A salt-tolerant growth-promoting Pseudomonas putida strain PLW1 and its application

Abstract

The invention relates to a salt-tolerant growth-promoting Pseudomonas putida PLW1 and an application thereof, belonging to the field of microbial technology. The Pseudomonas putida PLW1 was deposited in the China Center for Type Culture Collection on June 24, 2024, with a deposit number of CCTCC NO: M 20241352. The strain has the functions of decomposing organic phosphorus, decomposing inorganic phosphorus, producing lipase, producing siderophore and producing indoleacetic acid, and has good salt and alkali resistance. The bacterial agent prepared by using PLW1 can promote the germination of corn seeds and the growth of corn seedlings under salt stress, and has broad application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a salt-tolerant growth-promoting Pseudomonas putida PLW1 and an application thereof. Background Art

[0002] Soil salinization is one of the main manifestations of land degradation, which not only threatens the biosphere and ecological environment, but also causes resource destruction and agricultural production losses. Soil salinization can lead to serious degradation of soil structure and nutrient depletion, and can also lead to physiological dehydration of plants, while causing oxidative stress damage to plants, affecting their normal growth and development.

[0003] In recent years, due to the global nature of soil salinization, the management of soil salinization has been pushed to a new level and has become a hot topic of research at home and abroad. Corn is the most important food crop in my country and is of great significance to ensuring national food security. However, corn is a salt-sensitive crop, and soil salinization has greatly limited the improvement of corn yield. Therefore, improving the yield and quality of corn in saline-alkali land is one of the important tasks of agricultural scientific researchers in my country.

[0004] Compared with traditional physical and chemical methods, bioremediation has the advantages of not changing the ecological balance and having long-lasting effects, and is currently an important way to improve saline-alkali land. Plant growth promoting rhizobacteria (PGPR) are beneficial bacteria that can promote plant growth and inhibit plant diseases, and have great potential for application in saline-alkali land improvement and agricultural production.

[0005] For example, a patent application with publication number "CN117106614A" discloses a rhizospheric bacterium Qingdao Pseudomonas YIM B08402 and its application. The rhizospheric bacterium can grow in an environment of pH 6.0-8.0 and 0-6% NaCl, and has the functions of dissolving inorganic phosphorus, decomposing organic phosphorus, fixing nitrogen, producing iron carriers, secreting indoleacetic acid and degrading phenolic acid; however, the patent was not applied to tomato plants in saline-alkali environments during actual experiments, and it is impossible to determine whether the strain YIM B08402 has a growth-promoting effect on tomato plants under salt stress. The patent application with publication number "CN117106668A" discloses a salt-alkali tolerant growth-promoting Tianjin Pseudomonas P522 and its application. The strain can stably colonize in saline-alkali soil. At the same time, the strain also has the properties of producing ACC deaminase, indoleacetic acid, siderophore, inorganic phosphorus, and biofilm. It can significantly promote the seed germination and seedling growth of saline-alkali dryland crops and has good practical application value. However, the patent only proves that the strain P522 has a promoting effect on the seed germination and seedling growth of saline-alkali dryland crops, while corn is a typical water-loving crop, and the strain may not be suitable for corn.

[0006] At present, there is relatively little research on salt-tolerant growth-promoting strains in the existing technology, and the strain resources that can be used in actual production are still scarce. It is urgent to research and develop more salt-tolerant strains with growth-promoting functions to promote the growth of a variety of crops. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention provides a salt-tolerant growth-promoting Pseudomonas putida PLW1 and its application. The strain has the functions of degrading organic phosphorus, degrading inorganic phosphorus, producing lipase, producing siderophore and producing indoleacetic acid, and has good salt and alkali resistance. The bacterial agent prepared using PLW1 can promote the germination of corn seeds and the growth of corn seedlings under salt stress, and has broad application prospects.

[0008] The technical solution of the present invention is as follows:

[0009] A strain of Pseudomonas putida ( Pseudomonas putida ) PLW1, deposited on June 24, 2024 in China Center for Type Culture Collection, Wuhan University, Wuhan, China, with the deposit number CCTCC NO: M 20241352.

[0010] The 16S rDNA sequence of Pseudomonas putida PLW1 is shown in SEQ ID NO.1.

[0011] The method for culturing Pseudomonas putida PLW1 comprises the following steps:

[0012] (1) Pseudomonas putida PLW1 was inoculated into R2A solid culture medium and activated at 28-30°C to grow a single colony;

[0013] (2) The single colony in step (1) was inoculated into R2A liquid culture medium, and cultured at 28-30°C and 180-200 rpm with shaking to obtain a Pseudomonas putida PLW1 bacterial liquid.

[0014] The Pseudomonas putida PLW1 is used in decomposing organic phosphorus, decomposing inorganic phosphorus, producing lipase, producing siderophore, and producing indoleacetic acid.

[0015] The application of the Pseudomonas putida PLW1 in promoting corn seed germination.

[0016] Preferably, the Pseudomonas putida PLW1 promotes corn seed germination in a salt environment with a NaCl content of ≤200 mM.

[0017] The application of the Pseudomonas putida PLW1 in promoting the growth of corn seedlings.

[0018] Preferably, the Pseudomonas putida PLW1 promotes the growth of corn seedlings in a salt environment with a NaCl content of ≤100 mM.

[0019] A live bacteria preparation with the Pseudomonas putida PLW1 as an effective ingredient.

[0020] Beneficial effects of the present invention:

[0021] The Pseudomonas putida PLW1 provided by the present invention has strong salt-alkali resistance, can tolerate 6% (0.6 g / L) salt environment and alkaline environment with a pH of 10, and has the functions of decomposing organic phosphorus, decomposing inorganic phosphorus, producing lipase, producing siderophore and producing indoleacetic acid. The bacterial agent prepared by using PLW1 can promote the germination of corn seeds and the growth of corn seedlings under salt stress, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The growth of strain PLW1 on R2A solid medium containing different concentrations of NaCl.

[0023] Figure 2 This is the growth of strain PLW1 on R2A solid medium at different pH values.

[0024] Figure 3 This is the colony morphology of strain PLW1.

[0025] Figure 4 This is the result of testing the ability of strain PLW1 to degrade inorganic phosphorus.

[0026] Figure 5 This is the result of testing the ability of strain PLW1 to degrade organic phosphorus.

[0027] Figure 6 This is the result of testing the lipase production ability of strain PLW1.

[0028] Figure 7 This is the result of testing the iron carrier production ability of strain PLW1.

[0029] Figure 8 This is the result of testing the IAA production ability of strain PLW1.

[0030] Fig. 9 This is a diagram showing that strain PLW1 promotes corn seed germination under salt stress.

[0031] Fig.10 This is a graph showing that strain PLW1 promotes the growth of corn seedlings under salt stress. DETAILED DESCRIPTION

[0032] The following is explained in conjunction with specific embodiments:

[0033] Preparation of R2A liquid culture medium: 0.50 g yeast powder, 0.50 g peptone, 0.50 g casein hydrolysate, 0.50 g glucose, 0.50 g soluble starch, 0.30 g potassium dihydrogen phosphate, 0.05 g magnesium sulfate heptahydrate, 0.30 g sodium pyruvate, and 1000 mL distilled water.

[0034] Preparation of R2A solid culture medium: Add 15 g of agar to the R2A liquid culture medium.

[0035] Example 1: Isolation and screening of Pseudomonas putida PLW1

[0036] The samples were collected from saline-alkali land in Dongying City, Shandong Province, China. The specific separation and screening methods were as follows: 1 g of corn rhizosphere soil was weighed and placed in a conical flask containing 9 mL of sterile water, and the sample suspension was fully shaken; the suspension was gradiently diluted using the gradient dilution method, and 100 μL of the dilution was taken with a dilution of 10 -3 , 10 -4 , 10 -5 , 10 -6 The suspension was spread on R2A solid culture medium and cultured in a constant temperature incubator at 25°C. After a single colony grew out, it was picked out with a toothpick and purified on the culture medium using the three-zone line method. The purified strains were numbered and preserved.

[0037] The salt-tolerant and alkali-tolerant strains were screened using R2A solid culture medium with different NaCl concentrations and different pH values. The specific operation is as follows: the purified strains were streaked onto R2A solid culture medium containing 1% (0.1 g / L), 3% (0.3 g / L), 6% (0.6 g / L), and 9% (0.9 g / L) NaCl to test the salt tolerance of the strains. The results are as follows: Figure 1 As shown; the purified strain was streaked onto R2A solid culture medium with pH values ​​of 8.0, 9.0, and 10.0 to detect the strain's alkali resistance. The results are shown in Figure 2 shown.

[0038] like Figure 1~2 As shown, through the above separation and screening work, we finally screened out a strain with both salt and alkali resistance. This strain can tolerate 6% (0.6 g / L) salt environment and alkaline environment with pH 10, and was named "PLW1".

[0039] Example 2: Identification of Pseudomonas putida PLW1

[0040] The strain PLW1 obtained by the above screening was inoculated on R2A solid medium and cultured at 28°C for 24 hours, and the colony morphology was observed. Figure 3 as shown; Figure 3It can be seen that strain PLW1 is round on R2A solid medium, with a smooth and moist surface, milky white, opaque color, and neat edges.

[0041] The genomic DNA of PLW1 was extracted and PCR amplified using 16S rDNA universal primers. The amplification conditions were: 94℃, 4min; 94℃, 30s, 35 cycles; 55℃, 30s; 72℃, 30s; 72℃, 10min. The amplified 16S rDNA gene sequence was entrusted to Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results are shown in SEQ ID NO.1. The obtained 16SrDNA sequence was subjected to BLAST analysis with the existing sequences in the NCBI database, and strains with similar homology were selected. The phylogenetic tree was constructed using MEGA5.05 software. The results showed that the strain PLW1 obtained by the above screening was similar to Pseudomonas putida The strains were clustered in the same branch with a homology of 99.86%, so strain PLW1 was identified as Pseudomonas putida ( Pseudomonas putida ).

[0042] Pseudomonas putida ( Pseudomonas putida ) PLW1, deposited on June 24, 2024 in China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, with the deposit number: CCTCC NO: M 20241352.

[0043] Example 3: Cultivation of Pseudomonas putida PLW1

[0044] The specific cultivation methods are as follows:

[0045] (1) Pseudomonas putida PLW1 was inoculated into R2A solid culture medium and activated and cultured at 28°C for 24 h to grow a single colony;

[0046] (2) The single colony obtained in step (1) was inoculated into R2A liquid culture medium and cultured at 30°C and 200 rpm for 24 h to obtain a bacterial culture of Pseudomonas putida PLW1.

[0047] Experimental Example 1: Biological Function Detection of Pseudomonas putida PLW1

[0048] (1) The ability of strain PLW1 to degrade inorganic phosphorus was determined using an inorganic phosphorus bacterial culture medium. The specific method is as follows:

[0049] The strain PLW1 was streaked onto the Montkina inorganic phosphorus bacterial medium and cultured at 30°C for 3 days to observe the growth of the colonies.

[0050] The components of the Montgina inorganic phosphorus bacterial culture medium are as follows: 10 g glucose, 0.1 g ammonium sulfate, 5 g tricalcium phosphate, 0.3 g sodium chloride, 0.3 g magnesium sulfate heptahydrate, 0.3 g potassium chloride, 0.5 g yeast powder, 15 g agar, and 1000 mL distilled water.

[0051] Colony growth Figure 4 shown by Figure 4 It can be seen that PLW1 has a transparent degradation zone in the inorganic phosphorus bacterial culture medium, which indicates that the strain PLW1 has the ability to degrade inorganic phosphorus.

[0052] (2) The phosphate solubilization ability of strain PLW1 was determined using organophosphate bacterial culture medium. The specific method is as follows:

[0053] The strain PLW1 was streaked onto the Montkina organophosphate bacterial medium and cultured at 30°C for 3 days to observe the growth of the colonies.

[0054] The components of the Montgina organophosphorus bacterial culture medium are as follows: 10 g glucose, 0.5 g ammonium sulfate, 5 g tricalcium phosphate, 0.3 g sodium chloride, 0.3 g magnesium sulfate heptahydrate, 0.03 g manganese sulfate, 0.3 g potassium sulfate, 0.03 g ferrous sulfate, 0.2 g lecithin, 15 g agar, and 1000 mL distilled water.

[0055] Colony growth Figure 5 shown by Figure 5 It can be seen that PLW1 has a degradation zone in the culture medium of organophosphorus bacteria, which indicates that strain PLW1 has the ability to degrade organophosphorus.

[0056] (3) The lipase production ability of strain PLW1 was determined using lipase identification medium. The specific method is as follows:

[0057] The strain PLW1 was streaked onto the lipase identification medium and cultured at 28°C for 3 days to observe the growth of the colonies;

[0058] The components of the lipase identification medium are as follows: 10 g of tryptone, 5 g of yeast powder, 10 g of sodium chloride, 10 g of Tween80, 20 g of agar, and 1000 mL of distilled water.

[0059] Colony growth Figure 6 shown by Figure 6 It can be seen that PLW1 has a degradation zone in the lipase identification medium, which indicates that the strain PLW1 has the ability to produce lipase.

[0060] (4) The ability of strain PLW1 to produce siderophores was determined by CAS assay medium. The specific method is as follows:

[0061] The strain PLW1 was streaked onto CAS test medium and cultured at 28°C for 3 days to observe the growth of the colonies;

[0062] Among them, the components of the CAS detection medium are as follows: 60.5 mg of chrome azuro blue S, 72.9 mg of hexadecyltrimethylammonium bromide, 2.645 mg of ferric chloride hexahydrate, 295.25 mg of sodium dihydrogen phosphate dihydrate, 1213.5 mg of disodium hydrogen phosphate dodecahydrate, 125 mg of ammonium chloride, 37.5 mg of potassium dihydrogen phosphate, 62.5 mg of sodium chloride, 9 g of agar, and 1000 mL of distilled water.

[0063] Colony growth Figure 7 shown by Figure 7 It can be seen that PLW1 formed a transparent zone in the CAS detection medium, which indicates that strain PLW1 has the ability to produce siderophores.

[0064] (5) Determine the ability of strain PLW1 to produce indoleacetic acid (IAA). The specific method is as follows:

[0065] On the basis of LB liquid medium, L-tryptophan with a final concentration of 100 mg / L was additionally added, and the strain PLW1 was inoculated therein, and cultured at 30°C and 180 r / min for 4 days; after the culture was completed, 50 μL of the bacterial liquid was placed in a blank white porcelain plate, 50 μL of Salkowski colorimetric solution was added, and a white porcelain plate with 60 μL / L IAA solution added was used as a positive control; the white porcelain plate was kept away from light and reacted at room temperature for 30 minutes, and the color change was observed;

[0066] The components of the Salkowski colorimetric reagent are as follows: 1 mL of 0.5 mol / L FeCl3 solution, 50 mL of 35% (v / v) HClO4 solution.

[0067] The results are as follows Figure 8 As shown, the left side is the positive control; Figure 8 It can be seen that the color of the reaction solution after inoculation of strain PLW1 turned red, which indicates that PLW1 has the ability to produce IAA.

[0068] Experimental Example 2: Pseudomonas putida PLW1 promotes corn seed germination under salt stress

[0069] The strain PLW1 was inoculated into a triangular flask containing 50 mL of R2A liquid culture medium, and cultured at 30°C and 200 r / min for 24 h to obtain a seed solution; the seed solution was inoculated into 100 mL of R2A liquid culture medium at a volume percentage of 1%, and cultured at 30°C and 200 r / min for 24 h to obtain a fermentation liquid; the fermentation liquid was centrifuged at 5000 rpm for 20 min, the supernatant was discarded, and the bacterial precipitate was resuspended with sterile water to obtain a PLW1 bacterial suspension, and the OD of the PLW1 bacterial suspension was adjusted. 600 is 0.1.

[0070] Select corn seeds of uniform size and full grains, rinse them three times with sterile water, then soak them in 75% ethanol for 3 minutes, soak them in 5% sodium hypochlorite for 10 minutes, rinse them five times with 2 / ‰ mold inhibitor, and finally rinse them five times with sterile water to complete the disinfection of the corn seeds.

[0071] The surface-sterilized corn seeds were soaked in the above-mentioned PLW1 bacterial suspension for 2 hours as the experimental group (PLW1); at the same time, the surface-sterilized corn seeds were soaked in sterile water for 2 hours as the control group (CK); the corn seeds were transferred to a culture dish containing sterile filter paper, and 5 mL of NaCl solution with concentrations of 0 mM (sterile water), 50 mM, 100 mM, 150 mM, and 200 mM were added to the culture dish, and then placed in a 25°C culture room in the dark for culture. On the 7th day of culture, the germination of the corn seeds was observed and photographed, and the germination rate was calculated according to the following formula:

[0072] Seed germination rate (%) = (number of germinated seeds / number of test seeds) × 100

[0073] The germination of seeds on the 7th day of cultivation is shown in Table 1 and Fig. 9 shown.

[0074] Table 1. Germination rate of corn seeds under different treatments

[0075]

[0076] From Table 1 and Fig. 9 It can be seen that under salt stress of NaCl concentrations of 50mM, 100mM, 150mM, and 200mM, the germination rate of corn seeds treated with PLW1 bacterial suspension was significantly higher than that of the control group; especially under salt stress of NaCl concentration of 200mM, the germination rate of corn seeds in the control group was only 37.93%, while the germination rate of corn seeds treated with PLW1 bacterial suspension could reach 58.62%, which shows that strain PLW1 can promote the germination of corn seeds under salt stress.

[0077] Experimental Example 3: Pseudomonas putida PLW1 promotes the growth of corn seedlings under salt stress

[0078] The strain PLW1 was inoculated into a triangular flask containing 50 mL of R2A liquid culture medium, and cultured at 30°C and 200 r / min for 24 h to obtain a seed solution; the seed solution was inoculated into 100 mL of R2A liquid culture medium at a volume percentage of 1%, and cultured at 30°C and 200 r / min for 24 h to obtain a fermentation liquid; the fermentation liquid was centrifuged at 5000 rpm for 20 min, the supernatant was discarded, and the bacterial precipitate was resuspended with sterile water to obtain a PLW1 bacterial suspension, and the OD of the PLW1 bacterial suspension was adjusted. 600 is 1.

[0079] Corn seedlings with the same growth conditions were selected and randomly divided into 4 groups for different treatments, namely: salt-free sterile group (0mM NaCl + 50mL sterile water), salt-free bacteria group (0mM NaCl + 50mL PLW1 bacterial suspension), salt-free sterile group (100mM NaCl + 50mL sterile water), and salt-containing bacteria group (100mM NaCl + 50mL PLW1 bacterial suspension). Among them, the salt group was irrigated with 200mL of 100mM NaCl solution in the soil.

[0080] After the corn seedlings were cultured for 28 days, the growth of the seedlings was observed and photographed, and then the physiological plant height (maximum plant height after plant straightening) and root length of the corn seedlings in each treatment group were measured.

[0081] The growth of corn seedlings on the 28th day of cultivation is shown in Table 2 and Fig.10 As shown, Fig.10 In the salt-free group, the potted plants on the left were the salt-free and sterile group, and the potted plants on the right were the salt-free and sterile group; Fig.10 In the salt group, the potted plants on the left are the salt and sterile group, and the potted plants on the right are the salt and sterile group.

[0082] Table 2. Growth of corn seedlings under different treatments

[0083]

[0084] From Table 2 and Fig.10 It can be seen that under salt-free conditions or 100mM NaCl salt stress, the corn seedlings treated with PLW1 bacterial suspension grew better than those not inoculated with the bacteria; under 100mM NaCl salt stress, the plant height and root length of the corn seedlings treated with PLW1 bacterial suspension increased by 6.9% and 13.1% respectively compared with those not inoculated with the bacteria, which indicates that the strain PLW1 can promote the growth of corn seedlings under salt stress and improve the salt tolerance of corn plants.

Claims

1. A strain of Pseudomonas putida ( Pseudomonas putida ) PLW1, characterized in that It was deposited in the China Center for Type Culture Collection on June 24, 2024, address: Wuhan University, Wuhan, China, with the deposit number being CCTCC NO: M 20241352.

2. The method for culturing Pseudomonas putida PLW1 according to claim 1, characterized in that: The steps include: (1) Pseudomonas putida PLW1 was inoculated into R2A solid culture medium and activated at 28-30°C to grow a single colony; (2) The single colony in step (1) was inoculated into R2A liquid culture medium, and cultured at 28-30°C and 180-200 rpm with shaking to obtain a Pseudomonas putida PLW1 bacterial liquid.

3. The use of Pseudomonas putida PLW1 according to claim 1, characterized in that: It is used to decompose organic phosphorus, decompose inorganic phosphorus, produce lipase, produce siderophore, and produce indoleacetic acid.

4. The use of Pseudomonas putida PLW1 according to claim 1, characterized in that: Used to promote corn seed germination.

5. The use according to claim 4, characterized in that The Pseudomonas putida PLW1 promotes corn seed germination in a salt environment with a NaCl content of ≤200 mM.

6. The use of Pseudomonas putida PLW1 according to claim 1, characterized in that: Used to promote the growth of corn seedlings.

7. The use according to claim 6, characterized in that The Pseudomonas putida PLW1 promotes the growth of corn seedlings in a salt environment with a NaCl content of ≤100 mM.

8. A live bacteria preparation, characterized in that: The live bacteria preparation contains the Pseudomonas putida PLW1 described in claim 1 as an active ingredient.

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