Lysogenic bacteriophage community as well as extraction method and application thereof

Abstract

According to the lysogenic phage community and the extraction method and application thereof, bacterial and phage communities are separated from in-situ soil in a medium proportion through a tangential flow technology, single bacterial communities and single phage communities are obtained through enrichment and concentration, meanwhile, mitomycin C is added into the single bacterial communities for lysogenic induction, and the lysogenic phage communities are obtained. And then filtering and enriching by using the tangential flow system to obtain a high-flux lysogenic phage community, and finally obtaining a single bacterial community, a bacterial-phage mixed community and a bacterial-lysogenic phage mixed community. The phage transplantation technology can promote indigenous microorganisms to participate in the plant growth and development process, and the transplanted lysogenous phage can participate in host plant hormone anabolism by optimizing a microbial network structure and injecting auxiliary metabolism genes. The method provides a long-acting environment-friendly microorganism synergistic growth promoting technology for green agriculture development in China.

Description

Technical Field

[0001] The invention belongs to the technical field of bacteriophages, and in particular relates to a lysogenic bacteriophage community and an extraction method and application thereof. Background Art

[0002] The soil layer closely connected to the roots in the soil, which is physically, chemically and biologically different from the soil bulk, is called the rhizosphere, where plants increase the diversity and richness of molecules in the rhizosphere through root secretion sediments, thereby selectively recruiting soil microorganisms to shape the rhizosphere microbial community. Recently, the rhizosphere microbiome has been widely studied and reported, revealing the common patterns of microbial structure, composition, dynamics and function in different plant environments, as well as the role of microbiota in promoting plant nutrition and growth.

[0003] Cross-kingdom interactions between microbial groups can promote phages to reshape and regulate bacterial metabolism. Bacteriophages (referred to as phages) and host bacteria are typical examples of cross-kingdom interactions. Bacteriophages are a type of organism that survives by preying on living host bacteria. They are widely distributed in soil, air, and water. It is estimated that their total amount reaches 10 31 At the same time, phages have two lifestyles: lytic and lysogenic. Lysogenic means that phages integrate genetic material into the genome of host bacteria to form a structure called "prophage". This integration allows the phage genome to exist stably in the genome of the host bacteria, and the genetic material of the prophage can be transmitted along with the reproduction of bacteria. In this way, lysogenic phages can serve as a stable genetic carrier, carrying auxiliary metabolic genes (AMGs). In some cases, the prophage of lysogenic phages can re-enter the lytic cycle under certain conditions, release new phage particles, and infect other bacteria. This process allows phages with specific genetic information to spread to new host bacteria, thereby promoting horizontal gene transfer and reshaping host metabolism. Therefore, phages can reshape or accelerate host metabolism through the expression of auxiliary metabolic genes through lysogenic means, assist the host in the biosynthesis of plant hormones, and ultimately achieve the purpose of promoting plant growth.

[0004] Phage transplantation refers to an emerging biological technology that efficiently enriches the entire phage community in the environment, then transplants it into the designated target host environment and allows it to colonize and take effect. Soil phage transplantation is derived from the concept of fecal microbiota transplantation in the medical field to treat human diseases. The advantages of soil phage transplantation technology are: it can quantitatively identify the role and function of phages in the soil microbiome, making it easier to use the specificity and selectivity of phages to improve the soil environment, control diseases and increase plant productivity.

[0005] Through relevant literature review and patent search, no public publication or acceptance of the technology of promoting the growth and development of crops in farmland soil and enhancing agricultural yield by means of lysogenic phage community transplantation has been found. The existing method closest to the present invention is to use phage to inhibit the growth of plant harmful pathogens, thereby maintaining plant health. For example, invention publication number CN110679805A applies phage to plants or soil to inhibit the growth of pathogens, thereby improving plant health and growth status. By controlling pathogens, plants can better absorb nutrients and water, thereby promoting plant growth and development. Invention publication number CN106220428A prepares a plant disease control agent containing phage for controlling plant diseases and promoting plant growth. At the same time, the patent also covers the preparation method and application strategy of the control agent. Invention publication number CN109051089A uses the combination of different phages to improve the inhibitory effect on specific pathogens. Invention publication numbers CN103972752A, CN105202323A, and CN107179434A all only involve a bacteriophage targeting a specific plant pathogen and its application in plant disease control, so the broad spectrum is relatively weak. In addition to preparing bacteriophages as plant disease control agents, there are also a number of inventions that use soil bacteriophages to maintain plant health through other means, such as invention publication number CN110214764A, which optimizes bacteriophages through genetic engineering to make them more inhibitory to plant pathogens, thereby providing a method for modifying the plant disease control effect through bacteriophages, and this method involves genetic engineering methods with high costs. These methods will all point out that bacteriophages have the ability to promote the healthy growth of crops, but there is a general lack of applications for the synergistic interaction between lysogenic phages and hosts that are widely present in the soil to promote plant growth.

[0006] The main defects of the existing technology are: Since bacteriophages are highly specific to specific bacteria, but there are many types of bacteria in plant roots and soil, the effect of bacteriophages may be unstable or limited under different environmental conditions. In addition, the introduction of exogenous bacteriophages may interfere with the natural balance of soil microbial communities, leading to potential negative ecological consequences, such as effects on non-target bacteria, and there may be a risk of destroying the local soil microbial structure. And bacteriophages may have difficulty surviving or being effective in the soil for a long time, causing their effects on plant growth to weaken over time. At the same time, the production and application costs of bacteriophages may be high, so there are generally problems of high costs and lack of universality in large-scale agricultural production.

[0007] The main reasons for the defects are: Although people are gradually aware of the ecological status and influence of phages in the soil, researchers have paid relatively little attention to and research on the participation of soil phages in regulating host metabolism and promoting plant growth. First, because phages can only infect specific bacteria, and the pH, humidity, temperature and other environmental factors of the soil can affect the survival and activity of phages, which may cause them to lose their effectiveness under different environmental conditions, their applicability to diverse soil microbial communities is limited. Secondly, the introduction of exogenous phages may destroy the natural microbial balance in the soil, affect the growth and function of other beneficial microorganisms, and the addition of a single phage may be rapidly degraded in the soil or consumed by soil microorganisms and chemicals, thereby reducing its durability and effectiveness. Therefore, it is of great significance to extract highly lysogenic phage communities from in situ soils, develop highly efficient and enriched phage soil additives, promote plant growth, and reduce ecological risks of soil phage plant boosters. Summary of the invention

[0008] Technical problems solved: In view of the above-mentioned defects of the prior art, the invention provides a lysogenic phage community and its extraction method and application. The method first extracts, enriches and concentrates the bacteria and phage communities in the in-situ soil through membrane packages of different pore sizes, and then uses mitomycin C for lysogenic induction to obtain high-throughput lysogenic phages. Subsequently, the microbial extractant is injected into the target soil, and the lysogenic phage is injected into AMGs to collaborate with the host bacteria to reshape or accelerate the host metabolism. At the same time, the phage has the function of regulating the soil microbial community from the bottom up, further optimizing the indigenous bacterial symbiotic network and promoting the growth and development of crops. This technology is a lysogenic phage technology with good growth-promoting effect, low price and green friendliness.

[0009] Technical solution: A method for extracting lysogenic phage communities, comprising the following steps: uniformly mixing in situ soil with a buffer to obtain a soil suspension, centrifuging and standing the mixture to obtain a supernatant, and using a tangential flow system with different pore sizes to separate bacteria and phage communities respectively: first, the supernatant is filtered through a 0.2 μm filter membrane to obtain an enriched solution as a bacterial suspension, and then mitomycin C is added to the bacterial suspension to a final concentration of no more than 0.1 μg mL -1 , cultured under shaking at room temperature, and enriched again using a 30-100 kDa filter membrane to obtain a lysogenic phage community.

[0010] Preferably, the volume ratio of the in situ soil to the buffer is 1:1, and the buffer is Tris-HCl at pH 7.5, containing 68 mM NaCl and 10 mM MgSO4.

[0011] Preferably, the centrifugation condition is 4°C and 4500 G for 20 minutes.

[0012] The lysogenic phage community obtained by the above extraction method.

[0013] The application of the above lysogenic phage community in preparing products for enhancing plant growth.

[0014] The above-mentioned plants are Arabidopsis thaliana, Chinese cabbage, lettuce or chili pepper.

[0015] The soil conditioner for enhancing plant growth contains the lysogenic bacteriophage community and biologically active ingredients that are helpful for plant growth and development.

[0016] Soil conditioner application, injecting soil conditioner into target soil.

[0017] The working principle of the present invention is: 1. Bacteriophage is a kind of bacterial virus composed of protein capsid (60%) and internal nucleic acid genetic material (40%), without complete mature cell structure, and can be divided into two types: lytic and lysogenic; 2. Lysogenic phage can integrate its genome into the chromosome of the host bacteria to achieve horizontal gene transfer and affect the metabolic process of the host bacterial community. Auxiliary metabolic genes (AMGs) are gene fragments present in the phage genome, which are generally randomly packaged and assembled into the phage genome during the synthesis of phage particles. The auxiliary metabolic genes carried by phages can have an important impact on the metabolic process of host bacteria. These genes usually encode substances that can help bacteria utilize or process the substances required by phages and enhance the adaptability of bacteria. For example, phages may carry genes that promote the host bacteria's ability to metabolize specific nutrients, or help bacteria resist the host's immune response. The presence of these genes can improve the survival and reproduction ability of host bacteria, and may also affect the metabolic products and overall physiological state of host bacteria; 3. Broad-spectrum phages refer to phages that can infect two or more "species" with similar homology or host bacteria between different species, which is conducive to the horizontal transfer of genes by lysogenic phages and widely improves the synergistic metabolic potential of different hosts; 4. The selected phages "come from the soil and return to the soil" without any modification, which is environmentally friendly; 5. The length of phages is about 20 ~ 200μm, which is equivalent to hundreds and thousands of bacteria, so it can achieve extensive migration in the soil, which is conducive to saving application costs. 6. The phage community obtained by the tangential flow system is active and complete, and can quickly adapt to the working environment.

[0018] Beneficial effects: Aiming at the need of promoting plant growth, the present invention provides the application of lysogenic phage community transplantation in promoting plant growth and development. Its characteristics are: 1. The acquisition of complete active phage community weakens the problem of rapid degradation of single phage in soil, and optimizes the structure of microbial community from the perspective of interspecies cooperation; 2. A high proportion of lysogenic phage can promote host metabolic process through AMGs injection, effectively synthesize secretions to promote plant growth and development; 3. The materials for preparing microbial separation liquid are easy to obtain, easy to store, convenient to transport, simple to use and operate, and can effectively control costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of the application technology of phage community extraction to promote plant growth.

[0020] Figure 2 It is the number of Arabidopsis pods 21 days after the bacterial agent was injected into the soil of the experimental base.

[0021] Figure 3It is the average content of auxin in the soil of the experimental base 21 days after the bacterial agent was injected into the soil.

[0022] Figure 4 It is the change of bacterial α diversity in the soil of the experimental base 21 days after the injection of bacterial agent.

[0023] Figure 5 It is the fresh weight of the aboveground part of lettuce 28 days after the injection of bacterial agent into the soil at Fengqiu Station in Henan Province.

[0024] Figure 6 It is the average content of auxin in the soil at Fengqiu Station in Henan Province 28 days after the injection of bacterial agent.

[0025] Figure 7 It is the change of bacterial α diversity in the soil at Fengqiu Station in Henan Province 28 days after the injection of bacterial agent.

[0026] Figure 8 It is the fresh weight of the aboveground part of cabbage 28 days after the microbial agent was injected into the soil of the vegetable base in Zhangye City, Gansu Province.

[0027] Fig. 9 It is the average content of auxin in the soil of the vegetable base in Zhangye City, Gansu Province 28 days after the injection of bacterial agents.

[0028] Fig.10 It is the change of bacterial α diversity in the soil 28 days after the injection of microbial agent in the soil of the vegetable base in Zhangye City, Gansu Province.

[0029] Fig.11 It is the fresh weight of chili pepper in the soil of the vegetable base in Wuhan City, Hubei Province 90 days after the injection of bacterial agent.

[0030] Fig.12 It is the average content of auxin in the soil of the vegetable base in Wuhan City, Hubei Province 90 days after the injection of bacterial agents.

[0031] Fig.13 It is the change of bacterial alpha diversity in the soil of the vegetable base in Wuhan City, Hubei Province 90 days after the injection of microbial agents. DETAILED DESCRIPTION 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.

[0032] Embodiment 1: The soil for the test potting was collected from the soil of the experimental base. The plant was Columbia type Arabidopsis thaliana ( Arabidopsis thaliana (Columbia) Basic physical and chemical properties of soil: sand 20.1%, soil 36.8%, clay 37.4%, pH 7.18, total nitrogen 1.61 g·kg -1 , water-soluble nitrogen 1.55 g·kg-1 , total phosphorus 1.63 g·kg -1 , total potassium 16.48 g·kg -1 , CEC 12.76 cmol·kg -1 .

[0033] Weigh 1000.0 g of in situ soil and divide it into four 500 mL conical flasks. Add 250 mL of phage buffer (68 mM NaCl, 10 mM MgSO4, Tris-HCl pH 7.5) to the sample, mix thoroughly for 20 minutes and centrifuge. The supernatant obtained by centrifugation was filtered using a tangential flow filtration system. The enriched solution obtained by passing through a 0.2 μm filter membrane was the bacterial community, and the filtrate was further passed through a 30 kDa filter membrane to obtain the enriched solution of the phage community. Mitomycin C was then added to some single bacterial communities to a final concentration of 0.1 μg mL -1 After shaking culture at room temperature for 24 hours, the cells were enriched again using a 30 kDa filter to obtain a highly lysogenic phage community.

[0034] Four treatment groups were set up in the experiment: ① Control group (CK): 3 Arabidopsis plants were planted in each pot (0.5-1 cm soil was covered on the seeds, and the room temperature was 23 ± 1℃); ② Bacterial community transplantation only (B1): 100 mL of isolated bacterial community extract was inoculated on the basis of the control group; ③ Bacterial-phage mixed solution transplantation (B1V1): 100 mL of isolated bacterial community extract and 15 mL of isolated phage community extract were inoculated on the basis of the control group; ④ Bacterial-lysogenic phage mixed solution transplantation (B1P1): 100 mL of isolated bacterial community extract and 10 mL of isolated phage community extract were inoculated on the basis of the control group. After the 21st day of growth of Arabidopsis, the number of pods of Arabidopsis under the four treatments of CK, B1, B1V1, and B1P1 were 14.2 / plant, 19.4 / plant, 23.5 / plant, and 27.1 / plant, respectively ( Figure 2 The number of Arabidopsis pods in the B1P1 treatment group was significantly higher than that in the control group ( p <0.05). The average content of auxin in the soil of the treatment group and the control group was 0.06 μg·mL -1 , 0.13 μg·mL -1 , 0.18 μg·mL -1 , 0.26 μg·mL -1 ( Figure 3 ), the auxin content in the B1P1 treatment was significantly increased compared with the control group (p <0.05). At the same time, the 16S assay of rhizosphere soil revealed that the α diversity of bacteria in the four treatments of CK, B1, B1V1, and B1P1 were 35.31, 35.88, 36.26, and 36.72, respectively (Shannon index, Figure 4 The overall results showed that after 21 days of culture, the number of Arabidopsis pods increased by 12.9 per plant after mixed bacterial-lysogenic phage community transplantation compared with the treatment without phage transplantation. At the same time, the increase in microbial diversity in the B1P1 treatment indicated that phage community transplantation significantly increased the yield of Arabidopsis (increase in the number of pods) and was beneficial to the stability of the microbial community.

[0035] Embodiment 2: Target soil: Basic physical and chemical properties of soil: pH 6.8, organic matter 21.1 g kg -1 , total nitrogen 4.8 g·kg -1 , total phosphorus 2.1 g kg -1 The mechanical composition of the soil is 45.1% sand (sandy soil), 22.8% soil particles, and 32.1% clay particles.

[0036] The method for preparing highly lysogenic phage colonies is the same as that in Example 1.

[0037] Four treatment groups were set up in the experiment: ① Control group (CK): 3 lettuces were planted in each pot (0.5-1 cm soil was covered on the seeds, and the room temperature was 25 ± 1 ℃); ② Bacterial community transplantation only (B1): 100 mL of isolated bacterial community extract was inoculated on the basis of the control group; ③ Bacterial-phage mixture transplantation (B1V1): 100 mL of isolated bacterial community extract and 15 mL of isolated phage community extract were inoculated on the basis of the control group; ④ Bacterial-lysogenic phage mixture transplantation (B1P1): 100 mL of isolated bacterial community extract and 10 mL of isolated phage community extract were inoculated on the basis of the control group; The aboveground fresh weight of lettuce was measured after 28 days of growth. The average aboveground fresh weight of lettuce in the four treatments of CK, B1, B1V1, and B1P1 was 30.22 g / piece, 31.43 g / piece, 34.99 g / piece, and 39.28 g / piece, respectively. Figure 5 The aboveground fresh weight of lettuce in the B1P1 treatment group was significantly higher than that in the control group ( p <0.05). The average content of auxin in the soil was determined to be 0.06 μg·mL for the treatment group and the control group. -1 , 0.16 μg·mL -1 , 0.20 μg·mL -1 , 0.27 μg·mL -1 ( Figure 6The auxin content in the B1P1 treatment was significantly increased compared with the control group ( p <0.05). At the same time, the 16S assay of rhizosphere soil revealed that the α diversity of bacteria in the four treatments of CK, B1, B1V1, and B1P1 were 30.88, 31.05, 31.76, and 32.12, respectively (Shannon index, Figure 7 ). After 28 days of mixed bacterial-phage community transplantation, the fresh weight of the aboveground part of lettuce increased by 4.77 g / head compared with the treatment without phage transplantation. At the same time, the increase in microbial diversity in the B1P1 treatment indicated that phage community transplantation significantly increased the yield of lettuce (increase in aboveground fresh weight) and was beneficial to the stability of the microbial community.

[0038] Embodiment 3: Target soil: Vegetable base soil. Basic physical and chemical properties of soil: pH 5.8, organic matter 25.1 g kg -1 , total nitrogen 6.8 g·kg -1 , total phosphorus 2.7 g·kg -1 The mechanical composition of the soil is 39.1% sand (sandy soil), 22.8% soil particles, and 38.1% clay particles.

[0039] The method for preparing highly lysogenic phage colonies is the same as that in Example 1.

[0040] Four treatment groups were set up in the experiment: ① Control group (CK): 3 cabbages were planted in each pot (0.5-1 cm soil was covered on the seeds, and the room temperature was 20 ± 2℃); ② Bacterial community transplantation only (B1): 100 mL of isolated bacterial community extract was inoculated on the basis of the control group; ③ Bacteria-phage mixture transplantation (B1V1): 100 mL of isolated bacterial community extract and 15 mL of isolated phage community extract were inoculated on the basis of the control group; ④ Bacteria-lysogenic phage mixture transplantation (B1P1): 100 mL of isolated bacterial community extract and 10 mL of isolated phage community extract were inoculated on the basis of the control group; The aboveground fresh weight of cabbage was measured after 30 days of growth. The average aboveground fresh weight of cabbage in the four treatments of CK, B1, B1V1, and B1P1 was 35.46 g / head, 38.73 g / head, 48.84 g / head, and 42.13 g / head, respectively ( Figure 8 ) The aboveground fresh weight of cabbage in the B1P1 treatment group was significantly higher than that in the control group ( p <0.05). The average content of auxin in the soil was determined to be 0.03 μg·mL for the treatment group and the control group. -1 , 0.17 μg·mL -1 , 0.22 μg·mL -1 , 0.29 μg·mL-1 ( Fig. 9 The auxin content in the B1P1 treatment was significantly increased compared with the control group ( p <0.05). At the same time, the 16S test of rhizosphere soil revealed that the α diversity of bacteria in the four treatments of CK, B1, B1V1, and B1P1 were 31.78, 32.18, 32.69, and 32.99, respectively (Shannon index, Fig.10 ). After 28 days of mixed bacterial-phage community transplantation, the fresh weight of the aboveground part of cabbage increased by 6.67 g / head compared with the treatment without phage transplantation. At the same time, the increase in microbial diversity in the B1P1 treatment indicated that phage community transplantation significantly increased the yield of cabbage (increase in aboveground fresh weight) by increasing the content of auxin in the soil and was beneficial to the stability of the microbial community.

[0041] Embodiment 4: Target soil: Vegetable base soil. Basic physical and chemical properties of soil: pH 5.8, organic matter 25.1 g kg -1 , total nitrogen 6.8 g·kg -1 , total phosphorus 2.7 g·kg -1 The mechanical composition of the soil is 39.1% sand (sandy soil), 22.8% soil particles, and 38.1% clay particles.

[0042] The method for preparing highly lysogenic phage colonies is the same as that in Example 1.

[0043] Four treatments were set up in the experiment: ① Control group (CK): 3 chili peppers were planted in each pot (0.5-1 cm of soil was covered on the seeds, and the room temperature was 27 ± 2℃); ② Bacterial community transplantation only (B1): 100 mL of isolated bacterial community extract was inoculated on the basis of the control group; ③ Bacterial-phage mixture transplantation (B1V1): 100 mL of isolated bacterial community extract and 15 mL of isolated phage community extract were inoculated on the basis of the control group; ④ Bacterial-lysogenic phage mixture transplantation (B1P1): 100 mL of isolated bacterial community extract and 10 mL of isolated phage community extract were inoculated on the basis of the control group; The aboveground fresh weight of cabbage was measured on the 30th day of growth. The average aboveground fresh weight of lettuce in the four treatments of CK, B1, B1V1, and B1V10 were 10.10 g / head, 10.92 g / head, 11.82 g / head, and 13.13 g / head, respectively ( Fig.11 The fresh weight of pepper in the soil of the B1V1 treatment group was significantly increased compared with that of the control group ( p <0.05). The average content of auxin in the soil was determined to be 0.04 μg·mL for the treatment group and the control group. -1 , 0.20 μg·mL -1, 0.25 μg·mL -1 , 0.31 μg·mL -1 ( Fig.12 The auxin content in the B1V1 treatment was significantly increased compared with the control group ( p <0.05). At the same time, the 16S test of rhizosphere soil revealed that the α diversity of bacteria in the four treatments of CK, B1, B1V1, and B1P1 were 33.38, 33.95, 34.59, and 35.00, respectively (Shannon index, Fig.13 ). After 90 days of culture, the fresh weight of pepper increased by 3.03 g / piece compared with the treatment without phage transplantation. At the same time, the increase in microbial diversity in the B1P1 treatment indicated that phage community transplantation significantly increased the yield of cabbage (increase in fresh weight of aboveground parts) by increasing the content of auxin in the soil, and was beneficial to the stability of the microbial community.

Claims

1. A method for extracting a lysogenic phage community, characterized in that: The steps are as follows: the in situ soil and the buffer solution are mixed evenly to obtain a soil suspension, the supernatant is collected after centrifugation and allowed to stand, and the bacterial and phage communities are separated using tangential flow systems with different pore sizes: first, the supernatant is filtered through a 0.2 μm filter membrane to obtain an enrichment solution as a bacterial suspension, and then mitomycin C is added to the bacterial suspension to a final concentration of no more than 0.1 μg mL -1 , cultured under shaking at room temperature, and enriched again using a 30-100 kDa filter membrane to obtain a lysogenic phage community.

2. The method for extracting a lysogenic phage community according to claim 1, characterized in that: The volume ratio of the in situ soil to the buffer solution is 1:1, and the buffer solution is Tris-HCl at pH 7.5, containing 68 mM NaCl and 10 mM MgSO4.

3. The method for extracting a lysogenic phage community according to claim 1, characterized in that: The centrifugation conditions are 4°C and 4500 G for 20 minutes.

4. A lysogenic phage community obtained by the extraction method according to any one of claims 1 to 3.

5. Use of the lysogenic phage community according to claim 4 in preparing a product for enhancing plant growth.

6. The use according to claim 5, characterized in that: The plant is Arabidopsis thaliana, Chinese cabbage, lettuce or chili pepper.

7. A soil conditioner for enhancing plant growth, characterized in that: Contains the lysogenic phage community as claimed in claim 4, and biologically active ingredients that are helpful for plant growth and development.

8. The use of the soil conditioner according to claim 7, characterized in that: Inject soil conditioner into target soil.

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