CN116925979A - Bacillus bailii and application thereof in promoting plant growth - Google Patents

Bacillus bailii and application thereof in promoting plant growth Download PDF

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CN116925979A
CN116925979A CN202311086438.1A CN202311086438A CN116925979A CN 116925979 A CN116925979 A CN 116925979A CN 202311086438 A CN202311086438 A CN 202311086438A CN 116925979 A CN116925979 A CN 116925979A
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bacillus
cla178
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bailii
growth
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陈淋
刘云鹏
边禄森
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Experimental Center Of Forestry In North China Chinese Academy Of Forestry
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Experimental Center Of Forestry In North China Chinese Academy Of Forestry
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P21/00—Plant growth regulators
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00—Other organic fertilisers
    • C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus

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Abstract

The invention discloses bacillus beleiensis and application thereof in promoting plant growth, wherein bacillus beleiensis (Bacillus velezensis) CLA178 is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of 27115 in the year of 4 and 14 of 2023. The bacillus beleiensis (Bacillus velezensis) CLA178 has the characteristics of producing indoleacetic acid, siderophores, dissolving phosphorus and dissolving potassium, and can remarkably promote the growth of various plants including rose, sunflower and arabidopsis. The remarkable growth promoting property of the strain shows that the strain can be used as an excellent strain resource of a microbial fertilizer and has good application prospect.

Description

Bacillus bailii and application thereof in promoting plant growth
Technical Field
The invention relates to the technical field of microorganisms, in particular to bacillus beijerinckii and application thereof in promoting plant growth.
Background
Along with the better living standard of people, the requirements of people on agriculture and forestry products are high quality, green, organic and pollution-free. Since 2017, in order to realize green sustainable development, agricultural ecological civilization is built, and organic fertilizer is developed in agricultural rural areas to replace chemical fertilizer action, wherein the microbial organic fertilizer taking plant rhizosphere growth promoting bacteria as a main component can not only remarkably promote plant growth, but also is green and pollution-free, and is gradually favored by the masses.
Plant growth-promoting bacteria (Plant growth-promoting rhizobacteria) are a beneficial microorganism that can colonize and survive at Plant rhizosphere, and can improve Plant nutrient absorption, promote Plant growth, control soil-borne diseases, and enhance Plant stress resistance. The growth promoting mechanism of plant rhizosphere growth promoting bacteria is various and can be divided into direct action and indirect action. Direct action includes enhancing nutrient absorption by plants by activating the nutrients through dissolving phosphorus, dissolving potassium, fixing nitrogen, and producing siderophores; the hormone level in the plant body is regulated and controlled by secreting plant growth hormone, volatile substances and the like, so that the plant growth is promoted, and the water and nutrient absorption capacity of the plant is enhanced. Indirect effects include the production of antagonistic substances or the induction of plant system resistance to biological diseases.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: provides a strain of rhizosphere growth-promoting bacillus beijerinus and application thereof in promoting plant growth.
The technical scheme of the invention is as follows: bacillus bailii (Bacillus velezensis) CLA178 deposited at the China general microbiological culture Collection center, address: the preservation number of the Beijing city Chaoyang area North Chen Xili No.1 and 3 is CGMCC No.27115.
A formulation comprising bacillus beleiensis (Bacillus velezensis) CLA178 or a metabolite thereof as described above.
Use of bacillus beijerinus (Bacillus velezensis) CLA178 or a formulation as described above for promoting plant growth.
Further, the plant is rose, sunflower or arabidopsis.
Bacillus bailii (Bacillus velezensis) CLA178 of the invention is capable of producing indoleacetic acid with a yield of 18.82mg/L after 2 days of cultivation.
Bacillus bailii (Bacillus velezensis) CLA178 of the present invention had a siderophore production and was cultured for 2 days with a capacity to sequester elemental iron of 30.78%.
The bacillus belicus (Bacillus velezensis) CLA178 has remarkable phosphorus and potassium dissolving growth promoting property.
The bacillus beleiensis (Bacillus velezensis) CLA178 has obvious growth promoting effect, and after the strain is inoculated, the overground and underground biomass of roses, sunflowers and arabidopsis thaliana is obviously improved.
Compared with the prior art, the invention has the following beneficial effects:
the bacillus bailii (Bacillus velezensis) CLA178 has the growth promoting property of producing IAA and siderophores and dissolving phosphorus and potassium, and through a growth promoting effect test, the bacillus bailii can promote the growth of various plants, including rose, sunflower and arabidopsis. The strain improves the biomass of the aerial part and the underground part of rose by 20.23 percent and 27.29 percent respectively, improves the biomass of the aerial part and the underground part of sunflower by 40.64 percent and 52.25 percent respectively, and improves the biomass of the aerial part and the underground part of Arabidopsis by 5.09 times and 4.54 times respectively. The invention provides support for microbial fertilizer for promoting growth and increasing yield of agriculture and forestry.
Preservation information:
bacillus belicus (Bacillus velezensis) CLA178 was deposited at the China general microbiological culture Collection center (CGMCC; address: north Chen Xway No.1, 3, institute of microbiology, academy of sciences of China, post code: 100101) of China, 4 months and 14 days, with a deposit number of CGMCC No.27115.
Drawings
FIG. 1 shows colony morphology of Bacillus bailii CLA178 on LB solid medium;
FIG. 2 is a standard curve for IAA at various concentrations;
FIG. 3 shows the siderophore chromogenic response of Bacillus bailii CLA 178;
FIG. 4 is a phosphate solubilizing loop of Bacillus bailii CLA 178;
FIG. 5 is a morphology of Bacillus bailii CLA178 on potassium-solubilizing medium;
FIG. 6 is an effect of Bacillus bailii CLA178 on rose growth;
FIG. 7 is the effect of Bacillus belicus CLA178 on sunflower growth;
FIG. 8 is the effect of Bacillus bailii CLA178 on Arabidopsis growth.
Detailed Description
The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from commercial sources.
Example 1 screening and identification of Bacillus bailii CLA178
The sampling site in this embodiment is a Beijing city mountain area cynoglossum river town tree rose base.
(1) Separating and purifying strain
Collecting 10g of rose plant rhizosphere soil, adding the rose plant rhizosphere soil into a triangular flask containing 90mL of sterile water, and then vibrating the triangular flask at 30 ℃ and 170rpm for 60min to form a soil suspension stock solution; 1mL of the stock solution is taken and diluted to 10 in a gradient way -1 ，10 -2 ，10 -3 ，10 -4 ，10 -5 And are respectively and fully mixed. Respectively draw 100 mu L10 -3 ，10 -4 ，10 -5 The dilutions were plated on LB plates, 3 replicates per group, and the plates were placed in a 30℃incubator for 2 days. Different single colonies are selected according to the forms of the colonies for streak purification, the single colonies are placed in a 30 ℃ incubator for 2 days, then the single colonies in each culture dish are selected for streak purification again, the single colonies are placed in the 30 incubator for 2 days, and the single colonies are purified for multiple times to ensure that pure bacteria are separated from each culture dish.
(2) Screening of biocontrol Strain
The pathogenic bacteria agrobacterium tumefaciens is used as a target bacteria, and an antagonistic bacterium is screened by adopting a plate counter method, so that a bacterial strain CLA178 which is remarkably antagonistic to the agrobacterium tumefaciens is obtained.
(3) Identification of Strain morphology
Strain CLA178 was inoculated on LB solid medium and cultured at 30 ℃ for 2 days, as shown in fig. 1, the colony was flat, wrinkled on the surface, uneven in edge, white and sticky.
(4) Molecular identification of species
The total DNA of the strain is extracted, the 16S rDNA sequence is amplified and sequenced, and the sequencing result is shown as SEQ ID No.1 of the sequence table. By identification, it was determined that strain CLA178 belongs to Bacillus belicus (Bacillus velezensis) and is therefore renamed Bacillus belicus (Bacillus velezensis) CLA178.
(5) Preservation of strains
The bacillus beleiensis (Bacillus velezensis) CLA178 provided by the invention is preserved in China general microbiological culture Collection center (CGMCC) in 4 months and 14 days of 2023, and has the preservation number of CGMCC No.27115, namely, the address of the CGMCC No.1, 3, the institute of microbiology, the national academy of sciences, beijing, the Korea, and the post code of the CGMCC No.27115.
EXAMPLE 2 determination of IAA producing ability of Bacillus bailii
(1) Method for detecting IAA produced by bacterial strain
Kings medium (1L): 20g peptone, 1.725g K 2 HPO 4 ,1.5g MgSO 4 0.2g tryptophan, 15ml glycerol, pH 7-7.2, deionized water to a volume of 1L.
Salkowski reagent colorimetric solution formula: 1mL of 0.5M FeCl 3 With 50mL of 35% HClO 4 Mix well and mix it with the other ingredients.
IAA standard solution preparation: firstly, preparing an IAA standard stock solution with the concentration of 100mg/L, weighing 10mg of IAA, dissolving the IAA with a small amount of ethanol, and then, fixing the volume to 100mL with deionized water. Diluting IAA standard stock solution with deionized water to obtain IAA solutions with standard concentrations of 0, 5, 10, 20, 40, 60 and 80mg/L, respectively, and storing in dark place after preparation.
Collection of strain cultures: the activated CLA178 was transferred to LB liquid medium for overnight culture at 170rpm at 30℃and centrifuged at 6000rpm for 10min to collect the cells, which were resuspended in sterile PBS. Transfer to a media containing tryptophan in liquid medium at 1% inoculum size, 170rpm, shaking culture at 30℃for 2 days. And centrifuging at 8000rpm for 10min to collect thallus to obtain supernatant.
Detection of IAA content: taking 1mL of IAA standard solution with each concentration or supernatant after the bacteria culture is centrifuged, adding an equal volume of Salkowski reagent colorimetric solution, processing for 30min in a dark place, adding each processed reaction solution into a 96-well plate, and detecting the light absorption value with the wavelength of 530nm by using an enzyme-labeled instrument. And drawing an IAA standard curve (figure 2) by taking the IAA standard concentration as an abscissa and the absorbance value as an ordinate, and calculating the IAA content in the bacterial supernatant according to the IAA standard curve.
(2) IAA detection results produced by the strain
Bacillus bailii CLA178 is capable of producing IAA at 18.82mg/L.
EXAMPLE 3 determination of Bacillus bailii siderophore production ability
(1) Method for detecting siderophores produced by bacterial strains
Modified CAS liquid medium (PM 08020-1L, coolibo) including liquid basal medium, 10 Xbuffer and 10 XCAS assay, pH 6.6-7.0.
Liquid basal medium (1L): 100g glucose, 20g peptone, 0.5g MgSO 4 ·7H 2 O，0.5g CaCl 2 Deionized water was set to a volume of 1L.
10 x buffer: piperazine-1, 4-disulfonic acid (PIPES) was used as a buffer.
10 XCAS test fluid (1L): 0.06g of Chrome Azure (CAS), 0.0027g of FeCl 3 ·6H 2 O,0.073g cetyl trimethylammonium bromide (HDTMA), ionized water was set to a volume of 1L.
CAS liquid medium: the liquid basal medium was sterilized at 115℃for 20min, cooled to 50-60℃and then 10mL of sterile 10 Xbuffer and 10mL of sterile 10 XCAS test solution were slowly added to each 80mL of liquid basal medium.
The activated strain was inoculated in a 1% inoculum size into CAS liquid medium at 30℃and shaking at 170rpm for 2 days.
The bacterial liquid was centrifuged at 10000rpm for 10min, the supernatant was collected, and the color of the supernatant was observed, and the change from blue to orange was positive. The supernatant was examined for absorbance at 630nm (As) with an ultraviolet spectrophotometer, and the absorbance of the unsplit CAS liquid medium was used As a control (Ar), relative content of siderophores = [ (Ar-As)/Ar ] ×100%.
(2) Detection result of bacterial strain siderophore
After inoculating bacillus bailii CLA178 and culturing in CAS liquid medium for 2 days, the culture solution turns orange (figure 3), which shows that the strain can secrete siderophores; the relative content of siderophores of this strain was 30.77% by calculation of absorbance.
EXAMPLE 4 determination of phosphorus-solubilizing ability of Bacillus bailii
(1) Method for detecting bacterial strain phosphorus dissolution
Phosphate solubilizing medium: 10g glucose, 0.3g MgSO 4 ·7H 2 O，0.3g NaCl，0.5g(NH 4 ) 2 SO 4 ，0.03gMnSO 4 ·H 2 O,0.3g K 2 SO 4 ,0.03g FeSO 4 ·7H 2 O,5g Ca 3 (PO 4 ) 2 0.2g lecithin, 15g agar powder, distilled water to 1L, pH 6.8-7.2.
Bacillus beleiensis CLA178 was inoculated onto sterile phosphate solubilizing solid medium, cultured at 30 ℃ for 2-3 days, and whether there was a transparent ring around the colony was observed.
(2) Detection result of bacterial strain phosphorus dissolution
After inoculating bacillus bailii CLA178 on phosphate solubilizing medium and culturing at 30 ℃ for 2-3 days, a remarkable transparent circle is observed (figure 4), which shows that the strain has stronger phosphate solubilizing capability.
EXAMPLE 5 determination of the Potassium-decomposing Capacity of Bacillus bailii
(1) Detection method for bacterial strain potassium decomposition
The Alexander culture medium is used for detecting the potassium-decomposing capacity of the strain, and if the strain presents oil-drop-shaped colonies on the culture medium, the strain has the potassium-decomposing capacity.
Mountain Bao Luofu medium (Alexandrov, potassium-dissolving medium): 5g sucrose, 2g Na 2 HPO 4 ，0.5gMgSO 4 ·7H 2 O，0.005g FeCl 3 ，0.1g CaCO 3 1g of bauxite, 15g of agar, distilled water to 1L, pH7.0-7.4.
Bacillus belicus was inoculated into the culture medium of the asiatic mountain Bao Luofu, cultured at 30 ℃ for 2-3 days, and whether colonies on the culture medium were oil-drop-shaped or not was observed.
(2) Detection result of bacterial strain potassium decomposition
Bacillus belicus CLA178 was cultured on a Xylella media Bao Luofu at 30℃for 2-3 days, and colonies of the strain were found to be in the form of oil droplets (FIG. 5), indicating that the strain had a potassium-solubilizing function.
In conclusion, the results show (Table 1) that bacillus belicus CLA178 has four growth promotion characteristics of IAA production, siderophore dissolution, potassium dissolution, and the strain is an excellent strain for preparing microbial bacterial manure, and has good application prospect.
TABLE 1 Bacillus bailii CLA178 proliferative Properties
Note that: "+" indicates positive response; "-" indicates that the corresponding reaction is negative.
Example 6 Bacillus bailii Protoeffect assay
The roses and the sunflowers are important economic plants, have important application in food, gardening and medical treatment, have higher economic value, and the arabidopsis thaliana is also important as a model plant, so that the invention detects the growth promotion effect of the strain on the three plants.
(1) Strain-promoting effect detection method
Seedling raising of roses: cutting 1 year old Rosa spinosa twig, sterilizing the surface of the twig, cutting into small segments, cutting into sterilized wet culture medium (vermiculite: nutrient soil=1:1), and culturing in a light incubator at 28deg.C for 14 hr/10 hr darkness for 80 days.
Seedling raising of sunflowers: after surface sterilization of the seeds of short sunflower, they were placed in a seedling tray containing a moist sterile culture medium (vermiculite: nutrient soil=1:1) and incubated for 20 days at 28℃in an illumination incubator with 14h illumination/10 h darkness.
Seedling raising of arabidopsis thaliana: the surface of the Columbia wild arabidopsis seeds is sterilized and placed in a solid culture medium containing 1/2MS, vernalized for 2 days at 4 ℃ in a dark place, and then placed in a light incubator at 25 ℃ for 14h light/10 h dark for culturing for 14 days.
Preparation of a microbial inoculum: transfer activated CLA178 to LB liquid medium at 30deg.C, culturing overnight at 170rpm, centrifuging at 6000rpm to collect bacterial cells, and re-suspending to OD with sterile PBS 600 1.
Sunflower and rose seedlings were inoculated: the suspension of CLA178 bacteria was mixed at 5X 10 6 CFU/g soil was inoculated to sunflower and rose roots, respectively, and cultured in an illumination incubator at 28℃for 20 days and at 14 hours of illumination/10 hours of darkness, respectively.
Inoculating Arabidopsis thaliana: mu.L of the bacterial suspension is sucked, streaked under the root part of the Arabidopsis thaliana seedling, and the culture dish is placed in a light incubator with the temperature of 25 ℃ and the light/darkness of 14 hours for 14 days by taking sterile PBS as a control.
(2) Bacterial strain growth promoting effect
Fresh weights of aerial and underground portions of rose, sunflower and Arabidopsis were measured, respectively.
The results show that the CLA178 strain can obviously promote the biomass of the overground part and the underground part of roses (figure 6), sunflowers (figure 7) and arabidopsis thaliana (figure 8), and the strain has better growth promoting effect.

Claims (4)

1. Bacillus bailii (Bacillus velezensis) CLA178 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 27115 in the year 2023, month 4 and day 14.
2. A formulation comprising bacillus belgium (Bacillus velezensis) CLA178 or a metabolite thereof of claim 1.
3. Use of bacillus belgium (Bacillus velezensis) CLA178 of claim 1 or the formulation of claim 2 for promoting plant growth.
4. The use according to claim 3, wherein the plant is rose, sunflower or arabidopsis.
CN202311086438.1A 2023-08-28 2023-08-28 Bacillus bailii and application thereof in promoting plant growth Pending CN116925979A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117903986A (en) * 2024-01-18 2024-04-19 东北农业大学 Bacillus bailii, microbial inoculum, plant growth promoter, biological fertilizer and application
CN118126903A (en) * 2024-04-19 2024-06-04 青岛农业大学 Acid and alkali resistant multifunctional bacillus bailii, microbial agent and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117903986A (en) * 2024-01-18 2024-04-19 东北农业大学 Bacillus bailii, microbial inoculum, plant growth promoter, biological fertilizer and application
CN118126903A (en) * 2024-04-19 2024-06-04 青岛农业大学 Acid and alkali resistant multifunctional bacillus bailii, microbial agent and application thereof

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