A salt-alkali tolerant phosphate-dissolving growth-promoting bacterium and its application

Abstract

The invention discloses a salt-tolerant and alkali-tolerant phosphate-dissolving and growth-promoting bacterium and its application, which belongs to the field of biotechnology. The salt-tolerant and alkali-tolerant phosphate-dissolving and growth-promoting bacterium is named as Enterobacter holmesii ( Enterobacter wall ) P03, preserved in China Center for Type Culture Collection, with a preservation number of CCTCC No: M 20242250. The Enterobacter holliformis P03 provided by the present invention has good salt tolerance, acid and alkali tolerance, growth promotion, and phosphorus solubilization effects, grows well under high salinity, acidic conditions and alkaline conditions, and has good phosphorus solubilization ability. At the same time, indoleacetic acid can be produced during the growth process to promote plant growth.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a salt- and alkali-tolerant phosphate-dissolving growth-promoting bacterium and an application thereof. Background Art

[0002] Phosphorus is one of the essential elements for plant growth and plays an important role in plant growth, development and reproduction. Phosphorus in the soil is mainly inorganic phosphorus, accounting for 60%-80% of the total phosphorus content. It can be roughly divided into three forms: adsorbed phosphorus, mineral phosphorus and water-soluble phosphorus. Among them, the water-soluble phosphorus in inorganic phosphorus can be directly absorbed and utilized by plants, and its content is only 0.02%-0.2%. A large amount of phosphorus cannot be absorbed and utilized by plants. The main source of phosphorus utilization by plants is soil, which cannot meet the crop's demand for phosphorus. 95% of phosphorus is related to Ca 2+ , Fe 3+ 、Al 3+ It reacts with metal ions such as calcium phosphate, aluminum phosphate, and iron phosphate to exist in insoluble and invalid forms. With the development of agriculture, agricultural growers continue to apply large amounts of phosphate fertilizers to increase crop yields and economic benefits, resulting in a surplus of phosphorus in the soil. The utilization rate of phosphate fertilizers is only 10%-25%. Excessive phosphorus is converted into a slow-release state, Ca8-P, which not only increases planting costs and causes a huge waste of phosphorus resources, but also leads to the deterioration of the soil ecological environment. Therefore, improving the utilization rate of phosphorus in the soil is one of the important issues facing agricultural production.

[0003] Phosphate-solubilizing microorganisms (PSMs) are a type of microorganisms in the soil that can decompose insoluble phosphates. They release phosphorus by secreting phosphatases and organic acids (such as citric acid, gluconic acid, oxalic acid, etc.), which then become effective phosphorus that can be absorbed and utilized by plants. Phosphate-solubilizing microorganisms are widely distributed and rich in species, including bacteria, fungi, actinomycetes, etc., such as Bacillus ( Bacillus ), Pseudomonas ( Pseudomonas )、Arthrobacter( Arthrobacter ), Enterobacter spp. Enterobacter ) etc. Screening, developing and applying phosphate-solubilizing bacteria, using phosphate-solubilizing microorganisms to improve the utilization of phosphorus in the soil and promote plant growth, especially phosphate-solubilizing bacteria that can adapt to saline-alkali environments are of great significance for increasing the effective phosphorus content in saline-alkali soils. Summary of the invention

[0004] The purpose of the present invention is to provide a salt-alkali tolerant phosphate-dissolving growth-promoting bacterium to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A salt-tolerant and alkaline phosphate-solubilizing growth-promoting bacterium named Enterobacter hallii ( Enterobacter hormaechei )P03, deposited in China Center for Type Culture Collection, the deposit number is CCTCC No: M 20242250, the deposit address is Wuhan University, Wuhan, China, and the deposit time is October 21, 2024.

[0007] Another object of the present invention is to provide an application of the above-mentioned salt-alkali tolerant phosphate-solubilizing and growth-promoting bacteria in phosphate solubilization.

[0008] Another object of the present invention is to provide an application of the above-mentioned salt-alkali tolerant phosphate-solubilizing and growth-promoting bacteria in improving soil phosphorus pollution.

[0009] Another object of the present invention is to provide an application of the above-mentioned salt-alkali tolerant phosphate-solubilizing growth-promoting bacteria in promoting plant growth in salinized soil, acidic soil or alkaline soil.

[0010] Preferably, the plant comprises tomato.

[0011] Another object of the present invention is to provide an application of the above-mentioned salt-alkali tolerant phosphate-solubilizing and growth-promoting bacteria in planting crops in salinized soil, acidic soil or alkaline soil.

[0012] Preferably, the crop plants include tomatoes.

[0013] The present invention provides a salt-tolerant and alkali-tolerant phosphate-solubilizing and growth-promoting bacterium, named Enterobacter hollifolia P03, which has good salt tolerance, acid and alkali tolerance, growth promotion and phosphate-solubilizing effects, grows well under high salinity, acidic conditions and alkaline conditions, and has good phosphate-solubilizing ability. At the same time, it can produce indoleacetic acid during the growth process to promote plant growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a picture of the phosphate dissolution zone of strain P03 on the PKO culture plate.

[0015] Figure 2 Figure 2 shows the colony (A) and bacterial morphology (B) of strain P03.

[0016] Figure 3 The phylogenetic tree of strain P03 was constructed based on the 16S rDNA gene sequence; in the figure, the numbers on the branch nodes are the percentiles in the evolutionary tree analysis based on 1000 sample data.

[0017] Figure 4 This is a comparison chart of the soluble phosphorus content and pH value of the culture solution of strain P03 at different temperatures.

[0018] Figure 5 This is a comparison chart of the soluble phosphorus content and pH value of the culture solution of strain P03 at different initial pH values.

[0019] Figure 6 This is a comparison chart of the soluble phosphorus content and pH value of the culture solution of strain P03 under different inoculation amounts.

[0020] Figure 7 This is a comparison chart of the soluble phosphorus content and pH value of the culture solution of strain P03 under different salinities. DETAILED DESCRIPTION

[0021] The technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0022] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without making any creative work shall fall within the scope of protection of the present invention.

[0023] The following embodiments are examples of practical applications of the technical solutions of the present invention, but are not limited thereto. The reagents and experimental equipment involved are all commercially available products.

[0024] Example 1: This example provides a method for screening and identifying salt- and alkali-tolerant phosphate-solubilizing bacteria and growth-promoting bacteria, as follows:

[0025] 1. Soil sample collection: Soil samples were collected from a tomato greenhouse in Shouguang City, Shandong Province in October 2021. 0-20 cm rhizosphere soil was collected, placed in sterile ziplock bags, and brought back to the laboratory for storage at 4°C.

[0026] 2. Culture medium preparation: (1) Solid phosphate-solubilizing medium (PKO): 10 g glucose, 0.5 g ammonium sulfate, 2 g calcium phosphate, 0.3 g magnesium sulfate, 0.3 g manganese sulfate, 0.3 g potassium chloride, 0.3 g sodium chloride, 20 g agar powder, 1000 mL water. (2) Liquid phosphate-solubilizing medium: 10 g glucose, 0.5 g ammonium sulfate, 2 g calcium phosphate, 0.3 g magnesium sulfate, 0.3 g manganese sulfate, 0.3 g potassium chloride, 0.3 g sodium chloride, 1000 mL water. (3) NA medium: 5 g sodium chloride, 10 g peptone, 5 g beef extract powder, 20 g agar powder, 1000 mL water. (4) LB+L-tryptophan medium: 5 g yeast extract powder, 10 g peptone, 5 g sodium chloride NaCl, 0.1 g L-tryptophan, 1000 mL water, adjust the pH to 7-7.5.

[0027] 3. The screening of phosphate-solubilizing bacteria specifically includes the following steps:

[0028] (1) Isolation and purification of phosphate-solubilizing bacteria: The conventional dilution plate method was used to isolate soil phosphate-solubilizing bacteria. 1 g of soil sample was placed in 50 mL of sterile water and centrifuged at 180 rpm. -1After shaking for 30 min and standing for 5 min, the supernatant was diluted to 10 -6 , take 200 µL and spread it evenly on the PKO plate, place it in a constant temperature incubator at 27°C and culture it for 5 days, pick out the strain that produces the phosphate-dissolving circle for isolation and purification.

[0029] (2) Initial screening of phosphate-solubilizing bacteria: The purified phosphate-solubilizing strains were streaked on PKO culture plates and cultured at 27°C for 3 days before the colony diameter was measured ( d ) and the diameter of the phosphorus dissolution zone ( D ),calculate D / d , to determine the strain's ability to solubilize phosphate.

[0030] (3) Initial screening of growth-promoting bacteria: The purified phosphate-solubilizing strains were inoculated into LB+L-tryptophan liquid medium at 27°C and 180 rpm. -1 , shake and culture for 24 hours, then place 50μL of bacterial solution on a white ceramic plate, add an equal volume of Salkowski colorimetric solution to the bacterial solution, and place it away from light for 30 minutes to observe the color change. Those that turn red are positive and produce indoleacetic acid (IAA), and the uninoculated LB+L-tryptophan medium is used as the control. The darker the color, the more IAA is produced.

[0031] (4) Rescreening of phosphate-solubilizing bacteria: Select strains with strong phosphate-solubilizing ability from the initial screening and inoculate them into NA liquid culture medium at 180 r / min. -1 , 27℃ shaking culture for 24h. Take 1mL of bacterial solution and inoculate it into a triangular flask containing 50ml of PKO liquid culture medium. Shake and culture it for 2d under the same conditions. The blank control is the one without inoculation. After the culture is completed, the speed is 10000 r·min -1 After centrifugation for 15 min, the supernatant was taken and the soluble phosphorus content in the solution was determined by the molybdenum antimony colorimetric method.

[0032] (5) Rescreening of phosphate-solubilizing and growth-promoting bacteria: The strains with the highest IAA production in the initial screening were inoculated at 1% in LB-L-tryptophan liquid medium at 27°C and 180 rpm. -1 After 4 days of culture, the bacterial solution was rotated at 10000 r·min -1 Centrifuge for 10 minutes, take the supernatant and mix it with an equal volume of Salkowski colorimetric solution, keep it in the dark for 30 minutes, and measure the absorbance at 530nm. LB-L-tryptophan liquid medium is used as blank control.

[0033] By diluting and plating the soil samples, a strain was isolated and produced an obvious phosphate-dissolving zone on the PKO culture plate (such as Figure 1 The colony diameter of each strain was measured ( d ) and the diameter of the phosphorus dissolution zone ( D ),calculate D / dThe value of strain P03 D / d The maximum value was 6.5 (see Table 1), indicating that it had a strong ability to solubilize phosphorus. After further oscillation culture in PKO liquid medium for 2 days, the soluble phosphorus content in the solution was 385.42 mg·L -1 After strain P03 was cultured in LB+L-tryptophan medium and reacted with Salkowski colorimetric solution, it turned pink. The absorbance value was further measured at 530nm, and the IAA content in the solution was 3.41µg·ml -1 .

[0034] Table 1

[0035]

[0036] 4. Identification of phosphate-solubilizing and growth-promoting bacteria, as follows:

[0037] (1) Morphological and physiological and biochemical identification of strains: The selected phosphate-solubilizing bacteria were inoculated onto NA culture plates and cultured for 24 h. The morphological characteristics of the colonies were observed, and the physical and chemical properties such as Gram staining reaction, citrate utilization, indole production, malonate utilization, VP test, methyl red test, and maltose utilization were determined.

[0038] (2) Molecular biological identification: Total DNA was extracted using a bacterial DNA extraction kit, and 16S rDNA sequences were amplified using existing bacterial universal primers 27F and 1492R, which were sequenced by BGI. The sequences were aligned in the GenBank database, and a phylogenetic tree was constructed using MEGA7.0.

[0039] The colonies of strain P03 on LB solid culture plates are round, smooth, and milky white (e.g. Figure 2 The bacteria are short rod-shaped (as shown in A); Figure 2 The physiological and biochemical identification results of the strains are shown in Table 2.

[0040] Table 2

[0041]

[0042] The 16S rDNA sequence of strain P03 was compared with the GenBank database by Blast, and the sequences with higher homology were selected for sequence analysis using MEGA7.0. The phylogenetic tree was constructed using the NJ method (e.g. Figure 3 The results showed that the phosphate-solubilizing bacteria P03 and Enterobacter Enterobacter hormaechei strain DZXOTU4 (MT893338.1) had a sequence homology of 99.57%. Further combined with morphological characteristics and physiological and biochemical characteristics, strain P03 was identified as Enterobacter hallii ( Enterobacter hormaecheiThe strain was named Enterobacter holmesii ( Enterobacter hormaechei )P03, deposited in the China Center for Type Culture Collection on October 21, 2024, with the deposit number CCTCC No: M20242250, and the deposit address is Wuhan University, Wuhan, China.

[0043] Example 2: The characteristics of the above-mentioned Enterobacter holmesii P03 were studied as follows:

[0044] 1. Effect of temperature on the phosphate solubilization ability of the strain: The bacterial solution was inoculated into PKO liquid phosphate solubilization medium at a 1% inoculum and incubated in a constant temperature shaking incubator at 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, and 40℃ at 180 r·min -1 After 3 days of cultivation, the soluble phosphorus content and pH value of the culture solution were determined. Each treatment was repeated 3 times, and the uninoculated culture medium was used as the control.

[0045] Depend on Figure 4 It can be seen that strain P03 can grow in the range of 10-40℃ and has a phosphate-solubilizing effect. As the temperature rises, the soluble phosphorus content in the culture medium shows a trend of first increasing and then decreasing. The soluble phosphorus content in the culture medium at different temperatures is significantly different. Strain P03 has the strongest phosphate-solubilizing ability at 25℃, and the soluble phosphorus content in the culture medium reaches 520.25mg·L -1 , followed by 30℃, with soluble phosphorus content of 499.01 mg·L -1 After the culture was completed, the pH value of the culture solution gradually increased with the increase of temperature, and the pH value range was 3.23-3.82.

[0046] 2. Effect of initial pH value on the phosphate solubilization ability of the strain: The bacterial solution was inoculated with 1% inoculum into PKO liquid phosphate solubilizing medium with pH values ​​of 3, 4, 5, 6, 7, 8, and 9, respectively, at the optimal temperature and 180 r·min -1 After 3 days of cultivation, the soluble phosphorus content and pH value of the culture solution were determined. Each treatment was repeated 3 times, and the uninoculated culture medium was used as the control.

[0047] Depend on Figure 5 It can be seen that the strain can grow in the pH range of 3-9 and has good acid and alkali resistance. When the initial pH value is 4, the strain P03 has the strongest phosphate solubilization ability, and the soluble phosphorus content in the culture medium reaches 469.08 mg·L -1 , while at pH 9, the soluble phosphorus content is 306.2 mg·L -1 Except for the treatment with an initial pH value of 3, the final pH values ​​of the culture solutions of the other treatments were lower than the initial pH values. The higher the initial pH value, the higher the final pH value.

[0048] 3. Effect of inoculation amount on the phosphate solubilization ability of strains:

[0049] The bacterial solution was inoculated into PKO liquid phosphate-dissolving medium at 0.5%, 1%, 2%, 4%, and 6% inoculum, respectively, and the temperature was optimized at 180 r·min -1 After 3 days of shaking culture, the soluble phosphorus content and pH value of the culture solution were determined. Each treatment was repeated 3 times, and the uninoculated culture medium was used as the control.

[0050] Depend on Figure 6 It can be seen that when the inoculation amount was 2%, the strain P03 had the best phosphate solubilization ability, and the soluble phosphorus content in the culture medium reached 410.5 mg·L -1 , followed by 1% inoculation, with a soluble phosphorus content of 382.12 mg·L -1 Compared with the control group, the pH values ​​of the culture fluids with different inoculation amounts decreased significantly, ranging from 3.39 to 3.84. The pH value of the culture fluid with an inoculation amount of 2% was the lowest, and the pH value of the culture fluid with an inoculation amount of 6% was the highest.

[0051] 4. Effect of salinity on the phosphate solubilization ability of strains:

[0052] The bacterial solution was inoculated at a 1% inoculum volume until the salinity (NaCl concentration) reached 2 g·L -1 , 6g·L -1 , 8g·L -1 , 12g·L -1 , 16g·L -1 , 20g·L -1 , 24 g·L -1 In the PKO liquid phosphate-dissolving medium, the optimal temperature was 180 r·min -1 After 3 days of cultivation, the soluble phosphorus content and pH value of the culture solution were determined. The non-inoculated liquid phosphate-dissolving medium was used as the control, and each treatment was repeated 3 times.

[0053] Depend on Figure 7 It can be seen that the strain has a salt content of 0-24g·L -1 It can grow in a wide range and has the ability to solubilize phosphate. The salinity of the liquid phosphate-solubilizing medium is 8 g·L -1 At 4 ℃, strain P03 had the strongest phosphate solubilization ability, with the soluble phosphorus content reaching 361.01 mg·L -1 . Salinity is 24g·L -1 The soluble phosphorus content in the P03 culture medium was 205.54 mg·L -1 The pH value of the culture solution changed in the opposite direction to the soluble phosphorus content, showing a trend of first decreasing and then increasing.

[0054] Example 3: Exploring the effect of the above-mentioned Enterobacter holmesii P03 on the growth of tomato seedlings, as follows:

[0055] 1. Selection of test plants: The tomato variety used for the test was Yanyu.

[0056] 2. Preparation of fermentation liquid: Inoculate strain P03 at 1% inoculum into liquid NA medium and incubate at 25°C and 180 r·min -1 The culture was cultured in a constant temperature shaking incubator for 3 days, and the concentration of the bacterial solution was adjusted to 1×10 8 CFU, reserve.

[0057] 3. Effect of strain P03 on the growth of tomato seedlings: Select tomato seedlings with 2 leaves and 1 heart and consistent growth and transplant them into pots with a diameter of 12 cm, and plant one seedling in each pot. After 3 days of seedling acclimatization, apply 10 ml of fermented bacterial solution to each pot, and use an equal amount of sterile water as the control (CK). Each treatment was repeated 3 times, with 10 pots in each replicate. After 21 days of growth, the growth indicators such as plant height, stem diameter, fresh weight, dry weight, underground fresh weight, and underground dry weight of the tomato seedlings were measured, and the functional leaves were selected to determine the MDA, POD, SOD enzyme activities and chlorophyll content in the leaves.

[0058] Adding the fermented bacterial solution of strain P03 during the growth of tomato seedlings can significantly promote the growth of seedlings. As shown in Table 3, after applying the fermented bacterial solution, the plant height of the tomato seedlings was 43.9 cm, the stem diameter was 7 mm, the fresh weight was 14.41 g, the underground fresh weight was 1.24 g, the chlorophyll content was 4.09 mg / g, the POD activity was 8218.88 U / g, and the SOD activity was 78.41 U / g, which were significantly higher than the control, indicating that the addition of bacterial solution can significantly promote the growth of seedlings, increase the activity of POD and SOD enzymes, reduce the MDA content, and enhance the stress resistance of the seedlings.

[0059] Table 3

[0060]

[0061] 4. Effect of strain P03 on soil physical and chemical properties: The rhizosphere soil of potted tomatoes with different treatments was collected separately, and after thorough mixing, the available nitrogen, available phosphorus, available potassium, organic matter and other indicators in the soil were measured to analyze the effect of bacterial solution treatment on soil physical and chemical properties.

[0062] As shown in Table 4, applying bacterial solution to the soil can significantly increase the content of available phosphorus and total phosphorus in the soil, which are 5.14% and 8.18% higher than the control (CK), respectively. At the same time, the activity of soil acid phosphatase and alkaline phosphatase is significantly increased.

[0063] Table 4

[0064]

[0065] In summary, the Enterobacter holmii P03 provided by the embodiment of the present invention has good salt tolerance, acid and alkali resistance, growth promotion, and phosphate solubilization effects. -1It grows well under acidic conditions (pH=3) and alkaline conditions (pH=9), and has good phosphate-solubilizing ability. At the same time, it can produce indoleacetic acid during the growth process to promote plant growth.

[0066] Based on the above-mentioned ideal embodiments of the present invention, the relevant staff can make various changes and modifications without departing from the technical concept of the present invention through the above-mentioned description. The technical scope of the present invention is not limited to the contents of the specification.

Claims

1. A salt-alkali tolerant phosphate-solubilizing growth-promoting bacterium, characterized in that: Enterobacter hallii ( Enterobacter hormaechei )P03, deposited in China Center for Type Culture Collection, with the deposit number being CCTCC No: M 20242250, and the deposit date being October 21, 2024.

2. Use of the salt-alkali tolerant phosphate-solubilizing and growth-promoting bacteria as claimed in claim 1 in phosphate solubilization.

3. Use of the salt-tolerant and alkali-soluble phosphate-promoting bacteria as claimed in claim 1 in promoting plant growth in salinized soil, acidic soil or alkaline soil.

4. The use according to claim 3, characterized in that: The plants include tomatoes.

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