Saline-alkali tolerant phosphate-solubilizing growth-promoting bacterium and application thereof

Abstract

The invention discloses a salt and alkali tolerant phosphate-solubilizing growth-promoting bacterium and application thereof, and belongs to the technical field of biology, the salt and alkali tolerant phosphate-solubilizing growth-promoting bacterium is named as Enterobacter hormaechei P03 and preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC No: M 20242250. The enterobacter hormaechei P03 provided by the invention has good salt resistance, acid and alkali resistance, growth promotion and phosphate solubilizing effects, grows well under high salinity, acidic conditions and alkaline conditions, has better phosphate solubilizing capacity, and can generate indoleacetic acid in 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 and 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 the phosphorus is related to Ca 2+ 、Fe 3+ 、Al 3+ Phosphorus reacts with other metal ions, resulting in insoluble, ineffective forms such as calcium phosphate, aluminum phosphate, and iron phosphate. With the development of agriculture, farmers continue to apply large amounts of phosphate fertilizer to increase crop yields and economic benefits, resulting in a surplus of phosphorus in the soil. The utilization rate of phosphate fertilizer is only 10%-25%. Excess phosphorus is converted into the slow-release form Ca8-P, which not only increases planting costs and causes a significant waste of phosphorus resources, but also deteriorates the soil ecosystem. Therefore, improving the utilization rate of phosphorus in the soil is a key issue facing agricultural production.

[0003] Phosphate-solubilizing microorganisms (PSMs) are a type of microorganism found 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.), turning it into 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 spp. ( Pseudomonas ), Arthrobacter ( Arthrobacter ), Enterobacter spp. ( Enterobacter Screening, developing and applying phosphate-solubilizing bacteria can improve the utilization of phosphorus in the soil and promote plant growth. In particular, phosphate-solubilizing bacteria that can adapt to saline-alkali environments are of great significance for increasing the available phosphorus content in saline-alkali soils. Summary of the Invention

[0004] The purpose of the present invention is to provide a salt- and alkali-tolerant phosphate-dissolving and 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: A salt- and alkali-tolerant phosphate-dissolving 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.

[0006] Another object of the present invention is to provide an application of the above-mentioned salt- and alkali-tolerant phosphate-solubilizing and growth-promoting bacteria in phosphate solubilization.

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

[0008] Another object of the present invention is to provide a use of the above-mentioned salt- and alkali-tolerant phosphate-solubilizing and growth-promoting bacteria in promoting plant growth in salinized soil, acidic soil, or alkaline soil.

[0009] Preferably, the plant comprises tomato.

[0010] Another object of the present invention is to provide an application of the above-mentioned salt- and alkali-tolerant phosphate-solubilizing and growth-promoting bacteria in growing crops in salinized soil, acidic soil or alkaline soil.

[0011] Preferably, the crops include tomatoes.

[0012] The present invention provides a salt- and alkali-tolerant, phosphate-solubilizing and growth-promoting bacterium, named Enterobacter hallii P03, which has good salt tolerance, acid and alkali resistance, growth promotion and phosphate-solubilizing effects. It grows well under high salinity, acidic 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

[0013] Figure 1 This is a picture of the phosphate solubilization zone of strain P03 on the PKO culture plate.

[0014] Figure 2 Figures 2 and 3 show the colony (A) and bacterial morphology (B) of strain P03.

[0015] 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 phylogenetic tree analysis based on 1000 sample data.

[0016] Figure 4 This is a comparison chart of the soluble phosphorus content and pH value of the culture medium of strain P03 at different temperatures.

[0017] Figure 5 This is a comparison chart of the soluble phosphorus content and pH value of the culture medium of strain P03 at different initial pH values.

[0018] Figure 6This is a comparison chart of the soluble phosphorus content and pH value of the culture medium of strain P03 under different inoculation amounts.

[0019] Figure 7 This is a comparison chart of the soluble phosphorus content and pH value of the culture medium of strain P03 under different salinities. DETAILED DESCRIPTION

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction 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.

[0021] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work shall fall within the scope of protection of the present invention.

[0022] The following examples are implementation cases of the technical solutions of the present invention in actual applications, but are not limited thereto. The reagents and experimental equipment involved therein are all commercially available products.

[0023] Example 1: This example provides a method for screening and identifying salt- and alkali-tolerant phosphate-solubilizing bacteria and growth-promoting bacteria, as follows: 1. Soil sample collection: Soil samples were collected from a tomato greenhouse in Shouguang City, Shandong Province in October 2021. The 0-20 cm rhizosphere soil was collected, placed in a sterile ziplock bag, and brought back to the laboratory for storage at 4°C.

[0024] 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.

[0025] 3. Screening of phosphate-solubilizing bacteria specifically includes the following steps: (1) Isolation and purification of phosphate-solubilizing bacteria: The conventional dilution plate method was used to isolate phosphate-solubilizing bacteria from soil. 1 g of soil sample was placed in 50 mL of sterile water and stirred at 180 rpm. -1 After shaking for 30 minutes and letting it stand for 5 minutes, the supernatant was diluted to 10 -6, take 200µL and evenly spread it on the PKO plate, place it in a constant temperature incubator at 27℃ and culture it for 5 days, pick out the strain that produces the phosphate-dissolving circle for isolation and purification.

[0026] (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 phosphate dissolution zone ( D ),calculate D / d , to determine the phosphate solubilization ability of the strain.

[0027] (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 Incubate with shaking for 24 hours. Then, place 50 µL of the bacterial solution on a white ceramic plate. Add an equal volume of Salkowski colorimetric solution and incubate in the dark for 30 minutes to observe the color change. A reddish color indicates positive results, indicating indoleacetic acid (IAA) production. Uninoculated LB + L-tryptophan medium serves as a control. Darker colors indicate higher levels of IAA production.

[0028] (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 , shake culture at 27℃ for 24h. Take 1mL of bacterial solution and inoculate it into a flask containing 50ml of PKO liquid medium. Shake and culture it under the same conditions for 2d. The uninoculated bacteria were used as blank control. After the culture was completed, the speed was 10000 r·min -1 After centrifugation for 15 min, the supernatant was collected and the soluble phosphorus content in the solution was determined using the molybdenum antimony colorimetric method.

[0029] (5) Rescreening of phosphate-solubilizing and growth-promoting bacteria: The strains that produced more IAA in the initial screening were inoculated at a 1% inoculum into 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, incubate in the dark for 30 minutes, and measure the absorbance at 530 nm. Use LB-L-tryptophan liquid medium as a blank control.

[0030] 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 ), and the colony diameter of each strain was measured ( d ) and the diameter of the phosphate dissolution zone ( D ),calculate D / d The value of strain P03 D / dThe maximum value was 6.5 (see Table 1), indicating that it had a strong ability to solubilize phosphorus. After further 2-day shaking culture in PKO liquid medium, the soluble phosphorus content in the solution was 385.42 mg·L -1 After being cultured in LB + L-tryptophan medium, strain P03 reacted with Salkowski colorimetric solution and turned pink. The absorbance value was further measured at 530 nm, and the IAA content in the solution was 3.41µg·ml -1 .

[0031] Table 1

[0032] 4. Identification of phosphate-solubilizing and growth-promoting bacteria is as follows: (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 physicochemical properties such as Gram staining reaction, citrate utilization, indole production, malonate utilization, VP test, methyl red test, and maltose utilization were measured.

[0033] (2) Molecular biological identification: Total DNA was extracted using a bacterial DNA extraction kit, and 16S rDNA sequences were amplified using the existing bacterial universal primers 27F and 1492R. Sequencing was performed by BGI Biotechnology Co., Ltd. The sequences were aligned with the GenBank database, and a phylogenetic tree was constructed using MEGA7.0.

[0034] The colonies of strain P03 on LB solid culture plates are round, smooth, and milky white (e.g. Figure 2 As shown in A); the bacteria are short rods (as shown in Figure 2 The physiological and biochemical identification results of the strains are shown in Table 2.

[0035] Table 2

[0036] The 16S rDNA sequence of strain P03 was aligned with the GenBank database by blast comparison, and the sequences with higher homology were selected. The sequences were analyzed by MEGA7.0, and the phylogenetic tree was constructed by the NJ method (e.g. Figure 3 The results showed that the phosphate-solubilizing bacteria P03 and Enterobacter hallii Enterobacter hormaechei strain DZXOTU4 (MT893338.1) sequence homology reached 99.57%. Further identification based on morphological characteristics and physiological and biochemical properties identified strain P03 as Enterobacter hallii ( Enterobacter. hormaechei The strain was named Enterobacter hallii ( 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.

[0037] Example 2: The characteristics of the above-mentioned Enterobacter hallii P03 were studied as follows: 1. Effect of temperature on the phosphate solubilization ability of the strain: The bacterial solution was inoculated into PKO liquid phosphate solubilizing medium at a 1% inoculum volume and incubated in a constant temperature shaking incubator at 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, and 40℃, at 180 rpm. -1 After 3 days of 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.

[0038] 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 increases, 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 varies significantly. Strain P03 has the strongest phosphate-solubilizing ability at 25℃, with the soluble phosphorus content in the culture medium reaching 520.25 mg·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 medium gradually increased with the increase of temperature, and the pH value range was 3.23-3.82.

[0039] 2. Effect of initial pH value on the phosphate solubilization ability of the strain: 1% inoculum was inoculated 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 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.

[0040] 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 solubility ability, and the soluble phosphorus content in the culture medium reaches 469.08 mg·L -1 , while when the pH value is 9, the soluble phosphorus content is 306.2 mg·L -1 Except for the treatment with an initial pH 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.

[0041] 3. Effect of inoculation amount on the phosphate solubilization ability of the strain: 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 rpm. -1 After 3 days of shaking culture, the soluble phosphorus content and pH value of the culture solution were measured. Each treatment was repeated 3 times, and the uninoculated culture medium was used as the control.

[0042] Depend on Figure 6 It can be seen that when the inoculation amount was 2%, the strain P03 had the best phosphate solubility performance, 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 medium with different inoculation amounts decreased significantly, ranging from 3.39 to 3.84. The pH value of the culture medium with an inoculation amount of 2% was the lowest, and the pH value of the culture medium with an inoculation amount of 6% was the highest.

[0043] 4. Effect of salinity on the phosphate solubilization ability of strains: 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 culture, the soluble phosphorus content and pH value of the culture medium were determined. Uninoculated liquid phosphate-dissolving medium was used as a control, and each treatment was repeated three times.

[0044] 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 8g·L -1 The strain P03 had the strongest phosphate solubility ability, with the soluble phosphorus content reaching 361.01 mg·L -1 The 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 medium changed in the opposite direction to the soluble phosphorus content, showing a trend of first decreasing and then increasing.

[0045] Example 3: Exploring the effect of the above-mentioned Enterobacter hallii P03 on the growth of tomato seedlings, specifically as follows: 1. Selection of test plants: The tomato variety used for the test was Yanyu.

[0046] 2. Preparation of fermentation broth: strain P03 was inoculated into liquid NA medium at 1% inoculum, and the mixture was incubated at 25°C and 180 rpm. -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.

[0047] 3. Effect of strain P03 on tomato seedling growth: Tomato seedlings with two leaves and one heart, showing consistent growth, were transplanted into 12 cm diameter pots, with one plant planted per pot. After 3 days of acclimatization, 10 ml of the fermentation solution was applied to each pot. An equal amount of sterile water served as the control (CK). Each treatment was replicated three times, with 10 pots per replicate. After 21 days of growth, growth indicators such as plant height, stem diameter, fresh weight, dry weight, underground fresh weight, and underground dry weight were measured. Functional leaves were selected for determination of MDA, POD, and SOD enzyme activities, as well as chlorophyll content.

[0048] Adding a fermented liquid from strain P03 to tomato seedlings during their growth period significantly promoted seedling growth. Table 3 shows that after application, the tomato seedlings achieved a plant height of 43.9 cm, a stem diameter of 7 mm, a fresh weight of 14.41 g, a below-ground fresh weight of 1.24 g, a chlorophyll content of 4.09 mg / g, a POD activity of 8218.88 U / g, and a SOD activity of 78.41 U / g—all significantly higher than those in the control. This suggests that the addition of the liquid significantly promoted seedling growth, increased POD and SOD enzyme activities, reduced MDA content, and enhanced stress resistance during the seedling stage.

[0049] Table 3

[0050] 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 the bacterial solution treatment on the soil physical and chemical properties.

[0051] As shown in Table 4, applying bacterial solution to soil can significantly increase the available phosphorus and total phosphorus contents in the soil, which increased by 5.14% and 8.18% respectively compared with the control (CK), and also significantly increased the activities of soil acid phosphatase and alkaline phosphatase.

[0052] Table 4

[0053] 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. -1 ), it grows well under acidic conditions (pH=3) and alkaline conditions (pH=9), and has good phosphate solubilization ability. At the same time, it can produce indoleacetic acid during the growth process to promote plant growth.

[0054] Based on the above-mentioned ideal embodiment of the present invention, and through the above description, relevant personnel can make various changes and modifications without departing from the scope of the technical concept of the present invention. 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-tolerant phosphate-solubilizing growth-promoting bacteria as claimed in claim 1 in improving soil phosphorus pollution.

4. 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.

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

6. Use of the salt- and alkali-tolerant phosphate-solubilizing and growth-promoting bacteria as claimed in claim 1 in planting crops in salinized soil, acidic soil or alkaline soil.

7. The use according to claim 6, characterized in that: The crops include tomatoes.

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