CN118667729B - Pseudomonas with salt-tolerant nitrogen fixation and phosphorus dissolution capabilities and application thereof - Google Patents
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Abstract
The invention relates to pseudomonas with salt-tolerant nitrogen fixation and phosphorus dissolution capabilities and application thereof, and belongs to the technical field of agricultural microorganisms. The salt-tolerant growth-promoting bacteria with the capability of fixing nitrogen and dissolving phosphorus are Pseudomonas sp, the strain number is YJ42, the preservation number is CCTCC M2024914, and the salt-tolerant growth-promoting bacteria are preserved in the eight-path Lopa nationality China center of typical culture collection of Wuchang district in Wuhan, hubei province on the 13 th day of 2024. The strain is obtained from rhizosphere soil of halophyte suaeda salsa in yellow river delta saline-alkali soil, and is obtained after screening and culturing. The bacterial strain obtained by the invention has stronger nitrogen and phosphorus dissolving capacity, the bacterial liquid can obviously improve the salt tolerance of alfalfa in saline-alkali soil, promote the growth of alfalfa and increase the biomass of alfalfa, and the bacterial liquid can reduce the salinization degree of the soil and improve the saline-alkali soil while promoting the salt tolerance growth of alfalfa.
Description
Technical Field
The invention relates to the technical field of agricultural microorganisms, in particular to pseudomonas with salt-tolerant nitrogen fixation and phosphorus dissolution capabilities and application thereof.
Background
Soil salinization has different degrees of damage to agricultural productivity, ecological system diversity and the like, and not only reduces soil fertility, but also influences normal growth and development of plants. At the present stage, the application of the breeding, the culture and the planting of the salt-tolerant plant variety and the microorganism provides a opportunity for reasonably improving the salt stress. However, the use of microorganisms has gained much attention due to the long and uncertain variety breeding processes. The application of the microorganism refers to that the microorganism with important functions or the proper salt-tolerant strain is searched, and the prepared bacterial liquid or solid bacterial agent is added into saline soil or planted crops to form a plant-microorganism common restoration system to resist the salt stress problem.
Plant rhizosphere growth promoting bacteria (plant growth promoting rhizobacteria, PGPR) are a class of microorganisms capable of symbiotic with plants, which can promote the growth and development of plants through various ways, improve the stress resistance of plants, and are widely used for solving the problem of salt stress. The genera having salt tolerance have been studied recently mainly in Pseudomonas (Pseudomonas), bacillus (Bacillus), azospirillum (Azospirillum), etc. Pseudomonas is used as the PGPR with the largest population quantity and the most application prospect, and the adversity stress adaptability of plants can be improved through various ways. However, strain resources which can be practically applied to social production at present are still deficient, and particularly, the pseudomonas which can not only adapt to high salinity environment, but also effectively promote salt-tolerant growth of planted plants in saline-alkali soil and improve soil to achieve the aim of soil remediation and the application of the pseudomonas are still not seen.
Disclosure of Invention
Aiming at the defects and shortcomings of the prior art, the invention provides the pseudomonas strain with the salt-tolerant nitrogen fixation and phosphorus dissolution capabilities, which is convenient for promoting the salt-tolerant growth of saline-alkali soil plants and providing scientific support for the improvement and repair of the saline-alkali soil.
The invention aims to provide a Pseudomonas sp YJ42 strain which has good activity under the condition of high salinity and can promote the growth of crops to fix nitrogen and application thereof.
The invention also aims to provide a salt-tolerant growth-promoting microbial agent or a plant growth-promoting microbial agent.
It is still another object of the present invention to provide a method for promoting salt tolerant growth of plants in saline-alkali soil.
It is still another object of the present invention to provide a method for improving/remediating saline-alkali soil.
The invention also aims to provide application of the Pseudomonas sp YJ42 or bacterial liquid thereof in nitrogen fixation and/or phosphorus dissolution.
The above object of the present invention is achieved by the following technical solutions:
In a first aspect, the invention provides a Pseudomonas with salt tolerance, nitrogen fixation and phosphorus dissolution capabilities, wherein the Pseudomonas is screened from saline-alkali plant suaeda salsa rhizosphere soil in yellow river delta saline-alkali soil, and the Pseudomonas is deposited in eight paths of the China center for type culture collection of Lopa nationality in Wuchang district in Wuhan, hubei province on the 13 th month of 2024, with the nucleotide sequence shown as SEQ ID NO.1, and the preservation number is CCTCC M2024914.
The bacterial colony of YJ42 strain is light yellow, smooth and opaque, and has middle ring and regular edge, and its proper growth condition is 28 deg.C, pH range of 7.2-7.4 and inoculation amount range of 3-4%.
In a second aspect, the invention provides a salt-tolerant growth-promoting microbial agent or a plant growth-promoting microbial agent prepared from Pseudomonas sp YJ42 or a microbial liquid thereof.
Preferably, the concentration of the bacterial liquid is not lower than 4X 10 9 cfu/mL.
In a third aspect, the invention provides a method for promoting salt tolerant growth of plants in saline-alkali soil, comprising treating the plants with a Pseudomonas sp.
Preferably, the YJ42 strain concentration is not lower than 4X 10 9 cfu/mL, and the inoculation amount is 3-4%.
In a fourth aspect, the invention also provides a method for improving/repairing saline-alkali soil, which adopts Pseudomonas sp to treat the saline-alkali soil by using the YJ42 strain.
The YJ42 strain has good salt tolerance, and can promote plant growth or promote plant growth in a saline-alkali soil environment.
After the YJ42 strain is prepared into a microbial inoculum, alfalfa is planted on saline-alkali soil, and the microbial inoculum YJ42 is added, so that the YJ42 microbial inoculum can promote the growth of the alfalfa. Compared with the blank control, the plant height, root length and single plant dry weight of the alfalfa treated by YJ42 are improved. Wherein, under the salt gradient of 6 per mill, the plant height, root length and single plant dry weight of alfalfa are respectively improved by 79.60 percent, 6.86 percent and 174.38 percent. Therefore, the YJ42 microbial inoculum can be used for improving the soil fertility of the saline-alkali soil or improving/repairing the saline-alkali soil, and has important economic value and practical significance for promoting the growth of crops and improving the soil quality of the saline-alkali soil.
In a fifth aspect, the YJ42 strain can grow in an Ashby nitrogen-free culture medium inorganic phosphorus culture medium, and has nitrogen fixation and phosphorus dissolution performances, and can be used for fixing nitrogen and/or dissolving phosphorus in a saline-alkali soil environment.
Thus, the following applications of Pseudomonas sp YJ42 are within the scope of the present invention:
application of Pseudomonas sp YJ42 or bacterial liquid thereof in preparing salt-tolerant growth-promoting bacterial agent or plant growth-promoting bacterial agent.
Use of Pseudomonas sp YJ42 or a bacterial liquid thereof for promoting plant growth under promotion of plant growth or salt stress.
Application of Pseudomonas sp YJ42 or bacterial liquid thereof in improving/repairing saline-alkali soil.
Application of Pseudomonas sp YJ42 or bacterial liquid thereof in nitrogen fixation and/or phosphorus dissolution.
Application of Pseudomonas sp YJ42 or bacterial liquid thereof in nitrogen fixation and/or phosphorus dissolution in saline-alkali soil environment.
Application of Pseudomonas sp YJ42 or bacterial liquid thereof in preparation of preparation for improving nitrogen and phosphorus content of soil.
The embodiment of the invention has the beneficial effects that:
The Pseudomonas sp YJ42 with salt tolerance, nitrogen fixation and phosphorus dissolution capabilities can be prepared into a salt tolerance growth promoter or a plant growth promoter, and can be used for analyzing biological characteristics of salt tolerance and the like, and the strain has good activity when the salt concentration is less than 6 per mill and has important practical significance and value for improving the quality of saline-alkali soil and ecological environment;
The Pseudomonas sp YJ42 provided by the invention has no pollution and no public hazard in the use process, can obviously improve the salt tolerance of alfalfa while having the nitrogen fixation and phosphorus dissolution capabilities under salt stress, slows down the salt damage phenomenon, increases the biomass, promotes the alfalfa to grow, improves the physical and chemical properties of saline-alkali soil to a certain extent, and not only plays a role in increasing the yield, but also can improve the soil.
Drawings
FIG. 1 is a colony morphology of strain YJ42 provided in an example of the present invention;
FIG. 2 is a phylogenetic tree of strain YJ42 provided by an embodiment of the present invention;
FIG. 3 is a graph showing the optimal growth of strain YJ42 according to the example of the present invention;
FIG. 4 is a graph showing the results of the salt tolerance test of strain YJ42 provided in the examples of the present invention;
FIG. 5 is a graph showing the salt tolerance of strain YJ42 provided in the examples of the present invention;
FIG. 6 is a graph showing the results of the test of nitrogen and phosphorus fixation capacity of the strain YJ42 provided by the embodiment of the invention,
Wherein A is a growth chart of pseudomonas YJ42 in a nitrogen-free culture medium, and B is a growth chart of pseudomonas YJ42 in an inorganic phosphorus culture medium;
FIG. 7 shows plant heights of alfalfa under different treatments according to an embodiment of the present invention;
FIG. 8 shows root lengths of alfalfa under different treatments according to an embodiment of the present invention;
FIG. 9 shows the biomass of alfalfa under different treatments according to the examples of the present invention.
Detailed Description
Embodiments of the present embodiment will be described in more detail below with reference to the accompanying drawings. While certain embodiments of the present embodiments are illustrated in the accompanying drawings, it is to be understood that the present embodiments may be embodied in various forms and should not be construed as limited to the embodiments set forth herein, but rather, are provided to provide a more thorough and complete understanding of the present embodiments. It should be understood that the drawings and the embodiments of the present embodiments are presented for purposes of illustration only and are not intended to limit the scope of the embodiments.
In the embodiment of the invention, the strain of the salt-tolerant growth-promoting bacteria is called strain YJ42 for short. The salt-tolerant growth-promoting bacteria provided by the embodiment of the invention can be mixed with an organic fertilizer for application, can be independently applied, and can be used as a base fertilizer and a base fertilizer or can be used as an additional fertilizer.
The salt-tolerant growth-promoting bacterium provided by the embodiment of the invention is Pseudomonas sp, the strain number is YJ42, the preservation number is CCTCC M2024914, and the salt-tolerant growth-promoting bacterium is preserved in eight paths of the China center for type culture collection of the Lopa nationality in Wuchang district of Wuhan, hubei province on 13 days in 2024.
FIG. 1 is a colony morphology of strain YJ42 provided in an example of the present invention;
The figure shows the colony morphology of pseudomonas YJ42 after culturing at 28 ℃ for 48 h, the colony is light yellow, the surface is smooth and opaque, the middle of the colony is provided with a circle, and the edge is neat.
FIG. 2 is a phylogenetic tree of strain YJ42 provided by an embodiment of the present invention;
the figure shows a phylogenetic tree constructed by using MEGA after blast alignment by using strain sequences and NCBI of a website. As shown in FIG. 2, strain YJ42 is closely related to Pseudomonas (accession number: AM 990895.1).
FIG. 3 is a graph showing the optimal growth of strain YJ42 according to the example of the present invention;
The graph shows that the strain YJ42 is cultured at 28 ℃ in a shaking way at 180 r/min for 48 h, an appropriate amount of bacterial liquid is taken at intervals of 4h, the absorbance of a sample is measured at the wavelength of 600 nm, and each sample is measured in parallel three times. As shown in FIG. 3, 4-24 h is the log phase of growth of strain YJ42, which is increasing and stable after 24 h.
EXAMPLE 1 test of salt tolerance of Pseudomonas YJ42
The activated pseudomonas YJ42 is streaked and inoculated into NA culture media with salt contents of 0, 2, 4, 6, 8 and 10 per mill respectively, 5 repeats are arranged, and the culture is carried out in a biochemical incubator for 24 hours, the temperature is set to 28 ℃, and the salt tolerance is observed. As shown in FIG. 4, pseudomonas YJ42 grew well at salt contents of 0, 2, 4, 6, 8, and 10%.
In the embodiment, the NA culture medium has the formula and the dosage of 3.0 g parts of beef powder, 5.0 parts of tryptone, g parts of agar, 18.0 g of agar, pH=7.2-7.4 and 1000 parts of distilled water mL.
The activated pseudomonas YJ42 is inoculated into a 250 mL conical flask with the filling amount of 100 mL NB culture medium, placed in a controlled Wen Yaochuang shake culture 24h, the temperature is set to 28 ℃, the rotating speed is set to 180 r/min, and seed liquid is prepared for standby. The seed solution is respectively inoculated into NB culture media with different salt contents according to the inoculum size of 4 percent, 5 repetitions are arranged, the strain is subjected to shake culture at a temperature of Wen Yaochuang ℃ for 24h, the temperature is set to be 28 ℃, the rotating speed is set to be 180 r/min, the OD value is measured, and the salinity tolerance of the strain is determined. A salt tolerance graph as shown in FIG. 5 was obtained, in which the ordinate represents the bacterial concentration (OD 600) and the abscissa represents the salt content.
In the embodiment, the NB medium has a formula and an amount of 3.0 g g of beef powder, 5.0 g of tryptone, pH=7.2-7.4 and distilled water 1000 mL.
As shown in FIG. 5, the Pseudomonas YJ42 still maintains a high cell concentration at a salt content of 10%.
The results of FIG. 5 show that Pseudomonas YJ42 has a wide application range to NaCl, and can still maintain a high thallus concentration under the condition that the salt concentration reaches 10 per mill.
Example 2 test of Nitrogen and phosphorus fixation Capacity of Pseudomonas YJ42
The activated pseudomonas YJ42 was streaked onto Ashby nitrogen-free medium, 5 replicates were set, cultured in a biochemical incubator for 48h at 28 ℃ and observed for nitrogen fixation capacity. As shown in FIG. 6, A is that Pseudomonas YJ42 grows well in the culture medium, and has certain nitrogen fixation capacity.
In this example, the formulation and amount of the Ashby nitrogen-free medium were KH 2PO40.04 g,H2SO40.04 ml,NaCl 0.04 g,CaCO3 1.0.0 g, mannitol 2.0 g, caSO 4 0.02.02 g, agar 3.6 g, pH=7.0.+ -. 0.2, and distilled water 200 mL.
The activated pseudomonas YJ42 was streaked onto an inorganic phosphorus medium, 5 replicates were set, cultured in a biochemical incubator for 48h at 28 ℃ and observed for the presence of a transparent ring around the colonies. As shown in FIG. 6, the result shows that the Pseudomonas YJ42 grows well in the culture medium, has obvious transparent circles, and has certain phosphorus dissolving capacity.
In the embodiment, the formula and the dosage of the 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.
Example 3 application of Pseudomonas YJ42 to alfalfa growth in saline-alkaline soil
In this example, the salt-tolerant growth-promoting microbial agent is alfalfa salt-tolerant growth-promoting.
1. Preparation of Pseudomonas YJ42 microbial inoculum
The activated pseudomonas YJ42 is inoculated into a 250 mL conical flask with the filling amount of 100 mL NB culture medium, placed in a controlled Wen Yaochuang shake culture 24h, the temperature is set to 28 ℃, the rotating speed is set to 180r/min, and seed liquid is prepared. The seed liquid is respectively inoculated into 500 mL conical flasks with 300 mL NB percent of culture medium according to the inoculum size of 3-4 percent, and is subjected to shake culture at a temperature of 28 ℃ and a rotating speed of 180r/min under a control of Wen Yaochuang for 24 h. Then, the cultured fermentation broth was placed in a centrifuge for centrifugation and the supernatant was discarded, and the precipitated cells were collected, the centrifugation temperature was set at 4℃and the rotational speed was set at 10000 r/min.
The collected precipitated bacteria are resuspended in sterile water (OD 600 = 2 + -0.01) to obtain the salt-tolerant growth-promoting bacteria agent with the effective viable count of pseudomonas YJ42 reaching 4X 10 9 cfu/mL.
2. Protoxigenic effect of pseudomonas YJ42 on alfalfa under salt stress
To verify the nitrogen fixation and phosphorus dissolution capabilities of pseudomonas YJ42 in soil with different salt contents and the effect of promoting salt-tolerant growth of alfalfa, the soil used in the embodiment is mixed nutrient soil (nutrient soil: vermiculite: perlite=3:2:1) after high-temperature sterilization in a sterilizing pot. The test is provided with 4 salt gradients, namely 0 permillage, 2 permillage, 4 permillage and 6 permillage, and the test is provided with 2 treatments, namely a bacterial agent group added with YJ42 bacterial agent with the effective viable count of 4 multiplied by 10 9 cfu/mL and a control group not added with the bacterial agent, and each treatment is provided with 5 repetitions. Alfalfa seeds which are full in particles, uniform in size and free of attack by diseases and insects are selected to sprout in an incubator, the culture condition is set to be 16-h% in photoperiod, the temperature is 28 ℃, the dark treatment is 8-h, the temperature is 22 ℃, and the humidity is set to be 50-80%. Selecting alfalfa seedlings with consistent growth vigor after germination for 4 days, transplanting the alfalfa seedlings into a plastic flowerpot for experiment, planting 15 alfalfa seedlings in each pot, and thinning after the first multiple leaves of the alfalfa seedlings are fully unfolded, so that 10 alfalfa seedlings with uniform sizes are ensured to be in each pot.
Treatment of a microbial inoculum group, namely, pouring 100mL of YJ42 microbial inoculum with the effective viable count of 4 multiplied by 10 8 cfu/mL once on the 21 st day, the 28 th day and the 35 th day of the transfer of alfalfa seedlings into a flowerpot;
Treatment of the control group, 100mL pieces of sterile water were poured once on days 21, 28 and 35 of alfalfa seedlings transferred into the pots.
In this example, the growth conditions of alfalfa seedlings in the YJ42 microbial inoculum and the control group are shown in FIG. 7.
The plant height of the plant is measured after 7 days of the last YJ42 microbial inoculum and sterile water irrigation, as shown in FIG. 8, and the total biomass of the plant is measured after 7 days of the last YJ42 microbial inoculum and sterile water irrigation, as shown in FIG. 9.
The result shows that the plant height and the total biomass of the alfalfa are improved after the microbial inoculum is added. Wherein, under the salt gradient of 6 per mill, the plant height, root length and biomass of the alfalfa are 79.06 percent, 6.86 percent and 174.38 percent higher than those of the alfalfa which is not inoculated with bacteria respectively. The application of the YJ42 microbial inoculum can obviously improve the salt tolerance and growth promotion capability of the alfalfa under salt stress, slow down the salt damage phenomenon and increase the biomass, thereby promoting the growth effect of the alfalfa, improving the physicochemical property of the saline-alkali soil to a certain extent, and not only playing a role in increasing the yield, but also improving the soil.
The foregoing examples merely represent several embodiments of the present invention and are described in more detail and are not to be construed as limiting the scope of the claims. It should be noted that variations and modifications can be made by those skilled in the art without departing from the inventive concept, and are included within the scope of the present invention. Accordingly, the scope of the invention is to be determined by the following claims.
Claims (3)
1. The application of Pseudomonas sp YJ42 or bacterial liquid thereof in promoting alfalfa growth under salt stress is characterized in that Pseudomonas sp strain number is YJ42, the preservation number is CCTCC M2024914, and the Pseudomonas sp strain number is preserved in eight paths of the China center for type culture collection of Wuchang district, wuhan, hubei province on the 13 th day in 2024.
2. Use of Pseudomonas sp YJ42 according to claim 1 for the preparation of a salt tolerant alfalfa growth promoting agent.
3. The use of the alfalfa salt-tolerant growth-promoting microbial agent according to claim 2 in alfalfa salt-tolerant growth promotion.
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| WO2020187822A1 (en) * | 2019-03-18 | 2020-09-24 | Futureco Bioscience, S.A. | Pseudomonas sp. strain, composition comprising the same, and uses thereof |
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