Phage and Its Use in Compensating Soil Nitrogen Fixation

Abstract

A phage and its use in compensating soil nitrogen fixation: Klebsiella pneumoniae phage YSZKA, wherein the method has solved some problems of conventional azotobacters, such as high cost, interspecific competition and limited range of application. Through phage transplantation, the efficiency of microbial nitrogen fixation in soil can be improved, and thus, more nitrogen sources may be provided for the nitrogen cycle and plant growth in ecosystems. Also, a new solution is provided for the nitrogen sequestration process of soil in saline-alkali lands, tea plantations and pesticide-polluted sites in China.

Claims

1-5. (canceled)

6. A method for compensating soil nitrogen fixation efficiency comprising the step of adding Klebsiella pneumoniae phage YSZKA to soil.

7. The method of claim 6, wherein the soil is for planting soybean, tea or Chinese herbal medicine.

8. The method of claim 6, wherein the nitrogen-fixing capacity of the soil is improved by injecting Klebsiella pneumoniae phage YSZKA into the soils.

9. The method of claim 8, further comprising adding biological or chemical substances that contribute to soil improvement to the soil.

10. The method of claim 8, further comprising monitoring the change of nitrogen content in the soil and adjusting the amount of Klebsiella pneumoniae phage YSZKA added to the soil.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0017] FIG. 1 is a schematic diagram for the contribution of phage community transplantation to soil nitrogen fixation.

[0018] FIG. 2 is a TEM (transmission electron microscope) image of the phage YSZKA;

[0019] FIG. 3 shows a one-step growth curve of the phage YSZKA;

[0020] FIG. 4 shows the abundance of nitrogen-fixing gene nifH 45 days after microbial inoculant injection in the soil of a saline-alkali land in Nantong City, Jiangsu Province.

[0021] FIG. 5 shows the nitrogenase activity 45 days after microbial inoculant injection in the soil of a saline-alkali land in Nantong City, Jiangsu Province.

[0022] FIG. 6 shows the average concentration of organic nitrogen 120 days after microbial inoculant injection in the soil of a farmland in black soil region of Heilongjiang Province.

[0023] FIG. 7 shows the abundance of nitrogen-fixing gene nifH 50 days after microbial inoculant injection in the soil of a tea plantation in Hunan Province.

[0024] FIG. 8 shows the abundance of nitrogen-fixing gene nifK 50 days after microbial inoculant injection in the soil of a tea plantation in Hunan Province.

[0025] FIG. 9 shows the average concentration of organic nitrogen 50 days after microbial inoculant injection in the soil of a tea plantation in Hunan Province.

[0026] FIG. 10 shows the average concentration of organic nitrogen 35 days after microbial inoculant injection in a chlorobenzene pesticide-polluted soil in Anhui Province

[0027] FIG. 11 shows the relative expression level of nitrogen-fixing gene nifH 150 days after microbial inoculant injection in a Chinese herbal medicine planting soil in Longnan City, Gansu Province.

[0028] FIG. 12 shows the relative expression level of nitrogen-fixing gene nifK 150 days after microbial inoculant injection in a Chinese herbal medicine planting soil in Longnan City, Gansu Province.

[0029] FIG. 13 shows the average concentration of organic nitrogen 150 days after microbial inoculant injection in a Chinese herbal medicine planting soil in Longnan City, Gansu Province. Klebsiella pneumoniae phage YSZKA was collected in the China Center for Type Culture Collection (CCTCC) on Aug. 1, 2018, assigned a CCTCC NO: M 2018513, and classified as Klebsiella phage YSZKA. The address is China Center for Type Culture Collection, Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China.

DETAILED DESCRIPTION OF THE INVENTION

[0030] The following embodiments are not intended to limit the technical solution of the present invention in any way, and all technical solutions obtained through equivalent replacement or equivalent modification should be deemed as falling within the scope of protection of the present invention. Klebsiella pneumoniae phage YSZKA was assigned a CCTCC NO: M 2018513 The genes nifH and nifK refer to a nitrogen-fixing functional gene carried by the phages detected in soil and a nitrogen-fixing gene carried by in-situ host bacteria, respectively.

TABLE-US-00001 TABLE 1 Determination of optimal multiplicity of infection (OMOI) Multiplicity of Bacterial infection (MOI) Phage count count YSZKA titer (PFU CFU.sup.1) (PFU mL.sup.1) (CFU mL.sup.1) (PFU mL.sup.1) 100:1 1 10.sup.9 1 10.sup.7 4.1 10.sup.6 10:1 1 10.sup.8 1 10.sup.7 5.2 10.sup.6 1:1 1 10.sup.7 1 10.sup.7 1.1 10.sup.7 1:10 1 10.sup.6 1 10.sup.7 3.8 10.sup.7 1:100 1 10.sup.5 1 10.sup.7 3.6 10.sup.8

Example 1

[0031] In the test potting soil, which was collected from the soil of a saline-alkali land in Nantong City, Jiangsu Province, Rongtao No. 9 peas were planted. Basic physical and chemical properties of the soil: sand grains (sandy soil) 21.3 wt. %, soil grains 40.2 wt. %, clay grains 33.4 wt. %, pH 8.8, total nitrogen 1.6 g.Math.kg.sup.1, water-soluble nitrogen 1.8 g.Math.kg.sup.1, total phosphorus 1.2 g.Math.kg.sup.1, total potassium 15.5 g.Math.kg.sup.1, CEC 17.2 cmol.Math.kg.sup.1.

[0032] Two groups were set for the experiment: (1) control group (CK): 3 peas were planted in each pot (with 0.51 cm of soil covered on the seeds, room temperature 252 C.); (2) phage treatment group (V): based on the control group, 100 mL of phage suspension with a concentration of 107 PFU.Math.mL.sup.1 was inoculated. Soil was sampled on site after the 45th day of pea growth, and the abundance of nitrogen-fixing gene nifH of the soil under treatment of the Group CK and Group V was measured: Group CK: 4.7710.sup.52.2610.sup.4 copies.Math.g.sup.1, Group V: 8.3110.sup.81.1410.sup.8 copies.Math.g.sup.1 (FIG. 4). Compared with the control group, the abundance of nitrogen-fixing gene nifH in soil of the saline-alkali land was increased by 3.3 orders of magnitude (p<0.05). The nitrogenase activity in the soil was measured in the treatment group and control group, which was 6.9310.sup.46.1110.sup.3 moles.Math.mg.sup.1, and 3.3310.sup.32.3510.sup.2 moles.Math.mg.sup.1, respectively (FIG. 5). The results indicate that the addition of phage suspension has significant effect for enriching the microbial nitrogenase anabolism genes and promoting the soil nitrogen fixation.

Example 2

[0033] Target soil: Farmland soil for planting Mengdou 14 Northeast soybean in black soil region of Heilongjiang Province. Basic physical and chemical properties of the soil: pH 6.1, organic matter 28.1 g.Math.kg.sup.1, total nitrogen 5.8 g.Math.kg.sup.1, total phosphorus 4.1 g.Math.kg.sup.1, rapidly available phosphorus 92.8 g.Math.kg.sup.1, soil mechanical composition: sand grains (sandy soil) 20.8 wt. %, soil grains 45.1 wt. %, clay grains 34.1 wt. %. Organic nitrogen compounds related with soil nitrogen fixation: 834.2527.5 mg.Math.kg.sup.1.

[0034] Four groups were set for the experiment: (1) control group (CK): 3 soybeans were planted in each pot (with 0.51 cm of soil covered on the seeds, room temperature 202 C.); (2) phage YSZKA treatment group (P1): based on the control group, 100 mL of phage YSZKA having nitrogen-fixing function with a concentration of 10.sup.6 PFU.Math.mL.sup.1 was inoculated. Soil and soybean were sampled on site after the 120th day of soybean growth, and the average concentration of organic nitrogen of the soybean soil was measured in the control group: about 720.702.57 mg.Math.kg.sup.1; the average concentration of organic nitrogen of the group P1 treated with inoculated phage was increased to 836.165.83 mg.Math.kg.sup.1 (FIG. 6), indicating that the phage inoculation significantly promotes the nitrogen fixation process of soil microorganisms to a certain extent, and also helps to improve the diversity and stability of soil microbial ecological functions in the black soil region.

Example 3

[0035] The soil for the test potted plant was collected from the soil of a tea plantation in Hunan Province. Basic physical and chemical properties of the soil: pH 4.64, organic matter 38.2 g.Math.kg.sup.1, total nitrogen 4.1 g.Math.kg.sup.1, total phosphorus 0.6 g.Math.kg.sup.1, rapidly available phosphorus 35.2 g.Math.kg.sup.1, total potassium 12.4 g.Math.kg.sup.1, rapidly available potassium 53.2 g.Math.kg.sup.1, soil mechanical composition: sand grains (sandy soil) 18.3 wt. %, soil grains 38.4 wt. %, clay grains 44.3 wt. %. The average concentration of nitrogen-related metabolites in zero to one meter underground soil layer was 545.221.3 mg.Math.kg.sup.1. Two groups were set for the experiment: (1) control group (CK): three spinach were planted in each pot (with 0.51 cm of soil covered on the seeds, room temperature 182 C.); (2) Phage treatment group (B): based on the control group, phage YSZKA was applied. Soil and spinach were sampled on site after the 50th day of spinach growth, and the abundance of bacterial nitrogen-fixing gene nifH of the soil under the treatment of the Group CK and Group B was measured: 1.5010.sup.46.6110.sup.2 copies.Math.g.sup.1 and 1.6710.sup.61.2510.sup.5 copies.Math.g.sup.1 (FIG. 7), and the abundance of nitrogen-fixing gene nifK of the Group CK and Group B was 3.2510.sup.31.1810.sup.2 copies.Math.g.sup.1 and 8.2410.sup.51.1110.sup.4 copies.Math.g.sup.1 (FIG. 8), respectively. The concentration of organic nitrogen in the treatment group (Group B) (618.4317.03 mg.Math.kg.sup.1) with phage applied was significantly higher than that in Group CK (998.7714.97 mg.Math.kg.sup.1) (FIG. 9), indicating that phage transplantation can significantly regulate the effect of microbial nitrogen fixation in soil and promote plant growth.

Example 4

[0036] Target soil: A chlorobenzene pesticide-polluted site in Anhui Province. Basic physical and chemical properties of the soil: pH 8.3, organic matter 30.3 g.Math.kg.sup.1, total nitrogen 1.8 g.Math.kg.sup.1, available nitrogen 28.9 mg.Math.kg.sup.1, total phosphorus 0.2 g.Math.kg.sup.1, available phosphorus 16.1 mg.Math.kg.sup.1. Soil mechanical composition: sand grains (sandy soil) 9.5 wt. %, soil grains 62.8 wt. %, clay grains 27.7 wt. %. Soil nitrogen fixation related organic nitrogen compounds: 518.3725.7 mg.Math.kg.sup.1.

[0037] A total of four groups were set for the experiment: (1) control group (CK): three peanuts were planted in each pot (with 0.51 cm of soil covered on the seeds, room temperature 202 C.); (2) Phage YSZKA treatment group (V1): based on the control group, 100 mL of nitrogen-fixing functional phage YSZKA with a concentration of 10.sup.6 PFU.Math.mL.sup.1 was inoculated. Soil and peanuts were sampled on site after the 35th day of peanut growth, and the average concentration of organic nitrogen of the control group soil was measured: about 582.6018.40 mg.Math.kg.sup.1, and after treatment with inoculated phage V1, the average concentration of organic nitrogen was increased to 745.3213.59 mg.Math.kg.sup.1 (FIG. 10), indicating that the phage inoculation significantly promotes the nitrogen fixation effect of soil microorganisms, improves the soil nutrients, and enhances the diversity of soil microbial ecological functions.

Example 5

[0038] The soil for the test potted plant was collected from a Chinese herbal medicine planting soil in Longnan City, Gansu Province. Basic physical and chemical properties of the soil: pH 8.20, organic matter 32.7 g.Math.kg.sup.1, total nitrogen 1.1 g.Math.kg.sup.1, available nitrogen 35.2 mg.Math.kg.sup.1, total phosphorus 0.4 g.Math.kg.sup.1, available phosphorus 31.6 mg.Math.kg.sup.1. Soil mechanical composition: sand grains (sandy soil) 11.2 wt. %, soil grains 65.5 wt. %, clay grains 23.3 wt. %. The average concentration of nitrogen-related metabolites in zero to one meter underground soil layer was 613.213.3 mg.Math.kg.sup.1. Two groups were set for the experiment: (1) control group (CK): three tomatoes were planted in each pot (with 0.51 cm of soil covered on the seeds, room temperature 182 C.); (2) phage treatment group (T): based on the control group, phage YSZKA was applied. Soil and tomato were sampled on site after the 150th day of tomato growth, and the relative expression level of bacterial nitrogen-fixing gene nifH of the soil under the treatment of the Group CK and Group T was 59.009.75 RPM and 235.7221.06 RPM, respectively (FIG. 11); the relative expression level of nitrogen-fixing gene nifK of the Group CK and Group T was 150.2515.89 RPM and 910.9415.55 RPM, respectively (FIG. 12). The concentration of organic nitrogen in the treatment group (Group T) with phage applied (1,015.0813.90 mg.Math.kg.sup.1) was significantly higher than that in Group CK (630.6818.83 mg.Math.kg.sup.1) (FIG. 13), indicating that the phage transplantation can significantly regulate the expression of host bacterial nitrogen fixing genes to promote soil nitrogen fixation, and the technology has the advantages of high broad-spectrum, low ecological risk, environmental friendliness and strong controllability, so it is a promising technology for soil nitrogen fixation and optimization.