CN111560323A - Heat-resistant bacillus capable of promoting growth and dissolving phosphorus and application thereof - Google Patents
Heat-resistant bacillus capable of promoting growth and dissolving phosphorus and application thereof Download PDFInfo
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- CN111560323A CN111560323A CN201910081066.0A CN201910081066A CN111560323A CN 111560323 A CN111560323 A CN 111560323A CN 201910081066 A CN201910081066 A CN 201910081066A CN 111560323 A CN111560323 A CN 111560323A
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- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 60
- 229910052698 phosphorus Inorganic materials 0.000 title claims abstract description 45
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- 239000011574 phosphorus Substances 0.000 title claims abstract description 43
- 230000012010 growth Effects 0.000 title claims abstract description 34
- 230000001737 promoting effect Effects 0.000 title claims abstract description 26
- 241000012248 Bacillus toyonensis Species 0.000 claims abstract description 9
- 238000004321 preservation Methods 0.000 claims abstract description 8
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- 239000010452 phosphate Substances 0.000 claims description 13
- 238000012258 culturing Methods 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 9
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
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- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 239000011609 ammonium molybdate Substances 0.000 description 2
- 235000018660 ammonium molybdate Nutrition 0.000 description 2
- APUPEJJSWDHEBO-UHFFFAOYSA-P ammonium molybdate Chemical compound [NH4+].[NH4+].[O-][Mo]([O-])(=O)=O APUPEJJSWDHEBO-UHFFFAOYSA-P 0.000 description 2
- 229940010552 ammonium molybdate Drugs 0.000 description 2
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 2
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- UFBJCMHMOXMLKC-UHFFFAOYSA-N 2,4-dinitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O UFBJCMHMOXMLKC-UHFFFAOYSA-N 0.000 description 1
- JCRIDWXIBSEOEG-UHFFFAOYSA-N 2,6-dinitrophenol Chemical compound OC1=C([N+]([O-])=O)C=CC=C1[N+]([O-])=O JCRIDWXIBSEOEG-UHFFFAOYSA-N 0.000 description 1
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- 229940026189 antimony potassium tartrate Drugs 0.000 description 1
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- WBTCZEPSIIFINA-MSFWTACDSA-J dipotassium;antimony(3+);(2r,3r)-2,3-dioxidobutanedioate;trihydrate Chemical compound O.O.O.[K+].[K+].[Sb+3].[Sb+3].[O-]C(=O)[C@H]([O-])[C@@H]([O-])C([O-])=O.[O-]C(=O)[C@H]([O-])[C@@H]([O-])C([O-])=O WBTCZEPSIIFINA-MSFWTACDSA-J 0.000 description 1
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- 229910052816 inorganic phosphate Inorganic materials 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
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- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
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- 239000003895 organic fertilizer Substances 0.000 description 1
- VSIIXMUUUJUKCM-UHFFFAOYSA-D pentacalcium;fluoride;triphosphate Chemical compound [F-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O VSIIXMUUUJUKCM-UHFFFAOYSA-D 0.000 description 1
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- CCEKAJIANROZEO-UHFFFAOYSA-N sulfluramid Chemical group CCNS(=O)(=O)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)F CCEKAJIANROZEO-UHFFFAOYSA-N 0.000 description 1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention provides a heat-resistant Bacillus for promoting growth and dissolving phosphorus and application thereof, wherein the strain is Bacillus toyonensis FJAT-47648, has a scientific name of Bacillus toyonensis FJAT-47648, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and has a preservation number of CGMCC NO. 16252. The bacillus of the invention can effectively degrade inorganic phosphorus, improve the content of soluble phosphorus in soil, obviously promote the growth of crops, develop root systems of the crops, enhance the stress resistance of the crops, have high temperature resistance and be beneficial to the composting treatment of agricultural wastes.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a bacillus strain capable of promoting phosphorus decomposition and resisting heat and application thereof in plant growth.
Background
The phosphorus supply level of soil is one of the key factors influencing the growth of plants, 95 percent of phosphorus in the soil is in an invalid form, and the plants are difficult to directly absorb and utilize, so that the phosphorus deficiency phenomenon exists in 74 percent of cultivated land soil in China.
In a crop-microorganism interaction system, Plant growth-promoting rhizobacteria (PGPR) are colonized in the rhizosphere soil of crops, and can effectively decompose insoluble and fixed elements (phosphorus, potassium and the like) in the soil, promote the absorption of the crops on fertilizers and elements in the soil, and further promote the growth, yield increase, disease resistance and the like of the crops. Therefore, the microbial fertilizer with the efficient growth promoting function is screened and developed and applied to agricultural production, potential element resources of soil are fully utilized, and the microbial fertilizer has important significance for improving element shortage of soil such as phosphorus and potassium and the like, reducing environmental pollution and promoting agricultural sustainable development.
Disclosure of Invention
Therefore, a strain capable of promoting growth and dissolving phosphorus is needed to be provided, and the problem that elements such as phosphorus, potassium and the like in soil cannot be absorbed and utilized by plants is solved.
In order to achieve the purpose, the inventor provides the following technical scheme:
a heat-resistant bacillus capable of promoting growth and dissolving phosphorus, which is characterized in that: the Bacillus is Penicillium Tuwense FJAT-47648, has a scientific name of Bacillus toyonensis FJAT-47648, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, has a preservation number of CGMCC NO.16252, has a preservation date of 2018, 8 and 13 days, and has a preservation address of the institute of microorganisms of China academy of sciences, Beijing, China.
The colony morphology of the bacillus FJAT-47648 is as follows: medium size, round, light yellow in color, smooth edge, rough, flat, opaque, dry surface.
Wherein the fermentation method of the bacillus comprises the following steps: inoculating bacillus into an LB liquid culture medium, and performing shaking culture at 25-35 ℃ for 48-72h to obtain a seed solution; then inoculating the seed liquid into LB liquid culture medium, and culturing for 48-72h under shaking at 30-60 deg.C.
Further, the heat-resistant bacillus for promoting phosphorus decomposition is applied to degrading insoluble phosphate.
Further, the application of the heat-resistant bacillus for promoting growth and dissolving phosphorus in promoting plant seed germination is realized by the specific method that the bacillus fermentation liquor is prepared into the fermentation liquor with the concentration of 1 × 105-1.5×105cfu/mL bacterial suspension, soaking seeds for 2-3 days, placing at 25-30 deg.C, and illuminating for 16-20 h/day.
Further, the application of the heat-resistant bacillus for promoting the growth of phosphorus-dissolving in promoting the growth of plant seedlings is realized by the specific method that the bacillus fermentation liquor is prepared into the fermentation liquor with the concentration of 0.8 × 106-1.2×106cfu/mL of bacterial suspension; planting the plant seedlings in a planting matrix indoors for 6-8 days, and then irrigating roots by using bacterial suspension once every 5-7 days. Watering every 2-3 days during plant growth period, and watering plant nutrient solution (such as Hoagland solution) every 10-14 days to supplement plant growthThe required nutrient elements in the formula (I). The bacterial suspension, water and plant nutrient solution are alternately used.
The planting matrix comprises N, P, K and organic matters, wherein N, P, K mass percent is not less than 3%, 3545% of the organic matters, and the pH value of the matrix is 5.5-6.5. The planting substrate comprises a vegetable planting substrate.
A plant growth promoting microbial inoculum comprises the bacillus.
The invention has the beneficial effects that:
(1) the bacillus of the invention can effectively degrade inorganic phosphorus, promote insoluble phosphate to release phosphorus, and improve the content of soluble phosphorus in soil, thereby obviously promoting the growth of crops, enabling the roots of the crops to be developed, and enhancing the stress resistance of the crops.
(2) The bacillus of the invention can improve the activity of plant seeds, promote the rooting and germination of the seeds, obviously promote the growth of plant seedlings and effectively shorten the growth cycle of the plants.
(3) The bacillus of the invention can resist high temperature and is beneficial to compost fermentation.
Drawings
FIG. 1 shows colony morphology of Bacillus FJAT-47648, wherein the left image shows colony growth status of FJAT-47648 strain on a whole plate, and the right image shows a partial enlarged view of the colony on the left image.
FIG. 2 is a tree showing the results of identifying the 16S rRNA sequence of Bacillus FJAT-47648 according to the embodiment.
FIG. 3 shows the effect of different concentrations of Bacillus FJAT-47648 on the growth of tomato seeds.
FIG. 4 shows the effect of different concentrations of Bacillus FJAT-47648 on germination index and germination rate of tomato seeds.
FIG. 5 shows the effect of different concentrations of Bacillus FJAT-47648 on the root length, stem length and vigor of tomato seeds.
FIG. 6 shows the growth of tomato seedlings according to the embodiment, wherein the left side of the diagram is CK-W group and the back side is B-6 group.
FIG. 7 shows the effect of Bacillus FJAT-47648 on the plant height, root length and stem thickness of tomato seedlings.
FIG. 8 shows the effect of Bacillus FJAT-47648 on fresh weight of tomato plants according to an embodiment.
FIG. 9 is a graph showing the effect of Bacillus FJAT-47648 on tomato plant dry weight, according to an embodiment.
Detailed Description
To explain technical contents, achieved objects and effects of the technical solutions in detail, the following detailed description is given with reference to the accompanying drawings in combination with specific embodiments.
EXAMPLE 1 phosphate solubilization of Bacillus
1. Test materials
1.1 test strains
Test strains: the bacillus FJAT-47648 is separated from rhizosphere soil under Gekko national geological park grasses in Tengchong volcano geothermal energy, is frozen and preserved in glycerol at-80 ℃, is preserved in the China general microbiological culture Collection center, and has the preservation number of CGMCC NO. 16252.
1.2 culture Medium
Activating a culture medium: (1) LB solid medium (from Producer) recipe: 10g of tryptone, 10g of sodium chloride, 5g of yeast powder, 15g of agar and 1000mL of water, and the pH value is 7.0. (2) LB liquid medium (from Producer) recipe: 10g of tryptone, 10g of sodium chloride, 5g of yeast powder, 1000mL of water and pH 7.0.
Inorganic phosphorus medium (NBRIP medium): glucose 10g, Ca3(PO4)25g，MgCl2·6H2O 5g，KCl0.2g，MgSO4·7H2O 0.25g，(NH4)2SO40.1g, 1000mL of distilled water, natural pH.
Organophosphorus growth medium: 10g of glucose, 0.5g of ammonium sulfate, 0.5g of yeast extract powder, 0.3g of sodium chloride, 0.3g of potassium chloride, 0.3g of magnesium sulfate, 0.03g of ferrous sulfate, 0.03g of manganese sulfate, 0.2g of lecithin, 1.0g of calcium carbonate, 1000mL of distilled water, 15g of agar and pH 7.0-7.5.
1.3 preparation of test reagents
Molybdenum antimony storage solution: 153mL of concentrated sulfuric acid (analytically pure, density 1.84g/mL) was weighed out, slowly added to 400mL of distilled water, and stirred until cooled. 10g of ground ammonium molybdate is weighed and poured into the mixture, and stirred to dissolve. Then adding 100mL of 0.5% (5g/L) antimony potassium tartrate solution, cooling, adding water to dilute to 1000mL, shaking up, storing in a brown reagent bottle, wherein the stock solution contains 1% ammonium molybdate and 2.75moL/L sulfuric acid.
Molybdenum antimony color-developing resisting agent: 1.50g of ascorbic acid is weighed out and dissolved in 100mL of molybdenum antimony anti-storage solution, and the solution has short effective period and is suitable for being used along with the preparation.
5mg/L phosphorus standard solution: 0.4394g Potassium dihydrogen phosphate (KH) dried at 50 deg.C2PO4Analytically pure), 100mL of water, 5mL of concentrated sulfuric acid (preservative), and the volume of the water is up to 1L, the concentration of phosphorus is 100mg/L, and the solution can be stored for a long time. Sucking 5mL of the solution into a 100mL volumetric flask, and adding water to a constant volume, wherein the constant volume is a phosphorus standard solution with the concentration of 5mg/L, and the solution is not suitable for long-term storage.
2. Test method
2.1 determination of phosphate solubilizing ability
2.1.1 activation of the test strains
Taking out the test strain in a refrigerator at minus 80 ℃, streaking the test strain in an LB solid agar culture medium plate in a super clean bench when the test strain is warmed to room temperature, and inversely placing the test strain in a biological incubator for culturing for 2d at 30 ℃. And observing the colony morphology after 2d, and selecting a single colony for secondary streak culture to ensure that the activated colony has a single morphology. And (3) selecting a proper amount of single colony to be cultured in an LB liquid culture medium for 2d by a shaking table at 170rpm and 30 ℃ to obtain a seed solution.
2.1.2 liquid Shake flask fermentation
Diluting the seed liquid by 2 times, and detecting OD by an enzyme-labeling instrument600nmCombining with bacterial count under microscope, diluting properly, and adjusting bacterial density to 108cfu/mL (bacterial liquid OD after 2-fold dilution)600nmBetween 0.3 and 0.5), 200. mu.L of each was inoculated into a 50mL centrifuge tube containing 10mL liquid medium of organic and inorganic phosphorus, and shake-cultured at 230rpm and 30 ℃ for 6d, to which 200. mu.L of sterile water was added as a control, and two replicates were made for each test bacterium.
2.1.3 detection of effective phosphorus content in supernatant by MoSb antibody method
a. Preparation of supernatant
And centrifuging the fermentation liquor cultured for 6d at 1200rpm for 30min, taking supernatant, and discarding the precipitate.
b. Drawing of standard curve
Respectively and accurately sucking 0, 2, 4, 6, 8 and 10mL of 5mg/L phosphorus standard solution into a 50mL volumetric flask, diluting the solution to about 3/5 points of the total volume by using water, adding 2 drops of 2, 6-dinitrophenol as an indicator, adjusting 50mL/L dilute sulfuric acid (or hydrochloric acid) and 10 percent sodium hydroxide until the solution is just yellowish, accurately adding 5mL of molybdenum-antimony color-resisting agent, shaking up, adding water to fix the volume, and obtaining a standard solution series with phosphorus contents of 0.0, 0.2, 0.4, 0.8 and 1.0mg/L respectively. Shaking, standing at room temperature above 15 deg.C for 30 min. At a wavelength of 700nm, the absorbance was measured, and a standard curve was plotted with the absorbance as the ordinate and the phosphorus concentration (mg/L) as the abscissa.
c. Determination of available phosphorus content in supernatant
Transferring a proper amount of supernatant into a 50mL volumetric flask, diluting with water to about 3/5 parts of the total volume, adding 1-2 drops of dinitrophenol indicator, accurately adding 5mL of molybdenum-antimony color-resisting reagent, shaking up, adding water to a constant volume, keeping the temperature at room temperature above 15 ℃, and standing for 30 min. Reading the absorbance OD700nmThen, the corresponding phosphorus content is checked from the standard curve.
d. Calculating the effective dissolved phosphorus content in the supernatant
The effective phosphorus content p (mg/L) of the supernatant is equal to the concentration of the supernatant, the colorimetric volume and the fractional times of the total volume of the fermentation liquor
Wherein, the concentration of the supernatant fluid is: the concentration mg/L of phosphorus is searched from the standard curve;
colorimetric volume: fixing the volume to 50 mL;
dividing times are the total volume of the fermentation liquor/sampling volume.
Effective phosphorus-dissolving amount P (mg/L) ═ effective phosphorus content of strain supernatant-control supernatant phosphorus content
2.2 phospholytic bacteria morphology and 16s rRNA identification
The shape of the phosphate solubilizing bacteria: and streaking and inoculating the purified bacillus onto an LB plate, and culturing at constant temperature of 30 ℃ for 48 h. After the colonies grow out, the characteristics of the size, the color, the edge uniformity, the wettability and the like of the colonies are observed.
And (3) molecular identification: inoculating the pure strain to LB liquid culture medium, placing the pure strain in a shaking table at 30 ℃, culturing the pure strain to a logarithmic phase, extracting genome DNA of the strain FJAT-47648 by adopting a Tris-saturated phenol method, carrying out PCR amplification by adopting 16SrRNA gene universal primers 27F and 1492R, carrying out PCR reaction programs according to the literature of Zhengxuefang and the like (Zhengxuefang, Liubo, Zhuyangqing, and the like, screening and identifying the bacillus biocontrol strain of tomato bacterial wilt [ J ]. China biological prevention and control institute, 2016,32(5):657-665.), sending PCR products to Shanghai Boshang biotechnology Limited company for sequencing, completing sequence homology comparison by adopting EZTioCloud, analyzing sequences by MEGA 6.0.6 software and constructing a phylogenetic tree.
2.3 determination of high temperature resistance
Preparing a seed solution: picking single colony in LB culture medium, culturing at 30 deg.C at 170rpm for 48h, diluting with sterilized ultrapure water to OD600nm0.75 to 0.85 (bacterial density 10)8Around cfu/mL).
Inoculating 200 μ L of seed solution into 5mL LB liquid culture medium, culturing at 30 deg.C and 60 deg.C respectively at 170rpm for 48h with blank culture medium as control, repeating each strain for 3 times, and detecting OD with enzyme labeling instrument600nmThe value is obtained.
3. Test results
3.1 determination of phosphate solubilizing ability
The phosphate solubilizing ability of Bacillus FJAT-47648 on organic and inorganic phosphorus is shown in Table 1. Experimental results show that the bacillus FJAT-47648 has a relatively obvious phosphate solubilizing effect on inorganic phosphorus, and has a relatively poor phosphate solubilizing effect on organic phosphorus.
TABLE 1 phosphate solubilizing ability of Bacillus FJAT-47648
It can be seen that Bacillus FJAT-47648, a phosphate solubilizing (inorganic phosphate degrading) microorganism, can promote apatite Ca3(PO4)2When the insoluble phosphate releases phosphorus, the content of soluble phosphorus in the soil is improved, thereby obviously promoting the farmingThe growth of the plant can make the root system developed, which is helpful for enhancing the stress resistance of the crops.
3.2 identification of the Strain
3.2.1 Strain morphology
The colony morphology of FJAT-47648 is: medium size, round, light yellow in color, smooth edge, rough, flat, opaque, dry surface. The colony morphology is shown in FIG. 1.
Identification of 3.2.216S rRNA Gene
The nucleic acid sequence of the 16S rRNA gene of the strain FJAT-47648 is as follows:
ATACATGCAGTCGAGCGAATGGATTGAGAGCTTGCTCTCAAGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGAACTGCATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGAAAACCCTAGAGATAGGGCTTCTCCTTCGGGAGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCATCATTAAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATCTCATAAAACCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACCTTTATGGAGCCAGCCGC(SEQ ID NO:1)
comparing the 16S rRNA gene sequence SEQ ID NO:1 of the strain FJAT-47648 with an EZBIOCOcloud gene database, wherein the strain FJAT-47648 has the closest genetic relationship with Bacillus toyonensis, and the homology of the 16SrRNA gene is 100 percent, so the strain FJAT-47648 belongs to Bacillus toyonensis Va. Downloading 16S rRNA gene sequences of strains with higher homology, carrying out comparative analysis, constructing a phylogenetic tree, and forming the phylogenetic tree as shown in figure 2 when a neighbor-Joining method is adopted and the Bootstrap value is 1000 times. In the constructed phylogenetic tree, the strain FJAT-47648 and Bacillus toyonensis are gathered in the same branch.
3.3 high temperature resistance test
The results of the test of Bacillus FJAT-47648 at a high temperature of 60 ℃ are shown in Table 2. The experimental result shows that the bacterial density of the FJAT-47648 strain is 48 hours after the FJAT-47648 strain is cultured in the environment of high temperature 60 DEG C>3×107cfu/mL. The bacillus FJAT-47648 has better high temperature resistance, and is particularly beneficial to compost fermentation.
TABLE 2 test results of high temperature resistance of the strains
EXAMPLE 2 growth promoting action of Bacillus
1. Tomato growth promotion test method
Selecting single colony of test strain, inoculating into 250mL conical flask containing 100mL LB liquid culture, and culturing at 30 deg.C for 48h (counting bacteria under microscope, bacterial density up to 10)8cfu/mL or more). Test bacteria fermentation liquor is diluted according to gradient, bacteria liquid is diluted 1400 times, 1200 times, 1000 times, 800 times and 600 times, and clear water is used as a control (ck).
Selecting tomato seeds with relatively consistent growth vigor, placing the tomato seeds in a transparent culture box of 9cm with 2-3 layers of filter paper laid at the bottom, placing 15 mung bean seeds in a constant-temperature artificial climate box at 27 ℃, and illuminating for 16h and dark for 8 h. Tracking and observing the tomato germination condition, recording the germination number until no new germination grains appear in 3d continuously, measuring the germ length of the tomato germination radicle machine, and analyzing the promoting effect of the test bacteria on tomato seed germination.
The germination percentage is%
The germination index ∑ (Gt/Dt), where Gt is the number of germinating seeds at t d and Dt is the corresponding number of days of germination.
Vigor index is germination index multiplied by embryo root length (cm)
The test data adopts DPS software and a new double-polarization method (Duncan) to carry out the significance test of the data difference among treatments.
2. Tomato seedling growth promotion test method
Inoculating the test strain into LB liquid medium, culturing at 30 deg.C under 170rpm for 48 hr until the density is about 0.8 × 108-1.2×108CFU/mL(OD600nmAbout 0.8), diluting the bacterial liquid by 100 times, and placing the diluted bacterial liquid in a refrigerator at 4 ℃ for later use.
And (4) selecting tomato seedlings with similar growth vigor, and transplanting the tomato seedlings into flowerpots, wherein each pot contains 4 plants. Test group B-6 and control group CK-W were set, and 20 strains (5 pots) were placed in each treatment group. After indoor planting for 7d, performing fermentation liquor root irrigation treatment, and irrigating 200mL of fermentation liquor diluted by 100 times per basin every week. The plants were watered every 2 days during the growth period, and 100mL Hoagland's solution per pot was poured every 2 weeks.
The experiments were performed in a smart greenhouse. The temperature of day and night is respectively 25 deg.C/20 deg.C, the sunshine time is 14h, and the diameter of the basin is 14 cm. 1kg of vegetable planting substrate sterilized at 121 ℃ is filled in each pot.
After 35d, the plant height, stem thickness (stem diameter), root length, fresh weight, dry weight of root, fresh weight of aerial parts, dry weight of aerial parts of each group of tomato plants were measured.
3. Test results
3.1 growth-promoting results of tomato seeds
The experimental results show that the bacterial liquid with 1000 times dilution has better growth promotion effect, namely the bacterial concentration of the bacillus FJAT-47648 is about 1-1.5 × 105cfu/mL, germination index and seed vitality index with 1000-fold dilution concentration are obviously different from those of a control group, and are higher than those of other test groups in germination index, root length, stem length,Seed vigor indices were 111.11%, 125.42%, 113.50%, 115.84%, 142.36% of ck control, respectively. Therefore, tomato seeds are soaked in the fermentation liquor of the bacillus FJAT-47648, so that germination can be promoted and growth can be promoted.
TABLE 3 growth promoting ability of Bacillus FJAT-47648 on tomato seeds
Note: in the above table, abcd represents significant difference (P <0.05)
3.2 growth-promoting results of tomato seedlings
The growth promotion experiment results of the bacillus on tomato seedlings are shown in table 4, and fig. 6, fig. 7, fig. 8 and fig. 9. Experimental results show that after the bacillus FJAT-47648 bacterial solution is applied, the root weight, stem thickness, root length, root fresh weight, dry weight and overground part dry weight and fresh weight of tomato seedlings are all improved compared with those of a control group.
Compared with a clear water control group (CK-W), the tomato root length of the tomato of the treatment group (B-6) applied with the bacterial liquid is obviously increased by 46.39%, the plant height and the stem thickness have no obvious difference, and the dry weight of the root, the fresh weight of the overground part and the dry weight of the overground part are respectively and obviously increased by 60.26%, 54.17% and 74.76% compared with the control group. The fermentation liquor of the bacillus FJAT-47648 has a certain growth promotion effect on tomato seedlings, and is mainly shown in the promotion of the growth and development of the roots of the tomato seedlings.
TABLE 4 growth promoting action of Bacillus FJAT-47648 on tomato
Note: different lower case letters after the same column of data in the table indicate that the difference reaches a significance level (P < 0.05).
In the practical application process, the fermentation liquor can be diluted by at least 1000 times, namely the bacterial concentration is 1 × 105-1.5×105cfu/mL soaking plant seeds to promote germination and growth of the seeds; or the fermentation liquor diluted by 100 times, namely the bacteria concentration, is about 106And (3) irrigating roots by cfu/mL to treat the plant seedlings to promote the enrichment of nutrient substances in the plant root systems, so that the vigorous growth of the plant seedlings is promoted. On the other hand, the bacillus FJAT-47648 can resist high temperature, can be used as a microbial fermentation inoculant to be applied to composting treatment of agricultural wastes, and can produce a bio-organic fertilizer.
It should be noted that, although the above embodiments have been described herein, the invention is not limited thereto. Therefore, based on the innovative concepts of the present invention, the technical solutions of the present invention can be directly or indirectly applied to other related technical fields by making changes and modifications to the embodiments described herein, or by using equivalent structures or equivalent processes performed in the content of the present specification and the attached drawings, which are included in the scope of the present invention.
Sequence listing
<110> institute of agricultural biological resources of academy of agricultural sciences of Fujian province
<120> heat-resistant bacillus capable of promoting growth and dissolving phosphorus and application thereof
<130>47648
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>1424
<212>DNA
<213> Bacillus toyonensis (Bacillus toyonensis)
<400>1
atacatgcag tcgagcgaat ggattgagag cttgctctca agaagttagc ggcggacggg 60
tgagtaacac gtgggtaacc tgcccataag actgggataa ctccgggaaa ccggggctaa 120
taccggataa cattttgaac tgcatggttc gaaattgaaa ggcggcttcg gctgtcactt 180
atggatggac ccgcgtcgca ttagctagtt ggtgaggtaa cggctcacca aggcaacgat 240
gcgtagccga cctgagaggg tgatcggcca cactgggact gagacacggc ccagactcct 300
acgggaggca gcagtaggga atcttccgca atggacgaaa gtctgacgga gcaacgccgc 360
gtgagtgatg aaggctttcg ggtcgtaaaa ctctgttgtt agggaagaac aagtgctagt 420
tgaataagct ggcaccttga cggtacctaa ccagaaagcc acggctaact acgtgccagc 480
agccgcggta atacgtaggt ggcaagcgtt atccggaatt attgggcgta aagcgcgcgc 540
aggtggtttc ttaagtctga tgtgaaagcc cacggctcaa ccgtggaggg tcattggaaa 600
ctgggagact tgagtgcaga agaggaaagt ggaattccat gtgtagcggt gaaatgcgta 660
gagatatgga ggaacaccag tggcgaaggc gactttctgg tctgtaactg acactgaggc 720
gcgaaagcgt ggggagcaaa caggattaga taccctggta gtccacgccg taaacgatga 780
gtgctaagtg ttagagggtt tccgcccttt agtgctgaag ttaacgcatt aagcactccg 840
cctggggagt acggccgcaa ggctgaaact caaaggaatt gacgggggcc cgcacaagcg 900
gtggagcatg tggtttaatt cgaagcaacg cgaagaacct taccaggtct tgacatcctc 960
tgaaaaccct agagataggg cttctccttc gggagcagag tgacaggtgg tgcatggttg 1020
tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttgatct 1080
tagttgccat cattaagttg ggcactctaa ggtgactgcc ggtgacaaac cggaggaagg 1140
tggggatgac gtcaaatcat catgcccctt atgacctggg ctacacacgt gctacaatgg 1200
acggtacaaa gagctgcaag accgcgaggt ggagctaatc tcataaaacc gttctcagtt 1260
cggattgtag gctgcaactc gcctacatga agctggaatc gctagtaatc gcggatcagc 1320
atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgagagttt 1380
gtaacacccg aagtcggtgg ggtaaccttt atggagccag ccgc 1424
Claims (8)
1. A heat-resistant bacillus capable of promoting growth and dissolving phosphorus, which is characterized in that: the Bacillus is Penicillium Tuwense FJAT-47648, has a scientific name of Bacillus toyonensis FJAT-47648, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, has a preservation number of CGMCC NO.16252, has a preservation date of 2018, 8 and 13 days, and has a preservation address of the institute of microorganisms of China academy of sciences, Beijing, China.
2. The phosphate-solubilizing thermotolerant bacillus according to claim 1, wherein: the fermentation method of the bacillus comprises the following steps: inoculating bacillus into an LB liquid culture medium, and performing shaking culture at 25-35 ℃ for 48-72h to obtain a seed solution; then inoculating the seed liquid into LB liquid culture medium, and culturing for 48-72h under shaking at 30-60 deg.C.
3. Use of the phosphate-solubilizing thermotolerant bacillus strain of claim 1 for degrading a poorly soluble phosphate.
4. Use of the phosphorus-solubilizing thermotolerant bacillus of claim 1 for promoting germination of a plant seed.
5. The use of the Bacillus thermotolerant for promoting phosphorus release according to claim 4, wherein the Bacillus fermentation broth is formulated to a concentration of 1 × 105-1.5×105cfu/mL bacterial suspension, soaking seeds for 2-3 days, placing at 25-30 deg.C, and illuminating for 16-20 h/day.
6. Use of the phosphorus-solubilizing thermotolerant bacillus of claim 1 for promoting the growth of young plants.
7. The use of the P-solubilizing thermotolerant bacillus according to claim 6, wherein the bacillus fermentation broth is prepared to a concentration of 0.8 × 106-1.2×106cfu/mL of bacterial suspension; planting the plant seedlings in a planting matrix indoors for 6-8 days, and then irrigating roots by using bacterial suspension once every 5-7 days.
8. A plant growth promoting microbial inoculum is characterized in that: the microbial inoculum comprises the bacillus of claim 1.
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Application publication date: 20200821 |