CN114231425A - Phosphorus-dissolving potassium-solubilizing bacterium Aspergillus niger Z8 and application thereof - Google Patents
Phosphorus-dissolving potassium-solubilizing bacterium Aspergillus niger Z8 and application thereof Download PDFInfo
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- CN114231425A CN114231425A CN202210037031.9A CN202210037031A CN114231425A CN 114231425 A CN114231425 A CN 114231425A CN 202210037031 A CN202210037031 A CN 202210037031A CN 114231425 A CN114231425 A CN 114231425A
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- 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
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- C12N1/14—Fungi; Culture media therefor
Abstract
The invention discloses a phosphorus and potassium solubilizing bacterium Aspergillus niger Z8 and application, relates to the field of microbiology, and aims to provide a strain with a phosphorus and potassium solubilizing function. The phosphate-solubilizing potassium-solubilizing bacterium Aspergillus niger (Aspergillus niger) Z8 is preserved in China center for type culture Collection with the preservation address: china, wuhan university, date of preservation: 12/3/2021, accession number: CCTCC No. M20211526. It is used as promoter for dissolving phosphorus and potassium to promote crop growth. The invention is applied to the field of agriculture.
Description
Technical Field
The invention belongs to the field of microbiology, and particularly relates to a phosphorus-solubilizing potassium-solubilizing bacterium Aspergillus niger Z8 and application thereof.
Background
Phosphorus and potassium are essential nutrient elements for crop growth, and phosphorus can participate in important physiological and biochemical processes such as plant respiration, cell division, energy conversion, biosynthesis and the like. Potassium can activate various enzymes in plants, regulate photosynthetic rate, promote the production of Adenosine Triphosphate (ATP) and saccharides and regulate stomatal movement, and is an important signal factor for regulating adaptive response of plants to abiotic stresses such as drought, salinity, oxidation stress, apoptosis and the like. Phosphorus and potassium in soil are main sources of phosphorus and potassium required by plants, but the phosphorus and potassium in the soil have various forms, and the effectiveness of different forms on crops is different. More than 95% of phosphorus element in soil exists in ineffective forms such as insoluble phosphate and the like, and can not be directly absorbed and utilized by plants. More than 90% of potassium in soil exists in silicate minerals such as potassium feldspar and mica, which are insoluble and need to be weathered for a long time to release potassium.
In order to promote the growth of crops and improve the quality of agricultural products, phosphate fertilizers and potash fertilizers are applied to most of the crops, and the chemical fertilizers have the problems of low utilization rate, waste of mineral resources, acidification and hardening of soil, reduction of organic matter content, eutrophication of water bodies and the like. Phosphorus and potassium nutrients which can be directly utilized by crops in cultivated land soil in China are seriously deficient, and the ecological balance and the environmental protection are influenced while a large amount of chemical fertilizers are applied, so that the development of agriculture is restricted. The microbial fertilizer can promote plant growth, improve plant stress resistance, effectively relieve soil and water pollution, and has good application value and ecological benefit. The phosphorus-dissolving potassium-dissolving bacteria can convert insoluble or insoluble phosphorus and potassium in soil into effective phosphorus and potassium which can be absorbed and utilized by plants, but at present, the separation of strains which can be used as microbial fertilizers is less, the research mostly takes single function as the main part, and the research report on the strains which have the phosphorus-dissolving potassium-dissolving function is rare.
Disclosure of Invention
The invention aims to provide a bacterial strain with the functions of dissolving phosphorus and potassium. Provides a phosphorus-dissolving potassium-dissolving bacterium Aspergillus niger Z8 and application thereof.
The invention discloses a potassium phosphate solubilizing bacterium Aspergillus niger Z8, which is preserved in China center for type culture Collection with the preservation address: china, wuhan university, date of preservation: 12/3/2021, accession number: CCTCC No. M20211526.
The invention discloses application of a phosphorus-dissolving potassium-dissolving bacterium Aspergillus niger Z8, which is used as a promoter for dissolving phosphorus and potassium to promote crop growth.
Further, the crop is soybean.
Further, the preparation method of the accelerant comprises the following steps:
the concentration is 1 x 107cuf/mL of Aspergillus niger Z8 spore suspension is inoculated into Aspergillus niger fermentation liquor according to the inoculation amount of 1%, and the fermentation liquor is cultured for 5d at 28 ℃ to obtain the promoter; the Aspergillus niger fermentation liquid is: glucose 10g, (NH)4)2SO4 0.1g、MgCl2·6H2O 5g、Ca3(PO4)25g, 2.5g of potassium feldspar powder and 1000mL of distilled water; pH 7.0.
Further, the using method of the accelerator comprises the following steps:
placing soybean seeds in the same environment for culturing and sprouting, placing 3 soybean sprouts into a 50mL culture bottle, adding 30mL soil immersion liquid into each bottle, keeping the concentration of Aspergillus niger fermentation liquid at 1.0%, and maintaining illumination culture.
Further, the using method of the accelerator comprises the following steps:
soaking soybeans in sterile water for 2h, digging out cultivation pits in soil, adding a spore suspension, covering with a layer of soil, putting the soaked soybean seeds, covering with a layer of soil, and keeping the soil moist to finish the process; the spore suspension is 1 × 107cuf/mL of a spore suspension of Aspergillus niger Z8; wherein 2mL of spore suspension is added per 1 soybean seed.
Further, the using method of the accelerator comprises the following steps:
soaking soybeans in sterile water for 2 hours, digging out cultivation pits in soil, adding 2mL of spore suspension mixture, covering with a layer of soil, putting soaked soybean seeds, covering with a layer of soil, and keeping the soil moist to finish the process; the spore suspension mixture is prepared by mixing spore suspension with concentration of 1 × 107cuf/mL of spore suspension of Aspergillus niger Z8, calcium phosphate with mass concentration of 0.5% and potassium feldspar powder with mass concentration of 1.5%; wherein 2mL of spore suspension is added per 1 soybean seed.
Further, the using method of the accelerator comprises the following steps:
soaking soybeans in sterile water for 2h, digging out cultivation pits in soil, adding the small balls, covering with a layer of soil, putting the soaked soybean seeds, covering with a layer of soil, and keeping the soil moist to finish the process; the pellet is prepared from 1 × 107cuf/mL of spore suspension of Aspergillus niger Z8, sodium alginate with the mass concentration of 4.5%, bone charcoal powder with the mass concentration of 1% and silicon dioxide with the mass concentration of 2.5% are mixed, the mixed solution is dripped into a calcium chloride solution with the mass concentration of 1% to obtain small balls with the diameter of 3-5 mm, and the small balls are fixed in a refrigerator at 4 ℃ for 4 hours; wherein 2g of spore suspension is added per 1 soybean seed.
Further, the using method of the accelerator comprises the following steps:
soaking soybeans in sterile water for 2h, digging out cultivation pits in soil, adding the small balls, covering with a layer of soil, putting the soaked soybean seeds, covering with a layer of soil, and keeping the soil moist to finish the process; the slow releasing agent of spore suspension is prepared with spore suspension in the concentration of 1 × 107cuf/mL of Aspergillus niger Z8 spore suspension, 4.5% of sodium alginate by mass concentration, 1% of bone charcoal powder by mass concentration, 2.5% of silicon dioxide by mass concentration, 0.5% of calcium phosphate by mass concentration and 1.5% of potassium feldspar powder by mass concentration are mixed, the mixed solution is dripped into a 1% calcium chloride solution by mass concentration to obtain small balls with the diameter of 3-5 mm, and the small balls are fixed in a refrigerator at 4 ℃ for 4 hours; wherein 2g of spore suspension is added per 1 soybean seed.
The method takes the democratic demonstration area of the academy of agricultural sciences of Heilongjiang province as a sampling point, collects soybean rhizosphere soil, puts the soybean rhizosphere soil into a sterilization sample bag, uses an ice bag fresh-keeping ash-carrying laboratory, screens and air-dries the collected soil, and then separates, screens and purifies soybean rhizosphere microorganisms:
one, gradient dilution coating method
Weighing 10g of rhizosphere soil, putting the rhizosphere soil into a triangular flask containing 90mL of sterile distilled water, and performing shake culture at 28 ℃ and 150r/min for 1 d. The bacterial suspension is sucked by a pipette gun in a clean bench and sequentially diluted into sterilized test tubes filled with 9mL of distilled water, and the dilution gradient isAre respectively 10-2、10-3、10-4. Shaking the diluted test tube, sucking 100 μ L, adding into PDA solid culture medium, coating, waiting until the surface liquid of the solid culture medium is dry, placing the plate in a constant temperature incubator at 28 deg.C, culturing for 3-4d, and observing colony growth.
Secondly, separation and purification of fungi
Selecting a diluted coating flat plate with clear bacterial colonies, picking mycelium by using an inoculating loop in an ultraclean workbench, carrying out four-area lineation on a PDA solid culture medium, placing the flat plate in a constant-temperature incubator at 28 ℃ for culturing for 4-5d, and observing the growth condition of single bacterial colonies in four areas. Taking a four-area plate with clear single colony, selecting mycelium, carrying out one-area streaking on a PDA solid culture medium, putting the one-area streaking plate into a constant-temperature incubator at 28 ℃ for culturing for 4-5d, observing whether the strain is a purified strain after the strain grows out from the one-area streaking plate, and repeating the steps if the strain is not purified.
Screening of phosphate-solubilizing potassium-solubilizing bacteria
And (3) selecting a purified strain, inoculating the purified strain on a phosphorus-dissolved solid culture medium, and carrying out inversion constant-temperature culture at 28 ℃ for 5 d. Observing the growth state of the strain, measuring the diameter (D) of the transparent ring and the diameter (D) of the bacterial colony, calculating the ratio of D/D, and obtaining the strain with the transparent ring, namely the phosphorus-dissolving strain. Selecting purified phosphorus-dissolving strains, inoculating the strains on a potassium-dissolving solid culture medium, and carrying out inversion constant-temperature culture at 28 ℃ for 5 d. Observing the growth state of the bacillus subtilis, measuring the diameter (D) of the transparent ring and the diameter (D) of the bacterial colony, calculating the D/D ratio, if the transparent ring appears, indicating that the bacterial strain is a phosphate-solubilizing potassium-solubilizing bacterium, and screening out the bacterium with better effect according to the growth speed of the bacterial strain and the D/D ratio to carry out the following experiment.
A phosphate-and potassium-solubilizing liquid medium was placed in a 50mL Erlenmeyer flask, and the screened strains were used to prepare spore suspensions (concentration: 1X 10)7) Inoculating 1% of the extract into a phosphorus-potassium-dissolving liquid culture medium, performing shake culture for 8d, after the culture is finished, diluting a filtrate obtained by centrifugal (8000r/min, 10min) separation, measuring the content of soluble phosphorus in the liquid by a molybdenum-antimony colorimetric method, putting a solid phase (mineral and fungal hyphae) obtained by centrifugal back into a triangular flask, and adding 100mL of NH (NH) with the concentration of 1mol/L4Ultrasonic oscillation is carried out for 30min after Ac, secondary centrifugation is carried out (8000r/min,10min), and measuring the obtained filtrate and leachate by an inductively coupled plasma emission spectrometer (ICP-OES).
Identification of phosphate-solubilizing potassium-solubilizing bacteria
Observation of the morphological characteristics of strain Z8: the colony becomes black, thick velvet and round after being initially white; the diameter is 28-35 mm, the color of the mycelium is black, and the sporophyte is spherical and black brown; with a pale yellow exudate.
After the strain is activated, the strain is ground by liquid nitrogen, and then total DNA of the fungus is extracted by using PCR universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') of the ITS sequence of the fungus. The PCR amplification product is sent to Nanchangkong Biotech limited for sequencing. And performing BLAST comparison on the obtained sequencing result in a GenBank database, selecting a sequence with higher homology, and constructing a phylogenetic tree by applying MEGA7.0 software. Homology comparisons were made with GenBank sequences using Clustal software (FIG. 1). The strain Z8 has 100% homology with Aspergillus niger (Aspergillus niger) and the strain Z8 is Aspergillus niger (Aspergillus niger) as identified by morphology and ITS sequence.
The invention has the following beneficial effects:
the Aspergillus niger Z8 screened by the method has the functions of dissolving phosphorus and potassium, can promote the growth of soybeans, reduce the using amount of chemical fertilizers and improve the soil fertility, and has important significance in agricultural production.
Drawings
FIG. 1 is a phylogenetic tree diagram of strain Z8; in the figure, MK271296: Z8; KJ778683.1 Aspergillus niger;
FIG. 2 is a graph of phosphorus dissolution and potassium dissolution data of the Z8 strain.
Detailed Description
For the purpose of promoting a clear understanding of the objects, aspects and advantages of the embodiments of the invention, reference will now be made in detail to the embodiments of the present disclosure, and it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the disclosure.
The exemplary embodiments of the present invention and the description thereof are provided to explain the present invention and not to limit the present invention.
Examples
A water culture experiment is adopted to verify the growth promotion effect of Aspergillus niger Z8 on soybean, CK is set as a control group, only soil immersion liquid is added, a treatment group is implemented by the scheme of claim 5, and bacterial liquids with different concentrations are set for treatment.
Water culture experiments prove that (Table 1) Aspergillus niger Z8 can obviously promote the growth of soybeans, wherein the growth promoting effect is the best at the concentration of 1.0%.
TABLE 1 treatment of Soybean seedling Biomass with different bacterial solutions
A pot experiment is adopted to verify the growth promotion effect of Aspergillus niger Z8 on soybean, CK is set as a control group, soybean seeds are soaked in sterile water for 2 hours, 6 soybean seeds are planted in each pot, and the soil is kept moist.
Treatment group T1 was:
soaking soybeans in sterile water for 2h, digging out cultivation pits in soil, adding a spore suspension, covering with a layer of soil, putting the soaked soybean seeds, covering with a layer of soil, and keeping the soil moist to finish the process; the spore suspension is 1 × 107cuf/mL of a spore suspension of Aspergillus niger Z8; wherein 2mL of spore suspension is added per 1 soybean seed.
Treatment group T2 was:
soaking soybeans in sterile water for 2 hours, digging out cultivation pits in soil, adding 2mL of spore suspension mixture, covering with a layer of soil, putting soaked soybean seeds, covering with a layer of soil, and keeping the soil moist to finish the process; the spore suspension mixture is prepared by mixing spore suspension with concentration of 1 × 107 cuf/mL of a spore suspension of Aspergillus niger Z8, calcium phosphate with the mass concentration of 0.5% and potassium feldspar powder with the mass concentration of 1.5% are mixed to form the fertilizer; wherein 2mL of spore suspension is added per 1 soybean seed.
Treatment group T3 was:
soaking soybeans in sterile water for 2h, digging out cultivation pits in soil, adding immobilized pellets, covering with a layer of soil, putting the soaked soybean seeds, covering with a layer of soil, and keeping the soil moist to finish the process; the immobilized beads are prepared from the components with the concentration of 1 multiplied by 107cuf/mL of spore suspension of Aspergillus niger Z8, sodium alginate with the mass concentration of 4.5%, bone charcoal powder with the mass concentration of 1% and silicon dioxide with the mass concentration of 2.5% are mixed, the mixed solution is dripped into a calcium chloride solution with the mass concentration of 1% to obtain small balls with the diameter of 3-5 mm, and the small balls are fixed in a refrigerator at 4 ℃ for 4 hours; wherein 2g of spore suspension is added per 1 soybean seed.
Treatment group T4 was:
soaking soybeans in sterile water for 2h, digging out cultivation pits in soil, adding the small balls, covering with a layer of soil, putting the soaked soybean seeds, covering with a layer of soil, and keeping the soil moist to finish the process; the slow releasing agent of spore suspension is prepared with spore suspension in the concentration of 1 × 107cuf/mL of Aspergillus niger Z8 spore suspension, 4.5% of sodium alginate by mass concentration, 1% of bone charcoal powder by mass concentration, 2.5% of silicon dioxide by mass concentration, 0.5% of calcium phosphate by mass concentration and 1.5% of potassium feldspar powder by mass concentration are mixed, the mixed solution is dripped into a 1% calcium chloride solution by mass concentration to obtain small balls with the diameter of 3-5 mm, and the small balls are fixed in a refrigerator at 4 ℃ for 4 hours; wherein 2g of spore suspension is added per 1 soybean seed.
Potted plant experiments prove that (table 2), the Aspergillus niger Z8 can obviously promote the growth of soybean and obviously improve the plant height, root length, fresh weight, dry weight, soluble sugar and soluble protein content (p is less than 0.05) of soybean plants.
TABLE 2 Effect of Aspergillus niger on Soybean growth
The Aspergillus niger Z8 of this example was tested to dissolve phosphorus and potassium, and it was found that Aspergillus niger Z8 was found to have a good effect of dissolving phosphorus and potassium, the phosphorus dissolving amount was 363.17mg/L and the potassium dissolving amount was 104.63 mg/L. The Aspergillus niger Z8 screened by the method has the functions of dissolving phosphorus and potassium, can promote the growth of soybeans, reduce the using amount of chemical fertilizers and improve the soil fertility, and has important significance in agricultural production.
Claims (9)
1. A phosphorus-dissolving potassium-solubilizing bacterium Aspergillus niger Z8 is characterized in that the Aspergillus niger Z8 is deposited in China center for type culture Collection with the deposition address: china, wuhan university, date of preservation: 12/3/2021, accession number: CCTCC No. M20211526.
2. The application of the potassium phosphate solubilizing bacterium aspergillus niger Z8 as claimed in claim 1, which is used as an accelerant of the potassium phosphate solubilizing bacterium to promote the growth of crops.
3. The application of the potassium phosphate solubilizing bacterium aspergillus niger Z8 as claimed in claim 2, wherein the crop is soybean.
4. The application of the potassium phosphate solubilizing bacterium aspergillus niger Z8 as claimed in claim 2, wherein the preparation method of the accelerant is as follows:
the concentration is 1 x 107cuf/mL of Aspergillus niger Z8 spore suspension is inoculated into Aspergillus niger fermentation liquor according to the inoculation amount of 1%, and the fermentation liquor is cultured for 5d at 28 ℃ to obtain the promoter; the Aspergillus niger fermentation liquid is: glucose 10g, (NH)4)2SO4 0.1g、MgCl2·6H2O 5g、Ca3(PO4)25g, 2.5g of potassium feldspar powder and 1000mL of distilled water; pH 7.0.
5. The application of the potassium phosphate solubilizing bacterium aspergillus niger Z8 as claimed in claim 2 or 4, wherein the promoter is used by the following method:
placing soybean seeds in the same environment for culturing and sprouting, placing 3 soybean sprouts into a 50mL culture bottle, adding 30mL soil immersion liquid into each bottle, keeping the mass concentration of Aspergillus niger fermentation liquid at 1.0%, and maintaining illumination culture.
6. The application of the potassium phosphate solubilizing bacterium aspergillus niger Z8 as claimed in claim 2 or 4, wherein the promoter is used by the following method:
soaking soybeans in sterile water for 2h, digging out cultivation pits in soil, adding a spore suspension, covering with a layer of soil, putting the soaked soybean seeds, covering with a layer of soil, and keeping the soil moist to finish the process; the spore suspension is 1 × 107cuf/mL of a spore suspension of Aspergillus niger Z8; wherein 2mL of spore suspension is added per 1 soybean seed.
7. The application of the potassium phosphate solubilizing bacterium aspergillus niger Z8 as claimed in claim 2 or 4, wherein the promoter is used by the following method:
soaking soybeans in sterile water for 2 hours, digging out cultivation pits in soil, adding 2mL of spore suspension mixture, covering with a layer of soil, putting soaked soybean seeds, covering with a layer of soil, and keeping the soil moist to finish the process; the spore suspension mixture is prepared by mixing spore suspension with concentration of 1 × 107cuf/mL of spore suspension of Aspergillus niger Z8, calcium phosphate with mass concentration of 0.5% and potassium feldspar powder with mass concentration of 1.5%; wherein 2mL of spore suspension is added per 1 soybean seed.
8. The application of the potassium phosphate solubilizing bacterium aspergillus niger Z8 as claimed in claim 2 or 4, wherein the promoter is used by the following method:
soaking soybean in sterile water for 2 hr, digging out cultivation pit in soil, adding immobilized pellet, covering with a layer of soil, adding soaked soybean seed, and covering with a layer of soilKeeping the soil moist, and then completing; the immobilized beads are prepared from the components with the concentration of 1 multiplied by 107cuf/mL of spore suspension of Aspergillus niger Z8, sodium alginate with the mass concentration of 4.5%, bone charcoal powder with the mass concentration of 1% and silicon dioxide with the mass concentration of 2.5% are mixed, the mixed solution is dripped into a calcium chloride solution with the mass concentration of 1% to obtain small balls with the diameter of 3-5 mm, and the small balls are fixed in a refrigerator at 4 ℃ for 4 hours; wherein 2g of spore suspension is added per 1 soybean seed.
9. The application of the potassium phosphate solubilizing bacterium aspergillus niger Z8 as claimed in claim 2 or 4, wherein the promoter is used by the following method:
soaking soybeans in sterile water for 2h, digging out cultivation pits in soil, adding the small balls, covering with a layer of soil, putting the soaked soybean seeds, covering with a layer of soil, and keeping the soil moist to finish the process; the slow releasing agent of spore suspension is prepared with spore suspension in the concentration of 1 × 107cuf/mL of Aspergillus niger Z8 spore suspension, 4.5% of sodium alginate by mass concentration, 1% of bone charcoal powder by mass concentration, 2.5% of silicon dioxide by mass concentration, 0.5% of calcium phosphate by mass concentration and 1.5% of potassium feldspar powder by mass concentration are mixed, the mixed solution is dripped into a 1% calcium chloride solution by mass concentration to obtain small balls with the diameter of 3-5 mm, and the small balls are fixed in a refrigerator at 4 ℃ for 4 hours; wherein 2g of spore suspension is added per 1 soybean seed.
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