Pseudomonas with salt-tolerant nitrogen-fixing and phosphorus-dissolving capabilities and its application

Abstract

The present invention relates to a Pseudomonas with salt-tolerant nitrogen-fixing and phosphorus-dissolving abilities and its application, and belongs to the technical field of agricultural microorganisms. The salt-tolerant growth-promoting bacteria with nitrogen-fixing and phosphorus-dissolving abilities are Pseudomonas sp., with a strain number of YJ42 and a deposit number of CCTCC M 2024914, which was deposited in the China Center for Type Culture Collection, Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province on May 13, 2024. The strain obtained in the present invention is derived from the rhizosphere soil of the halophyte Suaeda salsa in the saline-alkali land of the Yellow River Delta, and is obtained after screening and culturing. The strain obtained in the present invention has a strong ability to fix nitrogen and dissolve phosphorus, and its bacterial solution can significantly improve the salt tolerance of alfalfa in saline-alkali land, promote its growth, and increase its biomass; the bacterial solution involved in the present invention can reduce the degree of salinization of the land while promoting the salt-tolerant growth of alfalfa, and improve the saline-alkali land.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural microorganisms, in particular to a Pseudomonas bacterium with salt-tolerant nitrogen-fixing and phosphorus-dissolving capabilities and an application thereof. Background Art

[0002] Soil salinization has varying degrees of damage to agricultural productivity and ecosystem diversity: it not only reduces soil fertility, but also affects the normal growth and development of plants. At present, the selection, cultivation, and planting of salt-tolerant plant varieties and the application of microorganisms provide an opportunity to rationally improve salt stress. However, due to the long and uncertain process of variety breeding, the application of microorganisms has received more extensive attention. The application of microorganisms refers to the use of microorganisms with important functions or the search for suitable salt-tolerant strains, making bacterial liquids or solid agents and adding them to saline soils or planted crops to form a plant-microorganism co-repair system to resist salt stress problems.

[0003] Plant growth promoting rhizobacteria (PGPR) are a type of microorganisms that can coexist with plants. They can promote plant growth and development and improve plant stress resistance in various ways, and are widely used to solve salt stress problems. At present, the most studied bacteria with salt tolerance are mainly Pseudomonas, Bacillus, Azospirillum, etc. Pseudomonas, as the PGPR with the largest population and the most application prospects, can improve the adaptability of plants to adverse stress in various ways. However, there is still a shortage of bacterial species that can be actually applied to social production. In particular, Pseudomonas that can not only adapt to high-salinity environments, but also effectively promote the salt-tolerant growth of plants planted in saline-alkali lands, and can improve soil for the purpose of soil remediation and their applications are still rare. Summary of the invention

[0004] In view of the defects and shortcomings of the prior art, the present invention provides a Pseudomonas strain with salt-tolerant nitrogen-fixing and phosphorus-dissolving capabilities, which is convenient for promoting the salt-tolerant growth of plants in saline-alkali land and providing scientific support for the improvement and restoration of saline-alkali land.

[0005] The purpose of the present invention is to provide a Pseudomonas sp. YJ42 strain which has good activity under the condition of high salt and can promote crop growth and nitrogen fixation and application thereof.

[0006] Another object of the present invention is to provide a salt-tolerant growth-promoting bacterial agent or a plant growth-promoting bacterial agent.

[0007] Another object of the present invention is to provide a method for promoting the salt-tolerant growth of plants in saline-alkali land.

[0008] Another object of the present invention is to provide a method for improving / repairing saline-alkali soil.

[0009] Another object of the present invention is to provide the use of Pseudomonas sp. YJ42 or its bacterial solution in nitrogen fixation and / or phosphorus solubilization.

[0010] The above object of the present invention is achieved through the following technical solutions:

[0011] In the first aspect, the present invention provides a Pseudomonas sp. with salt-tolerant, nitrogen-fixing and phosphorus-dissolving capabilities screened from the rhizosphere soil of the halophyte Suaeda salsa in the saline-alkali land of the Yellow River Delta. The strain number is YJ42, which was preserved in the China Type Culture Collection at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province on May 13, 2024, with the preservation number CCTCC M2024914, and its nucleotide sequence is shown in SEQ ID NO. 1.

[0012] The colony morphology of the YJ42 strain is light yellow, with a smooth and opaque surface, a circle in the middle of the colony, and neat edges; its suitable growth conditions are: temperature of 28°C, pH range of 7.2-7.4, and inoculum range of 3-4%.

[0013] In a second aspect, the present invention provides a salt-tolerant growth-promoting bacterial agent or a plant growth-promoting bacterial agent prepared from Pseudomonas sp. YJ42 or its bacterial solution.

[0014] Preferably, the bacterial solution concentration is not less than 4×10 9 cfu / mL.

[0015] In a third aspect, the present invention provides a method for promoting the salt-tolerant growth of plants in saline-alkali land, comprising treating the plants with Pseudomonas sp. YJ42 strain.

[0016] Preferably, the concentration of the YJ42 strain is not less than 4×10 9 cfu / mL, and the inoculum size was 3-4%.

[0017] In a fourth aspect, the present invention also provides a method for improving / repairing saline-alkali soil, using Pseudomonas sp. YJ42 strain to treat the saline-alkali soil.

[0018] The YJ42 strain of the present invention has good salt tolerance and can promote plant growth or promote plant growth in a saline-alkali soil environment.

[0019] After the YJ42 strain was prepared into a bacterial agent, alfalfa was planted on saline-alkali soil and the bacterial agent YJ42 was added, showing that the YJ42 bacterial agent can promote the growth of alfalfa. Compared with the blank control, the plant height, root length and dry weight of alfalfa treated with YJ42 were increased. Among them, under a salt gradient of 6‰, the plant height, root length and dry weight of alfalfa increased by 79.60%, 6.86% and 174.38% respectively. Therefore, the YJ42 bacterial agent can be used to improve the soil fertility of saline-alkali land or improve / repair saline-alkali soil, which has important economic value and practical significance for promoting crop growth and improving the soil quality of saline-alkali land.

[0020] In a fifth aspect, the YJ42 strain described in the present invention can grow in Ashby nitrogen-free medium inorganic phosphorus medium, proving that it has nitrogen fixing and phosphorus solubilizing properties and can be used for nitrogen fixing and / or phosphorus solubilizing in saline-alkali soil environments.

[0021] Therefore, the following applications of Pseudomonas sp. YJ42 are all within the scope of protection of the present invention:

[0022] Application of Pseudomonas sp. YJ42 or its bacterial solution in the preparation of salt-tolerant growth-promoting bacterial agents or plant growth-promoting bacterial agents.

[0023] Application of Pseudomonas sp. YJ42 or its bacterial solution in promoting plant growth or promoting plant growth under salt stress.

[0024] Application of Pseudomonas sp. YJ42 or its bacterial solution in improving / restoring saline-alkali land.

[0025] Application of Pseudomonas sp. YJ42 or its bacterial solution in nitrogen fixation and / or phosphorus solubilization.

[0026] Application of Pseudomonas sp. YJ42 or its bacterial solution in nitrogen fixation and / or phosphorus solubilization in saline-alkali soil environment.

[0027] Application of Pseudomonas sp. YJ42 or its bacterial solution in preparing a preparation for increasing the nitrogen and phosphorus content in soil.

[0028] Beneficial effects of the embodiments of the present invention:

[0029] The present invention provides a Pseudomonas sp. YJ42 with salt-tolerant nitrogen-fixing and phosphorus-dissolving capabilities, which can be prepared into a salt-tolerant growth promoter or a plant growth promoter. The biological characteristics of the strain, such as salt tolerance, are analyzed. The strain has good activity when the salt concentration is below 6‰, which has important practical significance and value for improving saline-alkali soil quality and ecological environment.

[0030] The Pseudomonas sp. YJ42 provided by the invention is pollution-free and harmless during use, can have both nitrogen fixation and phosphorus solubility under salt stress, and can significantly improve the salt tolerance of alfalfa, alleviate salt damage, increase biomass, promote the growth of alfalfa, and improve the physical and chemical properties of saline-alkali soil to a certain extent, thereby not only increasing production but also improving soil. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 A colony morphology diagram of strain YJ42 provided in an embodiment of the present invention;

[0032] Figure 2 A phylogenetic tree of strain YJ42 provided in an embodiment of the present invention;

[0033] Figure 3 The optimal growth curve diagram of strain YJ42 provided in the embodiment of the present invention;

[0034] Figure 4 A graph showing the salt tolerance test results of the strain YJ42 provided in an embodiment of the present invention;

[0035] Figure 5 A salt tolerance curve diagram of strain YJ42 provided in an embodiment of the present invention;

[0036] Figure 6 This is a graph showing the nitrogen-fixing and phosphorus-dissolving ability test results of the strain YJ42 provided in the embodiment of the present invention.

[0037] A is the growth graph of Pseudomonas YJ42 in nitrogen-free medium, and B is the growth graph of Pseudomonas YJ42 in inorganic phosphorus medium;

[0038] Figure 7 The plant height of alfalfa under different treatments provided by the embodiment of the present invention;

[0039] Figure 8 The root length of alfalfa under different treatments provided by the embodiment of the present invention;

[0040] Fig. 9 The biomass of alfalfa under different treatments provided in the embodiments of the present invention. DETAILED DESCRIPTION

[0041] Embodiments of the present embodiment will be described in more detail below with reference to the accompanying drawings. Although certain embodiments of the present embodiment are shown in the accompanying drawings, it should be understood that the present embodiment can be implemented in various forms and should not be construed as being limited to the embodiments set forth herein. Instead, these embodiments are provided to provide a more thorough and complete understanding of the present embodiment. It should be understood that the drawings and embodiments of the present embodiment are only for exemplary purposes and are not intended to limit the scope of protection of the present embodiment.

[0042] In the embodiment of the present invention, the salt-tolerant growth-promoting bacteria strain is referred to as strain YJ42. The salt-tolerant growth-promoting bacteria provided in the embodiment of the present invention can be mixed with organic fertilizer or applied alone, and can be used as base fertilizer, basal fertilizer or topdressing fertilizer.

[0043] The salt-tolerant growth-promoting bacteria provided in the embodiment of the present invention are Pseudomonas sp., with strain number YJ42 and preservation number CCTCC M 2024914, which was preserved in the China Type Culture Collection Center, Luojiashan, Bayi Road, Wuchang District, Wuhan City, Hubei Province on May 13, 2024.

[0044] Figure 1 A colony morphology diagram of strain YJ42 provided in an embodiment of the present invention;

[0045] The picture shows the colony morphology of Pseudomonas YJ42 after being cultured at 28℃ for 48 hours. The colonies are light yellow, with a smooth and opaque surface, a circle in the middle of the colony, and neat edges.

[0046] Figure 2 A phylogenetic tree of strain YJ42 provided in an embodiment of the present invention;

[0047] The figure shows a phylogenetic tree constructed using MEGA after blast comparison of strain sequences on the NCBI website. Figure 2 As shown, strain YJ42 is closely related to Pseudomonas (accession number: AM990895.1).

[0048] Figure 3 The optimal growth curve diagram of strain YJ42 provided in the embodiment of the present invention;

[0049] The figure shows the absorbance of strain YJ42 at 600 nm after culturing at 28°C and 180 r / min for 48 h. Appropriate amount of bacterial solution was taken every 4 h and each sample was measured in triplicate. Figure 3 As shown, 4-24 h is the logarithmic growth phase of strain YJ42, which shows an increasing trend and tends to be stable after 24 h of growth.

[0050] Example 1 Test of salt tolerance of Pseudomonas YJ42

[0051] The activated Pseudomonas YJ42 was streaked and inoculated into NA medium with salt contents of 0, 2, 4, 6, 8, and 10‰, with a total of 5 replicates. The culture was incubated in a biochemical incubator for 24 hours at 28°C to observe its salt tolerance. Figure 4 As shown, Pseudomonas YJ42 grew well at salt contents of 0, 2, 4, 6, 8, and 10‰, respectively.

[0052] In this embodiment, the formula and dosage of the NA medium are: 3.0 g beef powder, 5.0 g tryptone, 18.0 g agar, pH = 7.2-7.4, and 1000 mL distilled water.

[0053] The activated Pseudomonas YJ42 was inoculated into a 250 mL conical flask containing 100 mL NB medium, and placed in a temperature-controlled shaker for 24 h. The temperature was set to 28°C and the speed was set to 180 r / min. The seed solution was prepared for use. The seed solution was inoculated into NB medium with different salt contents at a 4% inoculation rate, with a total of 5 replicates. The culture was shaken in a temperature-controlled shaker for 24 h. The temperature was set to 28°C and the speed was set to 180 r / min. The OD value was measured to determine the salt tolerance of the strain. Figure 5 The salt tolerance curve shown in the figure, the ordinate is the bacterial concentration (OD600) and the abscissa is the salt content.

[0054] In this embodiment, the formula and dosage of the NB culture medium are: 3.0 g beef powder, 5.0 g tryptone, pH=7.2-7.4, and 1000 mL distilled water.

[0055] like Figure 5 As shown, Pseudomonas YJ42 still maintains a high bacterial concentration when the salt content is 10‰.

[0056] pass Figure 5 The results showed that Pseudomonas YJ42 had a wide adaptability to NaCl and could maintain a high bacterial concentration even when the salt concentration reached 10‰.

[0057] Example 2 Test of nitrogen fixation and phosphorus dissolving ability of Pseudomonas YJ42

[0058] The activated Pseudomonas YJ42 was streaked and inoculated into Ashby nitrogen-free medium, with a total of 5 replicates, and cultured in a biochemical incubator for 48 hours at 28°C to observe its nitrogen fixation ability. Figure 6 As shown, A indicates that Pseudomonas YJ42 grows well in the culture medium, proving that it has a certain nitrogen fixation ability.

[0059] In this embodiment, the formula and dosage of the above-mentioned Ashby nitrogen-free culture medium are: KH2PO4 0.04 g, H2SO4 0.04 ml, NaCl 0.04 g, CaCO3 1.0 g, mannitol 2.0 g, CaSO4 0.02 g, agar 3.6 g, pH = 7.0 ± 0.2, and distilled water 200 mL.

[0060] The activated Pseudomonas YJ42 was streaked and inoculated into an inorganic phosphorus medium, with a total of 5 replicates, and cultured in a biochemical incubator for 48 hours at 28°C to observe whether there was a transparent circle around the colony. Figure 6 As shown in FIG. 1 , B shows that Pseudomonas YJ42 grows well in the culture medium and has an obvious transparent zone, which proves that it has a certain ability to dissolve phosphate.

[0061] In this embodiment, the formula and dosage of the above-mentioned inorganic phosphorus culture medium are: MgCl2·6H2O 1.0 g, (NH4)2·SO40.02 g, MgSO4·7H2O 0.05 g, KCl 0.04 g, Ca3(PO4)21.0 g, glucose 2.0 g, agar 3.6-4.0 g, pH=7.0~7.5, and distilled water 200 mL.

[0062] Example 3 Application of Pseudomonas YJ42 to Alfalfa Growth in Saline-Alkaline Soil

[0063] In this embodiment, the salt-tolerant growth-promoting bacteria agent is used as an example to promote the salt-tolerant growth of alfalfa.

[0064] 1. Preparation of Pseudomonas YJ42 inoculant

[0065] The activated Pseudomonas YJ42 was inoculated into a 250 mL conical flask containing 100 mL NB medium, and placed in a temperature-controlled shaker for 24 h, with the temperature set at 28°C and the speed set at 180 r / min to prepare the seed solution. The seed solution was inoculated into a 500 mL conical flask containing 300 mL NB medium at a 3-4% inoculation rate, and was incubated in a temperature-controlled shaker for 24 h, with the temperature set at 28°C and the speed set at 180 r / min. The cultured fermentation broth was then placed in a centrifuge for centrifugation and the supernatant was discarded. The precipitated bacteria were collected, and the centrifugation temperature was set at 4°C and the speed was set at 10,000 r / min.

[0066] The collected precipitated cells were resuspended in sterile water (OD 600 =2±0.01), and the effective viable count of Pseudomonas YJ42 reached 4×10 9 cfu / mL of salt-tolerant growth-promoting bacteria.

[0067] 2. Effect of Pseudomonas YJ42 on the growth promotion of alfalfa under salt stress

[0068] In order to verify the nitrogen-fixing and phosphorus-dissolving ability of Pseudomonas YJ42 in soils with different salt contents and the effect of promoting the salt-tolerant growth of alfalfa, the soil used in this example is a mixed nutrient soil (nutrient soil: vermiculite: perlite = 3:2:1) sterilized at high temperature in a sterilizing pot. This experiment set up 4 salt gradients, namely 0‰, 2‰, 4‰ and 6‰; this experiment set up 2 treatments, namely adding 4×10 9 cfu / mL of YJ42 inoculant and a control group without the addition of the inoculant, with a total of 5 replicates for each treatment. Alfalfa seeds with full grains, uniform size and no pests were selected for germination in an incubator. The culture conditions were set as a photoperiod of 16 h, a temperature of 28 °C, a dark treatment of 8 h, a temperature of 22 °C, and a humidity of 50-80%. Alfalfa seedlings with uniform growth after germination for 4 days were selected and transplanted into plastic pots for experiments. Fifteen alfalfa seedlings were planted in each pot. Thinning was performed after the first compound leaf of the alfalfa seedling was fully expanded to ensure that there were 10 alfalfa seedlings of uniform size in each pot.

[0069] Treatment with the microbial agent group: 100 mL of water was applied once on the 21st, 28th, and 35th days after the alfalfa seedlings were transplanted into the pots. The effective number of live bacteria was 4×10 8 cfu / mL of YJ42 bacterial agent;

[0070] Treatment of the control group: Water the alfalfa seedlings with 100 mL of sterile water on the 21st, 28th and 35th days after they were transplanted into the pots.

[0071] In this embodiment, the growth conditions of alfalfa seedlings in the YJ42 bacterial agent group and the control group are as follows: Figure 7 shown.

[0072] Measure the plant height 7 days after the last YJ42 inoculant and sterile water irrigation. Figure 8 As shown, after the last YJ42 fungicide and sterile water irrigation for 7 days, the total biomass of the plants and other indicators were measured. Fig. 9 shown.

[0073] The results showed that the plant height and total biomass of alfalfa increased after the addition of the bacterial agent. Among them, under a salt gradient of 6‰, the plant height, root length and biomass of alfalfa were 79.06%, 6.86% and 174.38% higher than those of alfalfa not inoculated with bacteria, respectively. The application of YJ42 bacterial agent can significantly improve the salt tolerance and growth-promoting ability of alfalfa under salt stress, slow down the salt damage phenomenon, increase biomass, and thus promote the growth of alfalfa. It also improves the physical and chemical properties of saline-alkali soil to a certain extent, which not only increases production but also improves soil.

[0074] The above embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of patent protection. It should be noted that for ordinary technicians in this field, some modifications and improvements can be made without departing from the concept of the present invention, which are all included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. An application of Pseudomonas sp. YJ42 or its bacterial solution in promoting the growth of alfalfa under salt stress, characterized in that: The strain number of Pseudomonas sp. is YJ42, the preservation number is CCTCC M2024914, and it was deposited in the China Type Culture Collection Center, Luojiashan, Bayi Road, Wuchang District, Wuhan City, Hubei Province on May 13, 2024.

2. Use of Pseudomonas sp. YJ42 according to claim 1 in the preparation of alfalfa salt-tolerant growth-promoting bacterial agent.

3. Use of the alfalfa salt-tolerant growth-promoting bacterial agent according to claim 2 in promoting the salt-tolerant growth of alfalfa.

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