CN110791442A - Geobacillus altivelis with phosphate solubilizing function and application thereof - Google Patents

Abstract

The invention discloses a highland bacillus with a phosphate solubilizing function and application thereof, wherein the highland bacillus is preserved in China general microbiological culture Collection center in 2019, 3 and 18 months, the preservation number is CGMCC No.17338, and an LB liquid culture medium adopted for laboratory liquid culture and a Monkina inorganic phosphate solid culture medium adopted for phosphate solubilizing screening are adopted. Experiments show that the effective phosphorus concentration in the blank control solution without inoculation is far lower than that in the fermentation liquor of the bacillus altitudinis SM01, namely the bacillus altitudinis SM01 is a high-efficiency phosphate-solubilizing fungus.

Description

Geobacillus altivelis with phosphate solubilizing function and application thereof

Technical Field

The invention relates to the field of phosphate-solubilizing microbial fertilizers, in particular to bacillus altitudinis with a phosphate-solubilizing function and application thereof.

Background

The phosphorus element is the second important element for the growth and development of plants after the nitrogen element, accounts for about 0.2% of the dry weight of the plants, is a main component participating in energy metabolism, nucleic acid and cell synthesis and partial enzyme regulation, plays an important role in promoting the growth and development and metabolism of the plants, and is an irreplaceable component in an ecosystem. Statistics data show that 40% of cultivated lands in the world have phosphorus deficiency, and 74% of cultivated lands in China have phosphorus deficiency, and the important reason is that due to immobilization, phosphorus in the soil mainly exists in insoluble compounds, most of the phosphorus cannot be directly absorbed and utilized by plants, and only 0.1% of the phosphorus can be directly absorbed and utilized by the plants. Although the application of the phosphate fertilizer can relieve the deficiency of phosphorus to a certain extent, 75 to 90 percent of phosphorus in the phosphate fertilizer can be quickly adsorbed to the surface of soil particles or combined with metal ions (Fe, Al, Ca and the like) in the soil to generate insoluble phosphate. Therefore, even if a large amount of chemical phosphate fertilizer is applied, the problem of phosphorus deficiency of soil cannot be completely solved.

The phosphorus-dissolving microorganism can convert the indissolvable combined phosphorus in the soil into soluble phosphorus which can be absorbed and utilized by plants, and can improve the utilization rate of phosphorus elements in the soil. At present, the reported microorganisms with the phosphate solubilizing capability are various and comprise bacteria, fungi, actinomycetes and blue algae, wherein the number of the bacteria with the phosphate solubilizing capability accounts for 1 to 50 percent of the total number of the microorganisms in the soil, the number of the actinomycetes with the phosphate solubilizing capability accounts for 10 to 50 percent of the total number of the microorganisms in the soil, and the number of the fungi with the phosphate solubilizing capability only accounts for 0.1 to 0.5 percent of the total number of the microorganisms in the soil. The phosphate-solubilizing microorganisms of different species have great difference in phosphate-solubilizing ability and are mainly influenced by the genetic characteristics and growth environment of strains. The phosphorus-dissolving mechanism of the phosphorus-dissolving microorganisms is very complex, mainly organic acid acts, and some bacterial strains generate other chelate substances in the growth process, so that the phosphorus ore powder is dissolved. Besides the function of phosphate solubilizing, the phosphate solubilizing bacteria also have the functions of nitrogen fixation, plant hormone production, iron carrier production, antibiotic secretion and the like, thereby promoting the production of crops, increasing the yield of the crops and improving the disease resistance of the crops.

Disclosure of Invention

The invention aims to provide highland bacillus with the phosphate solubilizing function and application thereof, which can solve the problem that a large amount of phosphorus in soil cannot be directly absorbed and utilized by plants by utilizing the efficient phosphate solubilizing function of the highland bacillus, can effectively relieve the current situation of phosphorus deficiency in the soil by utilizing the phosphorus in a fixed state and an adsorption state, and has important practical significance for improving the utilization rate of phosphate fertilizer, reducing the environmental pollution caused by the use of the phosphate fertilizer and even improving the yield and the quality of crops.

The Bacillus altitudinis is preserved in China general microbiological culture collection and management Committee center at 18.3.2019, the preservation number is CGMCC No.17338, the preservation date is 18.3.2019, the Bacillus altitudinis is classified and named, the preservation unit is the China general microbiological culture collection and management Committee center, and the address is No. 3 North Chen Lu No.1 of the Korean district, Beijing, China.

Preferably, the laboratory liquid culture adopts LB liquid culture medium and the phosphate removing bacteria screening adopts Monkina inorganic phosphorus solid culture medium:

the LB liquid culture medium adopted by the laboratory liquid culture comprises the following components in percentage by mass:

5-15 g of tryptone;

3-16 g of yeast extract;

8-15 g of sodium chloride;

800-1200mL of distilled water;

the Monkina inorganic phosphorus solid culture medium formula adopted by screening the phosphate solubilizing bacteria comprises the following components in percentage by mass:

5-15 g of glucose;

(NH4)2SO4 0.3-0.6g；

NaCl 0.2-0.5 g；

KCl 0.2-0.5g；

MgSO4·7H2O 0.2-0.5g；

FeSO4·7H2O 0.02-0.05g；

MnSO4·4H2O 0.02-0.04 g；

Ca3(PO3)2 8-12 g；

800 ml of deionized water and 1300ml of deionized water.

Preferably, the laboratory liquid culture adopts LB liquid culture medium with pH adjusted to 6.8-7.0, and the phosphorus-solubilizing bacteria screening adopts Monkina inorganic phosphorus solid culture medium with pH adjusted to 6.9-7.1.

A microbial agent prepared by Bacillus altitudinis SM01 and having a phosphate solubilizing function, wherein the microbial agent comprises: the solid culture medium comprises a solid culture medium formula and a mass fermentation culture formula, wherein the solid culture medium formula and the mass fermentation culture formula have the same components, and comprise a solid material and an inorganic salt solution, and the mass ratio of the solid material to the inorganic salt is 98.7: 0.3; the solid material consists of rice hulls, corn flour, soybean meal and bran in a mass ratio of 50: 24: 20: 6; the inorganic salt comprises, by mass, 11% of monopotassium phosphate, 9% of magnesium sulfate, 15% of ammonium sulfate and 65% of light calcium carbonate.

Another technical problem to be solved by the present invention is to provide a method for preparing a geobacillus microbial agent with a phosphate solubilizing function, comprising the following steps:

s1: preparing a seed solution of the geobacillus;

s2: and (4) inoculating the seed liquid prepared by the S1 into a solid culture medium, and culturing at a constant temperature of 27-29 ℃.

S3: mixing the culture cultured in S2 with sterile water according to the mass ratio of 1: 15, filtering, inoculating the filtrate into a large amount of fermentation medium, and performing fermentation culture in a fermentation chamber at room temperature of 27-29 ℃ and relative humidity of more than 85%.

Preferably, the fermentation medium in S3 has the same composition as the solid medium.

Compared with the prior art, the invention has the beneficial effects that: experiments show that the effective phosphorus concentration in the blank control solution without inoculation is far lower than that in the fermentation liquor of the bacillus altitudinis SM01, namely the bacillus altitudinis SM01 is a high-efficiency phosphate-solubilizing fungus.

Drawings

FIG. 1 is a standard curve for determination of effective phosphorus in Bacillus altitudinis SM01 according to the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Referring to fig. 1, a bacillus altitudinis SM01 with phosphate solubilizing function, which has been deposited in the china general microbiological culture collection center at 18 months 3 and 2019 with the collection number of CGMCC No. 17338.

LB liquid culture medium adopted in the liquid culture of the laboratory and Monkina inorganic phosphorus solid culture medium adopted in the phosphorus removing bacteria screening: the pH value of an LB liquid culture medium adopted by the laboratory liquid culture is adjusted to 6.8-7.0, the pH value of an inorganic phosphorus solid culture medium of Monkina adopted by the phosphate solubilizing bacteria screening is adjusted to 6.9-7.1, and the LB liquid culture medium adopted by the laboratory liquid culture comprises the following components in percentage by mass:

5-15 g of tryptone;

3-16 g of yeast extract;

8-15 g of sodium chloride;

800-1200mL of distilled water;

the Monkina inorganic phosphorus solid culture medium formula adopted by screening the phosphate solubilizing bacteria comprises the following components in percentage by mass:

5-15 g of glucose;

(NH4)2SO4 0.3-0.6g；

NaCl 0.2-0.5 g；

KCl 0.2-0.5g；

MgSO4·7H2O 0.2-0.5g；

FeSO4·7H2O 0.02-0.05g；

MnSO4·4H2O 0.02-0.04 g；

Ca3(PO3)2 8-12 g；

800 ml of deionized water and 1300ml of deionized water.

The first embodiment is as follows:

taking raw materials:

preparing LB liquid culture medium adopted by laboratory liquid culture, wherein 5g of tryptone; 4g of yeast extract; 8g of sodium chloride; 850mL of distilled water; the pH was adjusted to 6.9.

Preparing a Monkina inorganic phosphorus solid culture medium, wherein the glucose is 10 g; (NH4)2SO 40.6 g;

NaCl 0.2 g；KCl 0.3 g；MgSO4·7H2O 0.3g；FeSO4·7H2O 0.03g；

MnSO 4.4H 2O 0.03.03 g; ca3(PO3) 210 g; 1000ml of deionized water, and the pH was adjusted to 7.0.

The raw materials are prepared into an LB liquid culture medium and a Monkina inorganic phosphorus solid culture medium.

Example two:

taking raw materials:

preparing LB liquid culture medium adopted by laboratory liquid culture, wherein the tryptone is 10 g; 5g of yeast extract; 10 g of sodium chloride; 1000mL of distilled water; the pH was adjusted to 7.

Preparing a Monkina inorganic phosphorus solid culture medium, wherein the glucose is 8 g; (NH4)2 SO40.5g;

NaCl 0.5g；KCl 0.4 g；MgSO4·7H2O 0.3g；FeSO4·7H2O 0.03g；

MnSO 4.4H 2O 0.03.03 g; ca3(PO3) 210 g; 1000ml of deionized water, and the pH was adjusted to 7.0.

The raw materials are prepared into an LB liquid culture medium and a Monkina inorganic phosphorus solid culture medium.

Example three:

taking raw materials:

LB liquid culture medium adopted for laboratory liquid culture is prepared, wherein 10 g of tryptone, 5g of yeast extract, 10 g of sodium chloride and 1000mL of distilled water are added, and the pH is adjusted to 7.0.

Preparing a Monkina inorganic phosphorus solid culture medium, wherein the glucose is 8 g; (NH4)2 SO40.5g;

NaCl 0.5g；KCl 0.4 g；MgSO4·7H2O 0.3g；FeSO4·7H2O 0.03g；

MnSO 4.4H 2O 0.03.03 g; ca3(PO3) 210 g; 1000ml of deionized water, and the pH was adjusted to 7.0.

The raw materials are prepared into an LB liquid culture medium and a Monkina inorganic phosphorus solid culture medium.

The bacillus altitudinis SM01 is respectively cultured in the culture media of the three examples, the colony shape of the strain of the bacillus altitudinis SM01 is circular, the middle part is transparent, the color is yellowish, the edges are irregular, and the culture medium prepared by the raw material ratio in the third example is the culture medium which is most suitable for the growth of the bacillus altitudinis SM 01.

The sequence determination result of the 16sDNA gene of the Bacillus altitudinis SM01 strain is as follows:

TCCAGTCGAGCGCACAGAAGAGAGCTTGCTCCCGGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGAGCTAATACCGGATAGTTCCTTGAACCGCATGGTTCAAGGATGAAAGACGGTTTCGGCTGTCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCAAGAGTAACTGCTTGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAAACTTGAGTGCAGAAGGGGAGAGTGGAATTCCAAGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTGCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACCCTAGAGATAGGGCTTTCCCTTCGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCTGCGAGACCGCAAGGTTTAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGCAACACCCGAAGTAGG。

the cell morphology of the Bacillus altitudinis SM01 and the pear flower experiment result are obtained at the same time, as shown in the following table 1

Experimental project	The result is	Experimental project	The result is
				Gram stain	Positive for	Sucrose	+
Cell shape	Rod-shaped	Lactose	+
				The diameter of the cells is more than 1 mu m	+	Cellobiose	+
Form spores	+	Cotton seed candy	+
				Movement property	+	Melibiose	－
Catalase reaction	+	Dextrin	+
				Galactitol	－	Starch	+
Sorbitol	－	Esculin	+
				Inositol	－	Urea	+
Mannitol	+	Salicin	+
				Glucose	+	Citric acid	+
Glucose gas production	+	Glucose phosphate	－
				Fructose	+	Acetate salt	+
Mannose	+	Nitrate reductase	－
				Rhamnose	－	6%NaCl	+
Xylose	+	8%NaCl	+
				Arabinose	－	10%NaCl	+

Table 1 cell morphology and results of physicochemical experiments for bacillus altitudinis SM 01.

Determination of phosphate-solubilizing ability of bacillus altitudinis SM 01:

the phosphate solubilizing capability of phosphate solubilizing bacteria is measured by adopting a Monkina inorganic phosphate liquid culture medium, the Geobacillus altivelis SM01 stored on a slope is inoculated into an LB liquid culture medium as a seed, the seed is subjected to shake culture for 12 hours in a constant temperature shaking table at 30 ℃ and 180 r/min, then the cultured bacteria liquid is inoculated into a 250 mL triangular flask containing 100 mL Monkina inorganic phosphate liquid culture medium according to the inoculation amount (v/v) of 1 percent, the blank control is obtained by inoculating the sterile LB liquid culture medium of 1 percent into the Monkina inorganic phosphate liquid culture medium, and the blank control is subjected to shake culture for 7 days in the constant temperature shaking table at 30 ℃ and 180 r/min.

And measuring the effective phosphorus content in the fermentation liquor by adopting a molybdenum-antimony colorimetric resistance method. Shaking up the fermentation liquor, taking 20 mL into a centrifuge tube, centrifuging at 8000 r/min for 10 min; sucking 2.5-10 mL of supernatant into a 50mL volumetric flask, adding distilled water until the volume is about 30 mL, adding 2 drops of 2, 6-dinitrophenol indicator, adding 1 drop of 1mol/L H2SO4 until the yellow color of the solution is just faded, adding 5 mL of molybdenum-antimony anti-reagent, fixing the volume to a scale, developing for 30 min at 25 ℃, and measuring the absorbance value on a 721 type ultraviolet-visible spectrophotometer with the wavelength set at 700 nm. And calculating the corresponding effective phosphorus content according to the standard curve.

Drawing a phosphorus standard curve: diluting 50 mg/L phosphorus standard solution by 10 times to 5 mg/L, respectively sucking 5 mg/L phosphorus standard solution 0, 2, 4, 6, 8, 10 mL to 50mL volumetric flasks, adding distilled water to the volume of about 30 mL, adding 2 drops of 2, 6-dinitrophenol indicator, dropwise adding 4 mol/L NaOH solution to the volumetric flask until the liquid appears yellowish, dropwise adding 1mol/L H2SO4 until the yellow color just disappears, then adding 5 mL of molybdenum-antimony anti-reagent, and fixing the volume to the scale with distilled water. The sample was developed at 25 ℃ for 30 minutes, and then the absorbance value was measured on a 721 type ultraviolet-visible spectrophotometer with the wavelength set at 700 nm together with the developing solution of the above sample. The phosphorus concentration is plotted as abscissa and the absorbance value is plotted as ordinate, as shown in FIG. 1.

After the determination, it was concluded that: the effective phosphorus concentration in the blank control solution without inoculation is far lower than that in the fermentation liquor of the bacillus altitudinis SM01, namely the bacillus altitudinis SM01 is a high-efficiency phosphate-solubilizing fungus.

A microbial agent prepared from Bacillus altitudinis SM01 and having phosphate solubilizing function, wherein the microbial agent comprises: the solid culture medium comprises a solid culture medium formula and a mass fermentation culture formula, wherein the solid culture medium formula and the mass fermentation culture formula have the same components, and comprise a solid material and an inorganic salt solution, and the mass ratio of the solid material to the inorganic salt is 98.7: 0.3; the solid material consists of rice hulls, corn flour, soybean meal and bran in a mass ratio of 50: 24: 20: 6; the inorganic salt comprises 11% of monopotassium phosphate, 9% of magnesium sulfate, 15% of ammonium sulfate and 65% of light calcium carbonate in percentage by mass.

A preparation method of a geobacillus microbial agent with a phosphate solubilizing function comprises the following steps:

the first step is as follows: preparing a seed solution of the geobacillus;

the second step is that: inoculating the seed solution prepared in the first step into a solid culture medium, and culturing at a constant temperature of 27-29 ℃.

The third step: mixing the culture obtained in the second step with sterile water according to the mass ratio of 1: 15, filtering, inoculating the filtrate to a large amount of fermentation medium, and performing fermentation culture in a fermentation chamber at room temperature of 27-29 ℃ and relative humidity of more than 85%. Wherein the fermentation medium and the solid medium have the same components and are a mixture of solid materials and inorganic salt solution.

In summary, the following steps: experiments show that the effective phosphorus concentration in the blank control solution without inoculation is far lower than that in the fermentation liquor of the bacillus altitudinis SM01, namely the bacillus altitudinis SM01 is a high-efficiency phosphate-solubilizing fungus.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.

Claims (6)

1. A Bacillus highland is SM01 with phosphate solubilizing function, which has been preserved in China general microbiological culture Collection center in 2019, 3 and 18 months, with the preservation number of CGMCC 17338.

2. The bacillus altitudinis SM01 with phosphate solubilizing function according to claim 1, comprising LB liquid culture medium used for laboratory liquid culture and Monkina inorganic phosphate solid culture medium used for phosphate solubilizing bacteria screening:

the LB liquid culture medium adopted by the laboratory liquid culture comprises the following components in percentage by mass:

5-15 g of tryptone;

3-16 g of yeast extract;

8-15 g of sodium chloride;

800-1200mL of distilled water;

the Monkina inorganic phosphorus solid culture medium formula adopted by screening the phosphate solubilizing bacteria comprises the following components in percentage by mass:

5-15 g of glucose;

(NH4)2SO4 0.3-0.6g；

NaCl 0.2-0.5 g；

KCl 0.2-0.5g；

MgSO4·7H2O 0.2-0.5g；

FeSO4·7H2O 0.02-0.05g；

MnSO4·4H2O 0.02-0.04 g；

Ca3(PO3)2 8-12 g；

800 ml of deionized water and 1300ml of deionized water.

3. The Bacillus altitudinis SM01 with phosphate solubilizing function according to claim 1, wherein the pH of LB liquid culture medium is adjusted to 6.8-7.0 in the laboratory liquid culture, and the pH of Monkina inorganic phosphate solid culture medium used in the phosphate solubilizing bacteria screening is adjusted to 6.9-7.1.

4. The microbial agent prepared by the bacillus altitudinis SM01 with the phosphate solubilizing function according to claim 1, wherein the microbial agent comprises the following components in percentage by weight: the microbial agent comprises: the solid culture medium comprises a solid culture medium formula and a mass fermentation culture formula, wherein the solid culture medium formula and the mass fermentation culture formula have the same components, and comprise a solid material and an inorganic salt solution, and the mass ratio of the solid material to the inorganic salt is 98.7: 0.3; the solid material consists of rice hulls, corn flour, soybean meal and bran in a mass ratio of 50: 24: 20: 6; the inorganic salt comprises, by mass, 11% of monopotassium phosphate, 9% of magnesium sulfate, 15% of ammonium sulfate and 65% of light calcium carbonate.

5. A preparation method of a geobacillus microbial agent with a phosphate solubilizing function comprises the following steps:

s1: preparing a seed solution of the geobacillus;

s2: inoculating the seed liquid prepared by S1 into a solid culture medium, and culturing at a constant temperature of 27-29 ℃;

s3: mixing the culture cultured in S2 with sterile water according to the mass ratio of 1: 15, filtering, inoculating the filtrate into a large amount of fermentation medium, and performing fermentation culture in a fermentation chamber at room temperature of 27-29 ℃ and relative humidity of more than 85%.

6. The method for preparing a Bacillus altitudinis microbial inoculum with phosphate solubilizing function according to claim 5, which comprises the following steps: the fermentation medium in S3 has the same composition as the solid medium.