Pseudomonas sp. And application thereof

Abstract

The invention belongs to the technical field of microorganisms, and relates to pseudomonas sp. And application thereof. The strain is named as Pseudomonas yangonensis JP2, is preserved in the China General Microbiological Culture Collection Center on June 24, 2024, is called CGMCC for short, is located in No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and has a biological preservation number of CGMCC No.31040. The invention also discloses a method for preparing the Pseudomonas yangonensis JP2 strain. Researches show that the strain has saline-alkaline tolerance, has the capacity of producing IAA and cellulase, and has an obvious promotion effect on the growth of wheat under saline-alkaline stress.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a strain of Pseudomonas yangonii and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Wheat (Triticum aestivum L.) is an annual or biennial herbaceous plant of the Poaceae family and the genus Triticeae. It is an important food crop. The prevention and development of saline-alkali soils has become an important research topic for the development of social economy and the promotion of sustainable development. The formation of most saline and alkaline soils is related to the Na content in the soil. + 、SO4 2- 、Cl-、CO3 2- The accumulation of salt and alkali stress has a great influence on the growth of wheat. Therefore, the degree of salinization is generally high, and plants can hardly survive in areas with severe saline-alkali soil. If wheat can be grown in saline-alkali land, the land utilization rate can be improved, the land utilization value can be increased, and the agricultural production efficiency can be improved. Therefore, promoting the growth of wheat under saline-alkali stress is the first problem to be solved in planting wheat in saline-alkali land. Summary of the invention

[0004] In order to overcome the deficiencies of the prior art, the present invention aims to provide a strain of Pseudomonas yangonii and its application. Studies have shown that the strain has salt-alkali tolerance, the ability to produce IAA and cellulase, and has a significant promoting effect on the growth of wheat under salt-alkali stress.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] First, a strain of Pseudomonas yangonensis, named Pseudomonas yangonensisJP2, was deposited on June 24, 2024 in the General Microbiology Center of the China Culture Collection Administration, referred to as CGMCC, with an address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and its biological deposit number is: CGMCC No.31040.

[0007] The strain provided by the present invention is cultured on LB medium at 28° C. for 24 hours, and the bacterial single colony is light yellow, transparent, moist on the surface, and smooth on the edge. For ease of presentation, the Pseudomonas yangonensis JP2 of the present invention is referred to as strain JP2 or JP2 in the present invention.

[0008] In a second aspect, a method for culturing the above-mentioned Pseudomonas yangonensis is provided, wherein the Pseudomonas yangonensis is obtained by culturing the above-mentioned Pseudomonas yangonensis.

[0009] In some embodiments, the culture medium used in the culture process is LB medium.

[0010] In some embodiments, the temperature during culturing is 26-30°C.

[0011] In a third aspect, a bacterial agent comprises the above-mentioned Pseudomonas yangonii or its culture, fermentation broth, metabolites, and lysates.

[0012] The culture of the present invention refers to the product obtained after culturing the strain.

[0013] In some embodiments, auxiliary materials are also included, which may be proteins, vitamins, trace elements, nucleotides, etc.

[0014] In a fourth aspect, a use of the above-mentioned Pseudomonas yangonii or bacterial agent in the production of indole-3-acetic acid (IAA).

[0015] In some embodiments, during the production of indole-3-acetic acid, LB medium containing L-tryptophan is used for culturing.

[0016] A fifth aspect provides a use of the above-mentioned Pseudomonas yangonii or bacterial agent in the production of cellulase.

[0017] In a sixth aspect, a use of the above-mentioned Pseudomonas yangonensis or bacterial agent in promoting wheat growth in a saline-alkali stress environment.

[0018] In a seventh aspect, a fertilizer for promoting wheat growth in a saline-alkali stress environment comprises the above-mentioned Pseudomonas yangonii or bacterial agent.

[0019] The beneficial effects of the present invention are:

[0020] The invention separates a strain of Pseudomonas yangonensis JP2 from rhizosphere soil of Suaeda salsa, determines its classification status by 16S rRNA sequencing and constructing a phylogenetic tree, finds that the strain JP2 can tolerate an alkaline environment with a pH value less than 11 and has strong alkaline resistance by a salt-alkali resistance test, finds that the strain JP2 has the ability to produce IAA by culturing in a liquid culture medium, and finds that after salt-alkali stress treatment, adding the strain JP2 can significantly promote the growth of wheat seeds, and the indexes such as the root length and the germination rate are significantly improved by a growth promotion test. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0022] Figure 1 This is a colony morphology diagram of strain JP2 in the embodiment of the present invention;

[0023] Figure 2 This is a bacterial phylogenetic tree of strain JP2 in the embodiment of the present invention;

[0024] Figure 3 This is a result diagram of the IAA production ability of strain JP2 in the embodiment of the present invention;

[0025] Figure 4 This is a result diagram of the cellulase production ability of strain JP2 in the example of the present invention;

[0026] Figure 5 The figure is a result diagram of the effect of seawater on wheat seed germination in the embodiment of the present invention, wherein A is the effect of seawater stress on the germination potential of wheat seeds, B is the effect of seawater stress on the germination rate of wheat seeds, C is the effect of seawater stress on the germination index of wheat seeds, and D is the effect of seawater stress on the vitality index of wheat seeds;

[0027] Figure 6 The figure is the result of the effect of strain JP2 on wheat seed germination under saline-alkali stress in the embodiment of the present invention, wherein A is the effect of strain JP2 on wheat seed germination potential under saline-alkali stress, B is the effect of strain JP2 on wheat seed germination rate under saline-alkali stress, C is the effect of strain JP2 on wheat seed germination index under saline-alkali stress, and D is the effect of strain JP2 on wheat seed vigor index under saline-alkali stress. DETAILED DESCRIPTION

[0028] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0029] Example

[0030] 1. Materials

[0031] Experimental samples: rhizosphere soil samples from Suaeda salsa in Weifang, Shandong.

[0032] Culture medium: LB medium.

[0033] Wheat variety: Jimai 22.

[0034] 2. Methods

[0035] 2.1 Isolation and culture of strains

[0036] The resuspended soil sample solution was diluted in physiological saline and applied to LB and 1 / 5LB culture medium. The culture was incubated at 28°C for 48-72 hours. The number of colonies on each culture medium was recorded. According to the growth characteristics of the colony, such as color, size, degree of protrusion, transparency, hardness, and whether the edge is regular, a single colony was picked, and the picked single colony was three-zoned on the corresponding culture medium and purified three times to obtain a pure culture strain, which was recorded as JP2.

[0037] 2.2 Identification of strain JP2

[0038] (1) Use a pipette to pick up the purified monoclonal clone and transfer it to liquid LB medium. Incubate it in a shaker at 170 rpm and 28°C for 24-48 hours until the bacterial solution becomes turbid. Then send it to the company for 16S rRNA sequencing.

[0039] (2) PCR system and reaction conditions are shown in Table 1. After the reaction, the PCR products were analyzed by agarose gel electrophoresis. The obtained fragments were sequenced and analyzed. The phylogenetic tree was constructed using MEGA software.

[0040] Table 1 PCR reaction system and reaction

[0041]

[0042]

[0043] The amplification used the bacterial universal primer 27F / 1492R, and the primer base sequence is as follows:

[0044] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', as shown in SEQ ID NO. 1;

[0045] 1492F:5'-TACGGCTACCTTGTTACGACTT-3', as shown in SEQ ID NO.2.

[0046] (3) The sequenced sequences were compared on the Ez-Taxon website (http: / / www.ezbiocloud.net / ) and the NCBI website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) to determine the taxonomic status of strain JP2.

[0047] 2.3 Strain preservation

[0048] The purified strain JP2 was streaked and shaken, mixed with sterile glycerol seed solution (50% glycerol), marked, and finally stored in a -80°C ultra-low temperature refrigerator (at least 3 backups for each strain).

[0049] 2.4 Determination of salt and alkali resistance

[0050] Single colonies were picked and cultured in LB liquid medium at 28°C and 170r / min for 48h to prepare mother solution, and 1% was inoculated into yeast extract tryptone liquid medium with pH 7 containing 0, 20, 40, 60, 80, 100, 120, 140g / L NaCl and pH 7, 8, 9, 10, 11, and yeast extract tryptone liquid medium. At the same time, no inoculation was used as a control, and cultured at 28°C and 170r / min for 48h. OD was measured. 600nm To reflect the salt-alkali tolerance of strain JP2.

[0051] 2.5 IAA production capacity determination

[0052] Add 1 mg / mL L-tryptophan to the liquid LB medium, repeat three times, inoculate 5% of the test strain, culture at 170 r / min and 28°C for 48 h, centrifuge at 4000 r / min for 10 min, aspirate 0.5 mL of the supernatant, add an equal volume of Salkowski colorimetric solution, keep it in the dark for 30 min, and observe its color change. The color change is the basis for qualitative judgment. The color is pink, and the redder the color, the higher the IAA content. At OD 530nm The absorbance was measured using an ultraviolet spectrophotometer, and the obtained bacterial absorbance was substituted into the IAA standard curve to obtain the IAA production of bacteria JP2.

[0053] 2.6 Determination of enzyme production capacity

[0054] The plate method was used to spot-inoculate the strain JP2 to be tested on the cellulose detection culture medium, and then stain it with Congo red to observe whether a transparent circle appeared, so as to judge whether the strain JP2 had the function of producing cellulase.

[0055] 2.7 Effects of saline-alkali stress on wheat seed germination

[0056] Wheat seed disinfection: Soak wheat seeds (Jimai 22) in 5% NaClO for 10 min, wash with sterile water for 4 to 5 times, and soak in sterile water for 24 h.

[0057] Preparation of germination bed: Drain the surface water of seeds with absorbent paper, select sterile culture dishes for experiments, and use filter paper with good water absorption and water retention as paper bed. Place the disinfected seeds on the filter paper in the sterilized culture dishes, 30 seeds in each culture dish; use seawater to simulate the saline-alkali stress environment, set up 6 gradients, and irrigate the wheat seeds with 0 (no addition), 0.4%, 0.8%, 1.2%, 1.6%, and 2% seawater solutions, respectively, and repeat 3 times for each treatment. Cultivate in an artificial climate incubator at 28°C, change the solution every day, record the seed germination, observe the seed germination, measure the seed germination length on the seventh day, and calculate the germination potential, germination rate, germination index, and vitality index.

[0058] 2.8 Growth-promoting effect on wheat

[0059] The strain JP2 was inoculated into LB liquid culture medium, cultured overnight at 28°C and 170rpm, and the bacteria were collected by centrifugation at 8000rpm for 5min. The supernatant was discarded, and the suspension was resuspended with sterile water. The concentration of the bacterial suspension was adjusted to OD=1, mixed with 3.2% seawater and added to the seed culture dish. The control group was added with 1.6% seawater. The number of germinations was recorded every day, and the seed germination length was recorded after seven days.

[0060] Experimental Results

[0061] The obtained strain JP2 was named Pseudomonas yangonensisJP2, and was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, referred to as CGMCC, on June 24, 2024. The address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and its biological deposit number is: CGMCC No.31040.

[0062] 1 Morphological identification of strain JP2

[0063] After culturing on LB medium at 28°C for 24 hours, the bacterial colonies were light yellow, transparent, moist on the surface, and smooth on the edges. Figure 1 shown.

[0064] 2 Identification of strain JP2

[0065] Through 16S rRNA sequencing, a gene sequence fragment of 886 bp was obtained. After sequence comparison, JP2 had the highest similarity with Pseudomonas yangonensis MK907288. In the phylogenetic tree constructed based on the 16S rRNA sequence, JP2 and Pseudomonas yangonensis clustered together, indicating that JP2 belongs to Pseudomonas yangonensis. Figure 2 shown.

[0066] 16S rRNA sequence of strain JP2:

[0067] ACACATGCAGTCGAGCGGATGAAGGGAGCTTGCTCCCTGATTTAGCGGC

[0068] GGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGATAACGTT

[0069] CCGAAAGGAACGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGA

[0070] CCTTCGGGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGG

[0071] TGAGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGAT

[0072] GATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCA

[0073] GCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCG

[0074] CGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGA

[0075] AGGGCGTTTGGCTAATATCCAAGCGTTTTGACGTTACCGACAGAATAAGC

[0076] ACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTGCAAGCG

[0077] TTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTCGTTAAGTTG

[0078] GATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCGA

[0079] GCTAGAGTACGGTAGAGGGTAGTGGAATTTCCTGTGTAGCGGTGAAATGC

[0080] GTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACTACCTGGACTGAT

[0081] ACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCC

[0082] TGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGAATCCTTGAGA

[0083] TTTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCC

[0084] GCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGA

[0085] GCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTGGCCTTGACA

[0086] TGCTGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCAGACAC

[0087] AGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGT

[0088] CCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTTATGGTGGG

[0089] CACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGAC

[0090] GTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTGCTACAATGG

[0091] TCGGTACAAAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAAC

[0092] CGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAA

[0093] TCGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCT

[0094] TGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCTCCAGAAGTAGCTAGTCTAACCTTCGGGGGACGGTACCACGGAGA, as shown in SEQ ID NO.3.

[0095] 3 Determination of salt-alkali tolerance of strain JP2

[0096] As shown in Tables 2 and 3, JP2 has average salt resistance, but good alkali resistance and can tolerate environments with a pH value less than 11.

[0097] Table 2 Salt tolerance of strain JP2

[0098]

[0099]

[0100] 4 IAA production capacity of strain JP2

[0101] like Figure 3 As shown, JP2 was able to produce IAA with a yield of up to 21.6 mg / L.

[0102] 5. Enzyme production ability of strain JP2

[0103] like Figure 4 As shown, JP2 has the ability to produce cellulase after Congo red staining.

[0104] 6 Effects of salt-alkali stress on wheat seed germination

[0105] Wheat can tolerate the salinity and alkalinity of seawater. Figure 5 As shown, it can be seen from the number of seed germinations and the length of sprouts that irrigation with 1.6% and 2% seawater can seriously inhibit the growth of wheat.

[0106] Effect of strain JP2 on promoting wheat seed growth under saline-alkali stress

[0107] The results of promoting Figure 6 As shown, after JP2 irrigation, the number of wheat germinations increased significantly compared with the control group, and the bud length increased compared with the control group, indicating that the bacterial suspension of strain JP2 can promote wheat growth and has a good growth-promoting effect.

[0108] in conclusion

[0109] The strain JP2 was identified as Pseudomonas yangonensis by 16S rRNA sequencing and phylogenetic tree construction. The salt-alkali tolerance experiment showed that the strain JP2 could tolerate NaCl concentrations below 10% and alkalis below pH=11, and had good alkali tolerance. The IAA production capacity test showed that JP2 could produce IAA up to 21.6 mg / L. The enzyme production capacity test showed that JP2 had the function of producing cellulase. The growth promotion experiment showed that the strain JP2 could significantly promote the growth of wheat seeds, and the root length, germination rate and other indicators were significantly improved.

[0110] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A strain of Pseudomonas yangonii, Its characteristics are: The strain was named Pseudomonas yangonensis JP2 and was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on June 24, 2024, referred to as CGMCC, with the address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. Its biological deposit number is: CGMCC No.31040.

2. A method for culturing Pseudomonas yangonii according to claim 1, Its characteristics are: The Pseudomonas yangonii is obtained by culturing.

3. The method for culturing Pseudomonas yangonii according to claim 2, Its characteristics are: The medium used in the culture process was LB medium; Alternatively, the temperature during the culture process is 26 to 30°C.

4. A bacterial agent, Its characteristics are: The method comprises the Pseudomonas yangonii or its culture, fermentation broth, metabolites and lysates as described in claim 1.

5. The bacterial agent according to claim 4, Its characteristics are: Also includes auxiliary materials.

6. Use of the Pseudomonas yangonii described in claim 1 or the bacterial agent described in claim 4 or 5 in producing indole-3-acetic acid.

7. The use according to claim 6, Its characteristics are: In the process of producing indole-3-acetic acid, LB medium containing L-tryptophan is used for cultivation.

8. Use of the Pseudomonas yangonii described in claim 1 or the bacterial agent described in claim 4 or 5 in producing cellulase.

9. Use of the Pseudomonas yangonii described in claim 1 or the bacterial agent described in claim 4 or 5 in promoting wheat growth under saline-alkali stress environment.

10. A fertilizer for promoting wheat growth in a saline-alkali stress environment. Its characteristics are: The method comprises the Pseudomonas yangonii described in claim 1 or the bacterial agent described in claim 4 or 5.

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