A kind of Citrobacter freundii having the ability to decompose phosphorus and potassium and its application

Abstract

The invention discloses a high-efficiency phosphorus-solubilizing Citrobacter freundii and its application. A strain ZSW‑4 with strong phosphorus-solubilizing ability was screened from the rhizosphere of soybean plants, and the strain was subjected to multiple compound mutagenesis using ultraviolet-plasma compound mutagenesis technology, and a strain with high phosphorus-solubilizing ability and stability was bred. The good strain ZSW‑4‑2‑5C has been preserved in the General Microorganism Center of China Committee for the Collection of Microorganisms, with the preservation number CGMCC No.8748. Inoculating crops such as corn, soybean, and mustard with liquid or solid bacterial agents containing the bacterium can promote the absorption of phosphorus in the soil by plants and increase the yield of crops.

Description

technical field

[0001] The invention relates to a highly efficient Citrobacter freundii ( Citrobacter fredndii ) and applications thereof, belonging to the technical field of agricultural microorganisms.

[0002] technical background

[0003] Phosphorus is one of the three major elements of plant nutrition, and insufficient supply of phosphorus has gradually become one of the most important factors limiting crop yield and quality. The total phosphorus content in the soil is sufficient, containing 0.02-0.5% total phosphorus, but most of them exist in the form of insoluble inorganic phosphates (mainly calcium phosphate, aluminum phosphate, iron phosphate, mainly calcium phosphate in alkaline soil) , only 1% exists in the form of soluble phosphorus salts that plants can directly absorb and utilize. In order to meet the needs of crop growth, nearly 30 million tons of phosphorus fertilizers are applied to the soil every year around the world, but 80% of them become ineffective p...

Examples

Embodiment 1

[0039] According to the screening method of the phosphorus-dissolving bacterial strain provided by the present invention, the bacterial strain with strong phosphorus-dissolving ability is selected, and the preparation steps are as follows:

[0040] 1) Use Citrobacter freundii ZSW-4 screened from the plant rhizosphere in the laboratory as the starting strain;

[0041] 2) Mutation breeding

[0042] (1) The Citrobacter freundii with phosphorus-solubilizing ability provided by the present invention is obtained through a mutagenesis selection method, which includes the following steps:

[0043] 1) Citrobacter freundii ZSW-4, which was screened from the plant rhizosphere in the laboratory, was used as the starting strain;

[0044] 2) Mutation breeding

[0045] (1) Prepare the single cell suspension of the starting strain ZSW-4

[0046] The starting strain ZSW-4 was inoculated in liquid medium A, cultured at 28°C, 180rpm for 16 hours, centrifuged, washed with sterile saline, place...

Embodiment 2

[0065] Example 2 Determination of ZSW-4-2-5C's ability to decompose calcium phosphate, lecithin and calcium phytate

[0066] Dispense 100ml of liquid culture medium B, C or D into 250ml Erlenmeyer flasks, and sterilize them for later use. Inoculate ZSW-4-2-5C in 50ml of liquid medium A, culture at 28°C for 36 hours, take 100μl (dilute A bacteria solution with liquid medium to make its OD600 0.7) and inoculate it in the above In the liquid culture medium B, C or D, each group was cultured in three parallels at 28°C and 150rpm for 7 days, and the bacteria liquid cultured on the above shaker was taken, centrifuged at 10,000rpm for 10 minutes, and the supernatant was taken and determined by molybdenum-antimony resistance method Phosphorus content in the supernatant. The experimental results show that: ZSW-4-2-5C has a calcium phosphate dissolving ability of 647.1±25.2 mg / l in liquid medium B, and a lecithin dissolving ability of 105±6.4 mg / l in liquid medium C. The ability to de...

Embodiment 3

[0071] Example 3 Determination of the ability of ZSW-4-2-5C to produce indole acetic acid (IAA)

[0072] Add sterile L-tryptophan with a concentration of 200μg·ml-1 to a 250ml Erlenmeyer flask containing 100ml of liquid medium A, inoculate ZSW-4-2-5C in 50ml of liquid medium A, at 28°C, After culturing at 150rpm for 20 hours, take 100μl (dilute the bacterial solution with medium A to make its OD600 0.7) and inoculate it into the above-mentioned liquid medium A containing L-tryptophan, set up three parallels, 28°C, 150 Rpm cultured for 72 hours, centrifuged at 4000 g for 10 min, and the concentration of IAA in the supernatant was determined by spectrophotometry. The experimental results showed that: ZSW-4-2-5C produced IAA concentration of 44.6±3.2mg / l.

technical field

[0001] The invention relates to a highly efficient Citrobacter freundii ( Citrobacter fredndii ) and applications thereof, belonging to the technical field of agricultural microorganisms.

[0002] technical background

[0003] Phosphorus is one of the three major elements of plant nutrition, and insufficient supply of phosphorus has gradually become one of the most important factors limiting crop yield and quality. The total phosphorus content in the soil is sufficient, containing 0.02-0.5% total phosphorus, but most of them exist in the form of insoluble inorganic phosphates (mainly calcium phosphate, aluminum phosphate, iron phosphate, mainly calcium phosphate in alkaline soil) , only 1% exists in the form of soluble phosphorus salts that plants can directly absorb and utilize. In order to meet the needs of crop growth, nearly 30 million tons of phosphorus fertilizers are applied to the soil every year around the world, but 80% of them become ineffective p...

Examples

Embodiment 1

[0039] According to the screening method of the phosphorus-dissolving bacterial strain provided by the present invention, the bacterial strain with strong phosphorus-dissolving ability is selected, and the preparation steps are as follows:

[0040] 1) Use Citrobacter freundii ZSW-4 screened from the plant rhizosphere in the laboratory as the starting strain;

[0041] 2) Mutation breeding

[0042] (1) The Citrobacter freundii with phosphorus-solubilizing ability provided by the present invention is obtained through a mutagenesis selection method, which includes the following steps:

[0043] 1) Citrobacter freundii ZSW-4, which was screened from the plant rhizosphere in the laboratory, was used as the starting strain;

[0044] 2) Mutation breeding

[0045] (1) Prepare the single cell suspension of the starting strain ZSW-4

[0046] The starting strain ZSW-4 was inoculated in liquid medium A, cultured at 28°C, 180rpm for 16 hours, centrifuged, washed with sterile saline, place...

Embodiment 2

[0065] Example 2 Determination of ZSW-4-2-5C's ability to decompose calcium phosphate, lecithin and calcium phytate

[0066] Dispense 100ml of liquid culture medium B, C or D into 250ml Erlenmeyer flasks, and sterilize them for later use. Inoculate ZSW-4-2-5C in 50ml of liquid medium A, culture at 28°C for 36 hours, take 100μl (dilute A bacteria solution with liquid medium to make its OD600 0.7) and inoculate it in the above In the liquid culture medium B, C or D, each group was cultured in three parallels at 28°C and 150rpm for 7 days, and the bacteria liquid cultured on the above shaker was taken, centrifuged at 10,000rpm for 10 minutes, and the supernatant was taken and determined by molybdenum-antimony resistance method Phosphorus content in the supernatant. The experimental results show that: ZSW-4-2-5C has a calcium phosphate dissolving ability of 647.1±25.2 mg / l in liquid medium B, and a lecithin dissolving ability of 105±6.4 mg / l in liquid medium C. The ability to de...

Embodiment 3

[0071] Example 3 Determination of the ability of ZSW-4-2-5C to produce indole acetic acid (IAA)

[0072] Add sterile L-tryptophan with a concentration of 200μg·ml-1 to a 250ml Erlenmeyer flask containing 100ml of liquid medium A, inoculate ZSW-4-2-5C in 50ml of liquid medium A, at 28°C, After culturing at 150rpm for 20 hours, take 100μl (dilute the bacterial solution with medium A to make its OD600 0.7) and inoculate it into the above-mentioned liquid medium A containing L-tryptophan, set up three parallels, 28°C, 150 Rpm cultured for 72 hours, centrifuged at 4000 g for 10 min, and the concentration of IAA in the supernatant was determined by spectrophotometry. The experimental results showed that: ZSW-4-2-5C produced IAA concentration of 44.6±3.2mg / l.

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