A bacteriophage combination agent for simultaneously improving salt resistance and nutrient expansion of saline-alkali land and its application

Abstract

A phage combination agent and its application for simultaneously improving the salt resistance and nutrient expansion of saline-alkali land, including two phages, namely, Pseudomonas aeruginosa phage φYSZPA, with a deposit number of CCTCC NO: M 2018515; a multivalent phage φYSZPK attacking Klebsiella pneumoniae and Pseudomonas aeruginosa, with a deposit number of CCTCC NO: M 2018516. The method has significant effects, low costs, environmental protection, simple operation, and is easy to promote and apply on a large scale. It provides a new idea and new technology for the improvement of saline-alkali land, so that the technology has a wide range of application prospects in coastal saline areas, plain saline areas, deserts and desert steppe saline areas.

Description

Technical Field

[0001] The present invention relates to the field of saline-alkali land soil improvement, and in particular to a bacteriophage combination agent for simultaneously improving the salt resistance and nutrient expansion of saline-alkali land and an application thereof. Background Art

[0002] The accumulation of soil salt can lead to crop losses or even complete crop failures, so utilizing saline-alkali land to develop its economic value and reduce its salinization has always been a research focus. Traditional saline-alkali land improvement methods, such as applying chemical modifiers or physical methods, are often costly, have limited effects, and may have a negative impact on the ecological environment. Therefore, how to efficiently and greenly improve the soil conditions of saline-alkali land and enhance its salt resistance and nutrient retention capacity has become an issue that needs to be addressed.

[0003] Soil microorganisms play an important role in maintaining the balance of ecosystems, regulating the geochemical cycle of elements, degrading pollutants, maintaining the energy flow, material cycle and information exchange of ecosystems. Existing studies have shown that bacteriophages have the potential to resist salt and alkali. Bacteriophages have two lifestyles: lytic and lysogenic. After entering the host, lytic phages replicate and reproduce, and finally lyse the host to release the reproduced phages, which can specifically lyse host bacteria, such as pathogens. At the same time, lytic phages can release the biological effective substances of the lysed host to the environment for reuse by surviving microorganisms through the "phage shunt" mechanism, while causing the pathogens to be lysed and the salt-resistant functional bacterial community to increase its advantage. In the lysogenic state, phages inject their own genetic material into bacteria and coexist with bacteria. Phages contain auxiliary metabolic genes (AMGs), which are unrelated to the replication and activity of the phages themselves and can directly participate in the functional metabolism of the host, such as carbon metabolism (carbon increase), nitrogen metabolism (nitrogen fixation), energy metabolism (salt-alkali resistance) and other processes.

[0004] In recent years, bacteriophages, as a biological agent that can target specific bacteria, have been widely studied for use in the agricultural field. Bacteriophages have the ability to cooperate with the host to resist salt and alkali while expanding nutrients: in saline-alkali environments, bacteriophages can specifically regulate soil microbial communities and improve the salt resistance of soil microorganisms. By inoculating bacteriophages with salt-alkali adaptability, the salt resistance of the soil can be effectively improved, and the accumulation of soil organic matter and the improvement of nutrient expansion capacity can be promoted. The host's ability to resist salt-alkali stress is enhanced by lysogenic AMGs, thereby promoting microorganisms to improve saline-alkali land. At the same time, the host secretes organic matter to increase soil carbon and nitrogen reserves, achieving the effect of nutrient expansion; lysing pathogens increases soil nutrient expansion through "phage diversion." Therefore, the present invention aims to provide an innovative method for improving the salt resistance and nutrient expansion capacity of saline-alkali land through phage inoculation.

[0005] Through patent search, no public publication or acceptance of biotechnology for improving saline-alkali soil and expanding nutrients by means of phage inoculation was found. The existing method closest to the present invention is: preparing microbial agents to promote saline-alkali soil improvement, such as Chinese Patent Application No. 202411429132.6, which is a microbial agent mainly based on Sphingomonas for saline-alkali land improvement; Chinese Patent Application No. 202411389862.8 is a microbial agent mainly based on halophilic Bacillus saccharolyticus, which regulates soil pH, enriches soil nutrient content through ACC deaminase, siderophore, and nitrogen fixation, phosphorus solubilization, and potassium solubilization functions present in the strain, and thereby improves plant salt-alkali tolerance, promotes plant growth, and increases yield by adjusting soil pH. Chinese patent applications No. 202111286529.0, 201510398462.8, 202310492489.8, 201810729034.2 and Chinese patent publication No. CN117209183A sequentially apply microbial agents with magnetic nanoparticles, porogens, hibiscus stone, plant ash and multi-source solid waste to reduce soil pH, improve soil fertility and improve saline-alkali land. The strains mentioned in the above documents are Klebsiella, Cyanobacterium fischeri, Rhodospirilla or lactic acid bacteria, and do not involve bacteriophage improvers. In addition to the improvement with microbial agents, there are also several patents involving the design of saline-alkali land improvement systems. For example, Chinese patent application No. 202310492489.8 is to optimize the soil structure of saline-alkali land by applying 36% to 37% corn straw, 18% to 19% wheat straw biochar, 9% to 10% iron-based biochar and 0.03% to 0.05% microbial agents, and the rest is cow dung. Chinese patent application No. 201710442928.9 improves saline-alkali land by spraying microbial agents and draining salt through irrigation to achieve an agricultural circular economy. These methods will all point out that microbial agents are an important way to improve saline-alkali land at present, but there is a general lack of applications for the widespread phage-host interaction in the soil to resist salt and alkali and expand soil nutrients.

[0006] The main defects of the existing technology are: Most of the existing microbial agents for saline-alkali land improvement are single bacterial groups, and there is a lack of technology for regulating saline-alkali land through bacteriophages; at the same time, most of the agents are single bacterial groups with low efficiency, ignoring the effect of the interaction of the overall microbial community. In addition, the artificially added functional bacteria have the risk of competing with the original native microbial community to destroy the original microbial community structure or produce secondary biological contamination in the process of promoting the improvement of saline-alkali land. Moreover, the artificial preparation process is complicated and the preparation cycle is long, while the research and development of saline-alkali land improvement systems generally have the problems of high cost and lack of universality. In terms of application scope, most technologies only improve saline-alkali land and cannot simultaneously carry out salt-alkali resistance and nutrient expansion work.

[0007] The main reasons for the defects are: (1) Most existing microbial agents only use a single bacterial strain. This single bacterial community cannot fully utilize the complex microbial ecosystem in saline-alkali land, cannot comprehensively improve the physical and chemical properties of the soil and the microbial community structure, and has limited improvement effects. (2) The synergistic effect of saline-alkali land microbial communities is crucial in improving soil quality. Different bacterial species in the microbial community enhance functions such as salt resistance, nitrogen fixation, and degradation of harmful substances through mutual cooperation. However, traditional microbial agents often ignore this synergistic effect between communities, resulting in the inability to achieve optimal results in complex environments. (3) Artificially added functional bacteria may compete with native microbial communities in the soil during the improvement of saline-alkali land, especially when the newly added strains overlap with the native microorganisms in terms of resource requirements or growth conditions, which may inhibit the normal functions of the original microbial community, destroy the ecological balance, and even affect the long-term health of the soil. (4) Current saline-alkali land improvement microbial agents often rely on specific bacterial species or specific process flows, which make their production costs high. At the same time, the saline-alkali land conditions in different regions (such as saline-alkali type, soil characteristics, climate, etc.) vary greatly, making the existing microbial agents less adaptable in other regions, lacking universality, and difficult to promote and apply on a large scale. Therefore, it is of great significance to screen salt-alkali resistant bacteriophages in situ to develop non-bacterial microbial salt-alkali improvers, expand soil nutrients, and reduce ecological risks. Summary of the invention

[0008] Technical problem to be solved: The present invention provides a phage combination agent and its application for simultaneously improving the salt resistance and nutrient expansion of saline-alkali land. The method optimizes the microbial community structure of saline-alkali land by selecting appropriate phages and performing reasonable inoculation, thereby improving the salt resistance of the soil, improving soil fertility, and promoting the accumulation of soil organic carbon, thereby improving the nutrient expansion capacity of saline-alkali land.

[0009] Technical solution: A phage combination agent for simultaneously improving the salt resistance and nutrient expansion of saline-alkali land, including two phages, namely Pseudomonas aeruginosa phage φYSZPA, with a preservation number of CCTCC NO:M 2018515 and a classification name of Pseudomonas aeruginosa phage φYSZPA; polyvalent bacteriophage φYSZPK attacking Klebsiella pneumoniae and Pseudomonas aeruginosa, deposit number: CCTCC NO:M 2018516, classification name Pseudomonas aeruginosa and Klebsiella phage φYSZPK.

[0010] Preferably, the lysogenic state of the polyvalent phage φYSZPK attacking Klebsiella pneumoniae and Pseudomonas aeruginosa is not less than 60%; the host of the Pseudomonas aeruginosa phage φYSZPA is a pathogen, and the lytic state of the obtained phage is not less than 80%.

[0011] Preferably, the concentration ratio of the Pseudomonas aeruginosa phage φYSZPA to the multivalent phage φYSZPK attacking Klebsiella pneumoniae and Pseudomonas aeruginosa in the above-mentioned combination is 2:1.

[0012] The application of the above-mentioned phage combination agent in simultaneously improving the salt resistance and nutrient expansion of saline-alkali land.

[0013] The above-mentioned salt resistance of saline-alkali land includes reducing the alkalinity in saline-alkali soil and reducing the salinity in saline-alkali soil, and the salinity includes chloride ions, sulfate ions or carbonate ions; the above-mentioned nutrient expansion includes increasing the organic matter content in saline-alkali soil or increasing the nitrogen content in saline-alkali soil.

[0014] The application method is to inject the phage combination into the target soil.

[0015] Specifically, after the phage combination agent is thawed, it is evenly added according to the mass ratio of soil to phage combination agent (500-1000):1, and the in-situ direct push injection equipment is used to continuously inject at a pressure of 0.1-2 MPa, with an injection volume of 1-5 m per hour. 3 .

[0016] The above-mentioned saline-alkali land includes slightly saline-alkali land with a soil salt content of 0.2% to 0.4% and moderately saline-alkali land with a soil salt content of 0.4% to 0.8%.

[0017] The above-mentioned saline-alkali land includes slightly saline-alkali land with a soil pH value of 8.0-9.0 and moderately saline-alkali land with a soil pH value of 9.0-9.5.

[0018] The working principle of the present invention is: 1. The bacteriophage is a mobile genetic element that cannot replicate itself but relies on host cells to reproduce. Its host is bacteria, and it has two life states: lysis and lysogeny; 2. In the lysogeny state, the bacteriophage injects its own genetic material into the bacteria and coexists with the bacteria. The bacteriophage contains auxiliary metabolic genes (AMGs), which are irrelevant to the replication and activity of the bacteriophage itself and can directly participate in the functional metabolism of the host, such as carbon metabolism (carbon increase), nitrogen metabolism (nitrogen fixation), energy metabolism (salt and alkali resistance) and other processes; 3. After the lytic phage enters the host, it replicates and reproduces, and finally lyses the host to release the propagated phage, which can specifically lyse the host bacteria, such as pathogens. At the same time, the lytic phage can release the biological effective substances of the lysed host into the environment for reuse by surviving microorganisms through the "viral shuttle" mechanism, while the pathogens are lysed and the salt-alkali resistance functional bacterial community advantage is increased; 4. Polyvalent phage refers to phages that can infect two or more host bacteria, which is conducive to the extensive use of the synergistic metabolic potential of phages; 5. The phages selected for phage inoculation technology are selected based on the natural (temperature, moisture, pH) and non-natural (heavy metals, pesticides, antibiotics) environmental conditions of the in-situ soil, and have the multivalent phages with salt-alkali resistance, nutrient expansion, and pathogen lysis functions. This phage can be better integrated into the microbial network of the region, minimize ecological risks, ensure the diversity and stability of local microbial ecological functions, and improve saline-alkali land at a lower cost.

[0019] Application effect: The method of the present invention can effectively regulate the microbial community in saline-alkali soil, improve the salt resistance of the soil, and promote the accumulation of soil organic matter by optimizing microbial metabolism, thereby improving the nutrient expansion capacity of saline-alkali land. At the same time, inoculating bacteriophages can reduce harmful pathogens in the soil, improve soil health, and promote sustainable agricultural development.

[0020] Beneficial effects: Aiming at the need to strengthen the improvement of saline-alkali land, the present invention provides a method for improving saline-alkali soil and expanding soil nutrients by inoculating with bacteriophages. It has the following advantages: 1. The bacteriophage inoculation method provided by the present invention has the advantages of being green, environmentally friendly and low-cost, and can significantly improve the salt resistance of saline-alkali land; 2. By adjusting the microbial community in saline-alkali land, the accumulation of soil organic carbon is improved, and the ability to expand soil nutrients is enhanced, which has good environmental benefits; 3. Bacteriophages weaken the interspecies competition problem existing in traditional salt-alkali resistant bacteria agents, and optimize the structure of microbial communities from the perspective of cross-border cooperation; 4. Bacteriophages are derived from in-situ soil, and there is no risk of biological contamination for themselves and their metabolites; 5. The method of the present invention is simple to operate and easy to promote and apply on a large scale, providing a new idea and new technology for the improvement of saline-alkali land, so that the technology has broad application prospects in coastal saline areas, plain saline areas, deserts and desert steppe saline areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the improvement effect of phage inoculation on salt resistance and nutrient expansion in saline-alkali land.

[0022] Figure 2 This is a transmission electron micrograph of Pseudomonas aeruginosa phage φYSZPA.

[0023] Figure 3 Transmission electron micrograph of the multivalent bacteriophage φYSZPK attacking Klebsiella pneumoniae and Pseudomonas aeruginosa.

[0024] Figure 4 pH changes in coastal saline-alkali soil before and after inoculation of bacteriophages.

[0025] Figure 5 Changes in chloride ion content in coastal saline-alkali soil before and after inoculation of bacteriophages.

[0026] Figure 6 Changes in organic matter content in coastal saline-alkali soil before and after inoculation of bacteriophages.

[0027] Figure 7 pH changes in soda saline soil before and after inoculation of bacteriophages.

[0028] Figure 8 Changes in carbonate ion content in soda saline-alkali soil before and after inoculation of bacteriophages.

[0029] Fig. 9 Changes in total nitrogen content in soda saline-alkali soil before and after inoculation of bacteriophages.

[0030] Fig.10 pH changes in sulfate-chloride saline soil before and after inoculation of bacteriophages.

[0031] Fig.11Changes in chloride ion content in sulfate-chloride saline-alkali soil before and after inoculation of bacteriophages.

[0032] Fig.12 Changes in sulfate ion content in sulfate-chloride saline-alkali soil before and after inoculation of bacteriophages. DETAILED DESCRIPTION

[0033] The following specific implementation methods do not limit the technical solutions of the present invention in any form, and all technical solutions obtained by equivalent replacement or equivalent transformation fall within the protection scope of the present invention.

[0034] A method for improving saline-alkali land with a combination agent composed of bacteriophages as the main component, comprising the following steps: the Pseudomonas aeruginosa phage φYSZPA, whose deposit number is CCTCC NO:M 2018515, was deposited in China Center for Type Culture Collection on August 1, 2018, address: Wuhan University, Wuhan, China; the multivalent phage φYSZPK attacking Klebsiella pneumoniae and Pseudomonas aeruginosa, whose deposit number is CCTCC NO:M 2018516, deposited on August 1, 2018 in China Center for Type Culture Collection, Wuhan University, Wuhan, China; the polyvalent phage φYSZPK attacking Klebsiella pneumoniae and Pseudomonas aeruginosa must have a lysogenic state ratio greater than 60%, carry salt-alkali resistance and nutrient expansion auxiliary metabolism genes, and is a polyvalent phage; the host of Pseudomonas aeruginosa phage φYSZPA is a pathogen, and the lytic state ratio of the obtained phage must be greater than 80%. The concentration of the two phages in the composition is 2:1. The two phages are mixed so that their concentrations in the composition are at a predetermined ratio.

[0035] After the phage suspension is thawed, it is evenly added in a soil to phage suspension mass ratio of 500:1-1000:1, and an in-situ direct push injection device is used to continuously inject at a pressure of 0.1-2 MPa, with an injection volume of 1-5m per hour. 3 .

[0036] The following is a further description of the embodiments of the present invention in conjunction with specific examples, but does not limit the protection scope of the present invention.

[0037] Example 1

[0038] A coastal saline-alkali land (coastal saline area) dominated by chloride was selected as an example. The crop planted was wheat. Basic physical and chemical properties of the soil: pH 8.54, organic matter 6.43 g kg -1 , total phosphorus 0.71 g·kg -1 , total potassium 18.74 g·kg -1 , chloride ion 14.93 g·kg -1 .

[0039] Two treatment groups were set up in the experiment: ① Control group (CK): 3 wheat plants were planted in each pot (2 cm of soil was covered on the seeds, and the room temperature was 25±2℃); ② Bacteriophage treatment (BV): 100 mL of 10 6 pfu·mL -1 The above-mentioned phage combination agent is a mixture of phage φYSZPK and phage φYSZPA in a ratio of 2:1. On-site soil sampling was carried out after the 70th day of wheat growth, and the soil pH under the CK and BV treatments was measured to be 8.50 and 7.98 respectively. The pH of the soil in the treatment group was reduced by 0.52 compared with the control group (p<0.05). The chloride ion content in the soil was measured to be 13.64 g·kg for the control group and the treatment group respectively. -1 , 8.89 g·kg -1 After phage inoculation, the soil organic matter content increased from 10.13 g·kg -1 Reach 16.44 g kg -1 This indicates that phage inoculation has significant effects on reducing alkali, reducing salt and increasing carbon in saline-alkali land.

[0040] Example 2

[0041] A carbonate-based soda saline-alkali land was selected as an example. The crop planted was soybean. Basic physical and chemical properties of the soil: pH 9.76, total nitrogen 0.45 g kg -1 , total phosphorus 0.60 g·kg -1 , total potassium 18.61 g·kg -1 , carbonate ion 6.46 g·kg -1 .

[0042] Two treatment groups were set up in the experiment: ① Control group (CK): 3 soybean plants were planted in each pot (2 cm of soil was covered on the seeds, and the room temperature was 25±2℃); ② Bacteriophage treatment (BV): 100 mL of 10 6 pfu·mL -1 The above-mentioned phage combination agent is a mixture of phage φYSZPK and phage φYSZPA in a ratio of 2:1. On-site soil sampling was carried out after the 70th day of soybean growth, and the soil pH under the CK and BV treatments was measured to be 9.67 and 9.13, respectively. The pH of the soil in the treatment group was reduced by 0.54 compared with the control group (p<0.05). The carbonate ion content in the soil was measured to be 6.31 g·kg for the control group and the treatment group, respectively. -1 , 5.74g·kg -1 After phage inoculation, the total nitrogen content in the soil increased from 1.02 g·kg -1 Reach 1.83 g kg-1 This indicates that phage inoculation has significant effects on reducing alkali, reducing salt and increasing nitrogen in saline-alkali land.

[0043] Example 3

[0044] A saline-alkali land dominated by sulfate and chloride in arid and semi-arid areas was selected. The crop planted was sorghum. Basic physical and chemical properties of the soil: pH 9.02, sulfate ion 5.47 g·kg -1 , chloride ion 12.16 g·kg -1 .

[0045] Two treatment groups were set up in the experiment: ① Control group (CK): 3 sorghum plants were planted in each pot (2 cm of soil was covered on the seeds, and the room temperature was 25±2℃); ② Bacteriophage treatment (BV): 100 mL of 10 6 pfu·mL -1 The above-mentioned phage combination agent is a mixture of phage φYSZPK and phage φYSZPA in a ratio of 2:1. On-site soil sampling was carried out after the 70th day of sorghum growth, and the soil pH under the CK and BV treatments was measured to be 8.81 and 8.16, respectively. The pH of the soil in the treatment group was reduced by 0.65 compared with the control group (p<0.05). The chloride ion content in the soil was measured to be 8.87 g·kg for the control group and the treatment group, respectively. -1 , 4.16 g·kg -1 After phage inoculation, the sulfate content in the soil increased from 9.24 g·kg -1 becomes 6.98 g kg -1 This indicates that phage inoculation has a significant effect on reducing alkali and salt in saline-alkali land.

Claims

1. A bacteriophage combination agent for simultaneously improving the salt resistance and nutrient expansion of saline-alkali land, characterized in that: There are two phages, namely Pseudomonas aeruginosa phage φYSZPA, with the deposit number: CCTCC NO:M 2018515, and the classification name Pseudomonas aeruginosa phage φYSZPA; polyvalent bacteriophage φYSZPK attacking Klebsiella pneumoniae and Pseudomonas aeruginosa, deposit number: CCTCC NO:M 2018516, classification name Pseudomonas aeruginosa and Klebsiella phage φYSZPK.

2. The phage combination agent for simultaneously improving the salt resistance and nutrient expansion of saline-alkali land according to claim 1, characterized in that: The lysogenic state of the multivalent phage φYSZPK that attacks Klebsiella pneumoniae and Pseudomonas aeruginosa accounts for no less than 60%; the host of the phage φYSZPA is a pathogen, and the lytic state of the obtained phage accounts for no less than 80%.

3. The bacteriophage combination agent for simultaneously improving the salt resistance and nutrient expansion of saline-alkali land according to claim 1, characterized in that: The concentration ratio of the Pseudomonas aeruginosa phage φYSZPA to the multivalent phage φYSZPK attacking Klebsiella pneumoniae and Pseudomonas aeruginosa in the combination agent is 2:

1.

4. Use of the bacteriophage combination agent according to any one of claims 1 to 3 in simultaneously improving the salt resistance and nutrient expansion of saline-alkali land.

5. The use according to claim 4, characterized in that: The salt resistance of saline-alkali land includes reducing the alkalinity in saline-alkali soil and reducing the salinity in saline-alkali soil, and the salinity includes chloride ions, sulfate ions or carbonate ions; the nutrient expansion includes increasing the organic matter content in saline-alkali soil or increasing the nitrogen content in saline-alkali soil.

6. The use according to claim 4, characterized in that: Inject the phage combination into the target soil.

7. The use according to claim 5, characterized in that: After the phage combination agent is thawed, it is evenly added according to the mass ratio of soil to phage combination agent (500-1000):

1. Use an in-situ direct push injection device to continuously inject at a pressure of 0.1-2 MPa, with an injection volume of 1-5 m per hour. 3 .

8. The use according to claim 5, characterized in that: The saline-alkali land includes slightly saline-alkali land with a soil salt content of 0.2% to 0.4% and moderately saline-alkali land with a soil salt content of 0.4% to 0.8%.

9. The use according to claim 5, characterized in that: The saline-alkali land includes light saline-alkali land with a soil pH value of 8.0-9.0 and moderate saline-alkali land with a soil pH value of 9.0-9.5.

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