyang, institute of microbiology, academy of China, zip code 100101, accession number CGMCC No. 21386.
The preparation method of the paenibacillus bacteria comprises the following steps: selecting healthy stipa purpurea with roots, cutting fresh roots of the stipa purpurea into small sections with the length of 0.2-0.5 cm, firstly carrying out surface disinfection for 30 s by using 75% ethanol by volume concentration, and then carrying out surface disinfection for 5 min by using 3% sodium hypochlorite by mass concentration. After disinfection, washing the mixture with sterilized distilled water for three times, and placing the mixture on sterile filter paper to absorb water; then placing on an NA plate culture medium, and culturing at 28 +/-2 ℃ in a dark place; after the macroscopic bacteria grow on the plate, picking a few of bacteria with a sterile toothpick and transferring the bacteria to a new NA plate culture medium for purification, and if the bacteria are not polluted, transferring the bacteria to an NA slant culture medium for scribing and storing for a short time at 4 ℃ or transferring the bacteria to a mixed solution of an NA liquid culture medium and glycerol for storing for a long time at-70 ℃.
Wherein: the NA plate culture medium is prepared by adding 10g of peptone, 3g of beef powder, 5g of sodium chloride and 15g of agar into 1000mL of water, uniformly mixing, adjusting the pH value to 7.2-7.4, and sterilizing at 121 ℃ for 30 min.
The NA liquid culture medium is prepared by adding 10g of peptone, 3g of beef powder and 5g of sodium chloride into 1000mL of water, uniformly mixing, adjusting the pH value to 7.2-7.4, and sterilizing at 121 ℃ for 30 min.
The macroscopic bacterial colonies are colony-forming units, and CFU is the colony with the average diameter of more than or equal to 1 mm.
The counting method of the macroscopic bacterial colonies refers to a concentration plate method, namely, the macroscopic single scattered bacterial colonies in a culture dish with the diameter of 90 mm have the number of 100-150 CFU.
The mixed solution of the NA liquid culture medium and the glycerol is a solution obtained by uniformly mixing the NA liquid culture medium and the glycerol according to the volume ratio of 3: 1.
The application of the paenibacillus bacteria comprises the following steps: sterilizing Paenibacillus (A), (B) and (C)Paenibacilluspabuli) B03 diluting the bacterial liquid by 100 times to obtain the bacterial liquid as the plant growth promoter of the stipa capillata.
The experimental procedures used in the following examples are conventional unless otherwise specified.
Example 1 isolation of plant root bacteria Using conventional isolation methods
Strain B03 was isolated from the root of the purple-flower stipa root in Kangle Cogba village (38 ° 49 '29 "N, 99 ° 54' 44" W; elevation 2815 m) of Zhangye, Gansu province. The method comprises the following steps: selecting healthy stipa purpurea with root plants, cutting fresh roots of the stipa purpurea into small sections (0.2-0.5 cm) for disinfection, and disinfecting the surfaces of the small sections with 75% ethanol for 30 s; sterilizing the surface of 3% sodium hypochlorite for 5 min, washing the sample with sterilized distilled water for three times, placing the sample on sterile filter paper to absorb water, placing the sample on an NA plate culture medium, performing dark culture at 28 +/-2 ℃, picking a small number of bacterial colonies after macroscopic bacterial colonies grow out on the plate, transferring the bacterial colonies onto a new NA plate culture medium to perform purification, transferring the bacterial colonies onto an NA inclined plane culture medium without pollution, and storing the bacterial colonies at 4 ℃ for a short time.
The strain provided by the invention is paenibacillus (B), (C)Paenibacilluspabuli) B03. The strain B03 has been preserved in China general microbiological culture Collection center (CGMCC) at 12 months and 17 months in 2020The hidden center address is: beijing, Ind. region of the republic of Yangyang, institute of microbiology, academy of China, zip code 100101, accession number CGMCC No. 21386.
EXAMPLE 2 cultivation of the bacterium and preparation of the metabolite
1. Primary strain culture: transferring and activating the strain B03 preserved on the slant culture medium to an NA plate culture medium, culturing at 28-30 ℃ in the dark, and taking the strain after the macroscopic bacteria grow out as a first-class strain.
2. And (3) secondary strain culture: inoculating the activated primary strain to an NA liquid culture medium, placing the NA liquid culture medium on a shaking table with the temperature of 28-30 ℃ and the rotating speed of 180 rpm for dark culture, regularly observing and recording, stopping shaking culture after the culture solution becomes turbid (about 72 hours), and transferring the NA liquid culture medium to a 4 ℃ refrigerator for short-term storage.
3. Preparation of fermentation liquors with different concentrations: the metabolite of the strain B03 is prepared into 100-fold and 200-fold diluted solution by adopting water as a diluent, and the metabolite of the sterilized strain B03 is prepared into 100-fold and 200-fold diluted solution.
Example 3 growth promoting Activity of Paenibacillus B03 metabolite
1. Growth promoting activity of fermentation liquor with different concentrations on the stipa purpurea:
in the embodiment, the grass seeds of the stipa filiformis are used as experimental materials, a germination test adopts a paper germination (TP) method, grass seedlings with relatively consistent growth vigor are selected and placed in a 6-hole plate for an activity test, the 6-hole plate is taken, then fermentation liquor and sterilized fermentation liquor are subjected to 3 gradient tests according to stock solution, dilution by 100 times and dilution by 200 times, 8 seedlings are placed in each hole, water is used as a control, and 3 parallel treatments are respectively arranged (see figure 1). Culturing in artificial climate box (light 10 h/dark 14h, temperature: 25 deg.C) for 7d, taking out, and measuring fresh weight, root length and stem length of seedling. The experimental results were statistically analyzed using Sigmaplot 12.0 software.
2. As a result:
the test groups are compared with the ck group, see Table 1 and FIGS. 2-4. The experimental result shows that compared with the ck group, the growth promotion effect of the sterilized solution diluted by 100 times by the paenibacillus B03 is the best, the fresh weight, the root length and the stem length of the sterilized solution are respectively 102.58%, 163.11% and 73.72% of the ck control, the three indexes have significant difference with the ck control, and the growth promotion effect of the inactivated solution diluted by 100 times by the paenibacillus B03 is the best.
TABLE 1 growth promoting Activity of metabolites of Strain B03 at different concentrations on Philippine Imperata
Claims (9)
1. A paenibacillus bacterium having growth-promoting activity, characterized by: the strain is paenibacillus (Paenibacilluspabuli) B03 with the preservation number of CGMCC No. 21386.
2. The method for producing a Paenibacillus bacterium having growth promoting activity according to claim 1, wherein: selecting healthy stipa purpurea with roots, cutting fresh roots of the stipa purpurea into small sections with the length of 0.2-0.5 cm, washing the small sections with sterilized distilled water for three times after disinfection, and placing the small sections on sterile filter paper to absorb water; then placing on an NA plate culture medium, and culturing at 28 +/-2 ℃ in a dark place; after the macroscopic bacteria grow on the plate, picking a few of bacteria with a sterile toothpick and transferring the bacteria to a new NA plate culture medium for purification, and if the bacteria are not polluted, transferring the bacteria to an NA slant culture medium for scribing and storing for a short time at 4 ℃ or transferring the bacteria to a mixed solution of an NA liquid culture medium and glycerol for storing for a long time at-70 ℃.
3. The method for producing a Paenibacillus bacterium having growth promoting activity according to claim 2, wherein: the disinfection means that fresh roots of the cut Philippine fescue are firstly disinfected for 30 s by ethanol with the volume concentration of 75 percent and then disinfected for 5 min by sodium hypochlorite with the mass concentration of 3 percent.
4. The method for producing a Paenibacillus bacterium having growth promoting activity according to claim 2, wherein: the NA plate culture medium is prepared by adding 10g of peptone, 3g of beef powder, 5g of sodium chloride and 15g of agar into 1000mL of water, uniformly mixing, adjusting the pH value to 7.2-7.4, and sterilizing at 121 ℃ for 30 min.
5. The method for producing a Paenibacillus bacterium having growth promoting activity according to claim 2, wherein: the NA liquid culture medium is prepared by adding 10g of peptone, 3g of beef powder and 5g of sodium chloride into 1000mL of water, uniformly mixing, adjusting the pH value to 7.2-7.4, and sterilizing at 121 ℃ for 30 min.
6. The method for producing a Paenibacillus bacterium having growth promoting activity according to claim 2, wherein: the macroscopic bacterial colonies are colony-forming units, and CFU is the colony with the average diameter of more than or equal to 1 mm.
7. The method for producing a Paenibacillus bacterium having growth promoting activity according to claim 6, wherein: the counting method of the macroscopic bacterial colonies refers to a concentration plate method, namely the macroscopic single scattered bacterial colonies in a culture dish with the diameter of 90 mm have the number of 100-150 CFU.
8. The method for producing a Paenibacillus bacterium having growth promoting activity according to claim 2, wherein: the mixed solution of the NA liquid culture medium and the glycerol is a solution obtained by uniformly mixing the NA liquid culture medium and the glycerol according to the volume ratio of 3: 1.
9. The use of a growth-promoting bacterium of the genus Paenibacillus as claimed in claim 1, wherein: sterilizing Paenibacillus (A), (B) and (C)Paenibacilluspabuli) B03 diluting the bacterial liquid by 100 times to obtain the bacterial liquid as the plant growth promoter of the stipa capillata.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112899188A (en) * | 2021-01-29 | 2021-06-04 | 西南大学 | Microbial agent for promoting crop root development and preparation and application thereof |
| CN117247869A (en) * | 2023-09-26 | 2023-12-19 | 中国科学院兰州化学物理研究所 | Paenibacillus with plant growth regulating activity and preparation and application thereof |
| CN117305163A (en) * | 2023-09-26 | 2023-12-29 | 中国科学院兰州化学物理研究所 | Bacillus pumilus with plant growth regulating activity, and preparation and application thereof |
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