A salt-alkali tolerant, phosphate-solubilizing, growth-promoting Enterobacter cloacae strain and its application

Abstract

The invention discloses a salt-alkali tolerant phosphate-solubilizing growth-promoting Enterobacter cloacae and an application thereof, which belongs to the field of biotechnology. The salt-alkali tolerant phosphate-solubilizing growth-promoting Enterobacter cloacae is named Enterobacter cloacae ( Enterobacter cloacea ) JP2022044, deposited in China Center for Type Culture Collection, with a deposit number of CCTCC No: M 20242251. The Enterobacter cloacae JP2022044 provided by the present invention has good salt-alkali resistance and phosphate solubilization effect, grows well in a saline-alkali environment and has efficient phosphate solubilization ability, and can significantly promote plant growth.

Description

Technical Field

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

[0002] Phosphorus is the second largest nutrient element for plant growth and development. It plays a vital role in the growth, metabolism and reproduction of plants. It is directly involved in nucleic acid synthesis, cell division and new tissue growth, and can also regulate protein synthesis and energy transfer. The main source of phosphorus required by plants is soil. There are more than 170 different phosphorus-supplying minerals in the soil. Although there is a considerable amount of total phosphorus in the soil, the soluble phosphorus content is less than 1%, which cannot meet the phosphorus needs of crops. When plants are deficient in phosphorus, they show symptoms such as overall dwarfing of the plant, dark green or bronze leaves, purple spots on the edges of the leaves, underdeveloped root system, delayed flowering and fruiting, and weakened resistance. At present, about 67% of the soil in my country is deficient in phosphorus, and 70% of the cultivated land soil is polluted by phosphorus. The use of a large amount of phosphorus fertilizer in agricultural production will lead to excessive phosphorus in the soil. The excess phosphorus reacts with metal ions and is converted into insoluble phosphorus, which cannot be absorbed and utilized by plants, resulting in waste of phosphorus resources and secondary salinization of the soil. Improving the utilization rate of insoluble phosphorus in the soil and reducing the amount of phosphorus fertilizer are important issues facing agricultural production.

[0003] Phosphorous Solubilizing Microorganisms (PSM) are microorganisms that can convert insoluble phosphorus into soluble phosphorus. They are widely distributed in the soil and include various microbial groups such as phosphate-solubilizing bacteria, phosphate-solubilizing fungi, phosphate-solubilizing actinomycetes, and cyanobacteria. Currently, there are 36 known genera of phosphate-solubilizing microorganisms, including Bacillus ( Bacillus ), Pseudomonas spp. Pseudomonas ), Erwinia ( Erwinia ) and other 26 genera, with a content between 1%-50%, and a large number; while the types and numbers of phosphate-solubilizing fungi are much less than those of phosphate-solubilizing bacteria, with a content between 0.1%-0.5%, including Aspergillus ( Aspergillus )、Penicillium( Penicillium )、Rhizopus spp. Rhizopus ) etc.; there are fewer species of phosphate-solubilizing actinomycetes, mainly including Streptomyces ( Streptomyces ), Micromonospora ( Micromonospora ) etc. Using phosphate-solubilizing microorganisms to improve the utilization of insoluble phosphorus in the soil and promote plant phosphorus absorption, screening and developing and utilizing efficient phosphate-solubilizing bacteria is of great significance to improving the effectiveness of phosphorus in salinized soil and saline-alkali soil and improving the soil ecological environment. Summary of the invention

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

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

[0006] A salt-alkali tolerant, phosphate-solubilizing, growth-promoting Enterobacter cloacae strain was named Enterobacter cloacae ( Enterobacter cloacea ) JP2022044, deposited in China Center for Type Culture Collection, the deposit number is CCTCC No: M 20242251, 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 Enterobacter cloacae 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 Enterobacter cloacae 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 and growth-promoting Enterobacter cloacae in promoting plant growth in saline-alkali 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 Enterobacter cloacae in planting crops in a saline-alkali environment.

[0012] Preferably, the crop plants include tomatoes.

[0013] The present invention provides a salt-alkali tolerant, phosphate-solubilizing and growth-promoting Enterobacter cloacae, which has good salt-alkali resistance and phosphate-solubilizing effects, grows well in a saline-alkali environment (salinity of 6%, pH=12) and has efficient phosphate-solubilizing ability, and can significantly promote plant growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The colony (A) and bacterial morphology (B) of strain JP2022044.

[0015] Figure 2 To construct the phylogenetic tree of strain JP2022044 based on the 16S rDNA gene sequence.

[0016] Figure 3 These are pictures of the growth of Enterobacter cloacae JP2022044 on culture plates with different salinity levels.

[0017] Figure 4 This is a comparison chart of the colony diameters of Enterobacter cloacae JP2022044 on culture plates with different salinities.

[0018] Figure 5 These are pictures of the growth of Enterobacter cloacae JP2022044 on plates with different pH values.

[0019] Figure 6 This is a comparison chart of the colony diameters of Enterobacter cloacae JP2022044 on plates with different pH values.

[0020] Figure 7 This is the growth curve of Enterobacter cloacae JP2022044.

[0021] Figure 8 This is a graph showing the effect of different culture times on the phosphate solubilization effect of Enterobacter cloacae JP2022044.

[0022] Fig. 9 This is a graph showing the effect of different salt concentrations on the phosphate solubilization effect of Enterobacter cloacae JP2022044.

[0023] Fig.10 This is a graph showing the effect of different pH values ​​on the phosphate solubilization effect of Enterobacter cloacae JP2022044.

[0024] Fig.11 This is a graph showing the effect of inoculation size on the phosphate solubilization effect of Enterobacter cloacae JP2022044. DETAILED DESCRIPTION

[0025] 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.

[0026] 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.

[0027] 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.

[0028] Example 1: This example provides a method for separating and purifying Enterobacter cloacae, which is as follows:

[0029] On March 12, 2022, saline-alkali soil was collected from saline-alkali land in Shouguang City, Shandong Province, and the strains were isolated using the dilution plate method. The soil samples were diluted to 10 -5 , 10 -6 , 10 -7times, take 100 μL of the supernatant and spread it evenly on the PKO culture plate, culture it at 27℃ for 3 days, pick a single colony with a transparent circle and streak it multiple times, pick a single colony with consistent colony morphology, inoculate it on the NA slant and culture it at 27℃ for 3 days to obtain the strain JP2022044, which was stored in a refrigerator at 4℃.

[0030] Example 2: The strain JP2022044 obtained in Example 1 above was identified as follows:

[0031] (1) Observation of colony and bacterial morphology: Inoculate the purified strain JP2022044 onto NA medium and culture at 27°C for 48 hours. Observe the colony morphology and growth. Pick colonies for Gram staining and observe the size and morphology of the bacteria. The colonies on NA medium are off-white, round, mucous, with a smooth, shiny surface and neat edges. The bacteria are short rod-shaped, with a size of (1.1-3.1)µm×(0.4-1.2)µm, and Gram staining is negative (for details, see Figure 1 as shown).

[0032] (2) Molecular biology and phylogenetic analysis: The molecular biological identification of strain JP2022044 used 16S rDNA gene fragments. The primers for amplification of the 16S rDNA gene fragments were the existing universal primers 27F and 1492R. The primers used for PCR amplification were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The total DNA of strain JP2022044 was extracted and PCR amplified using 27F / 1492R as primers. The PCR amplification product was sent to BGI Genomics Co., Ltd. for sequencing, and the sequence size was 1446 bp. The sequencing results were subjected to BLAST sequence homology analysis and the related gene sequences were downloaded. The phylogenetic tree was constructed by the maximum likelihood method, such as Figure 2 shown.

[0033] The 16S rDNA gene sequence of strain JP2022044 was compared with several Enterobacter cloacae in the database by BLAST analysis. Enterobacter cloacea ) sequence homology reached more than 99%. The phylogenetic tree results showed that the 16S rDNA gene sequence of strain JP2022044 was similar to that of Enterobacter cloacae ( Enterobacter cloacea ) were clustered into one branch and the strain was identified as Enterobacter cloacae ( Enterobacter cloacea The strain was named Enterobacter cloacae ( Enterobacter cloacea ) JP2022044, deposited in the China Center for Type Culture Collection on October 21, 2024, with the deposit number CCTCC No: M20242251, and the deposit address is Wuhan University, Wuhan, China.

[0034] Example 3: The salt-alkali resistance of Enterobacter cloacae JP2022044 was determined as follows:

[0035] 1. Effect of salinity on the growth of Enterobacter cloacae JP2022044: Enterobacter cloacae JP2022044 was inoculated on NA medium plates with NaCl contents of 0%, 2%, 4%, 6%, and 8%, 0.5 µL was inoculated at each point, and cultured in a 27°C constant temperature incubator for 3 days. The colony growth was observed and the colony diameter was determined by the cross method. Three plates were inoculated for each concentration.

[0036] The results are as follows Figure 3 and Figure 4 As shown in the figure, it can be seen that Enterobacter cloacae JP2022044 can grow on the medium with NaCl concentration of 0%-6%, but not on the medium with NaCl concentration of 8%. On the medium with NaCl concentration of 0%, the maximum colony diameter is 8.83mm, and the smallest colony diameter on the medium with NaCl concentration of 6% is 4.67mm ( Figure 4 ). The above results show that Enterobacter cloacae JP2022044 has good salt tolerance.

[0037] 2. Effect of different pH values ​​on the growth of Enterobacter cloacae JP2022044: Enterobacter cloacae JP2022044 was inoculated on NA medium plates with pH values ​​of 7, 8, 9, 10, 11, and 12, with 0.5 µL inoculated at each point. The plates were cultured in a constant temperature incubator at 27°C for 3 days, the colony growth was observed, and the colony diameter was determined by the cross method. Three plates were inoculated for each pH value.

[0038] The results are as follows Figure 5 and Figure 6 As shown in the figure, it can be seen that strain JP2022044 can grow on culture plates with different pH values. The colony diameter on the culture plate with a pH value of 7 is the largest, reaching 8.58mm; the colony diameter on the culture plate with a pH value of 12 is the smallest, reaching 5.92mm ( Figure 6 ). The above results show that Enterobacter cloacae JP2022044 has good alkali resistance.

[0039] Example 4: The growth curve of Enterobacter cloacae JP2022044 was measured as follows:

[0040] (1) Enterobacter cloacae JP2022044 was inoculated into liquid NA culture medium and cultured at 27°C and 180 rpm for 24 h for activation and set aside.

[0041] (2) Inoculate 1% of the activated bacterial solution into 50 mL of liquid NA culture medium in a conical flask, for a total of 12 flasks. Incubate the flask at 27°C and 180 rpm with shaking. Take out one flask every 2 hours and measure the absorbance of the bacterial solution at a wavelength of 600 nm. Continue measuring for 24 hours.

[0042] The results are as follows Figure 7 As shown in the figure, it can be seen that Enterobacter cloacae JP2022044 grows rapidly in NA medium. After 12 hours of culture, the absorbance value at 600nm reaches the maximum, and OD 600 is 2.067. As the culture time increases, the OD 600 Gradually decreased, OD 600 The results showed that Enterobacter cloacae JP2022044 grew rapidly, had strong reproductive capacity, could reproduce in large quantities in a short time, and was easy to ferment.

[0043] Example 5: The phosphate solubilization effect of Enterobacter cloacae JP2022044 was measured, as follows:

[0044] 1. Effect of culture time on the phosphate solubilization effect of strain JP2022044: Enterobacter cloacae JP2022044 was inoculated into liquid PKO medium at a ratio of 1%, and cultured at 27°C and 180 rpm with shaking. The uninoculated liquid PKO medium was used as the control. The bacterial solution was taken out at 1d, 2d, 3d, 4d, 5d, 6d, and 7d of culture, centrifuged at 10,000 rpm for 15 min, and the supernatant was taken. The soluble phosphorus content in the solution was determined by the molybdenum antimony colorimetric method. Each treatment was repeated 3 times.

[0045] The results are as follows Figure 8 As shown in the figure, the soluble phosphorus content in the culture fluid of Enterobacter cloacae JP2022044 at different culture times showed a trend of first increasing and then decreasing, and the soluble phosphorus content at different culture times was significantly different. The soluble phosphorus content in the culture fluid was the highest at 5 days of culture, reaching 569.66 mg / L, followed by 539.80 mg / L at 4 days of culture.

[0046] 2. Effect of salinity on the phosphate solubilization effect of Enterobacter cloacae JP2022044: Liquid PKO culture medium with salinity (NaCl content) of 0, 2%, 4%, 6%, and 8% was prepared respectively, and Enterobacter cloacae JP2022044 was inoculated into the above culture medium at an inoculation rate of 1%, and cultured at 27°C and 180rpm for 3 days. The liquid PKO culture medium without bacteria at the corresponding salt concentration was used as the control. After the culture, the soluble phosphorus content in the solution was determined by the molybdenum antimony colorimetric method. Each treatment was repeated 3 times.

[0047] The results are as follows Fig. 9As shown in the figure, it can be seen that the effective phosphorus content in the culture solution of Enterobacter cloacae JP2022044 gradually decreases with the increase of salinity, and it cannot grow at a salinity of 8%, and the difference between different salinity treatments is significant. Enterobacter cloacae JP2022044 has the strongest phosphorus solubility in the salt-free medium, with a soluble phosphorus content of 536.43 mg / L, and the soluble phosphorus content in the medium with a salinity of 6% is only 263.69 mg / L.

[0048] 3. Effect of pH on the phosphate solubilization effect of Enterobacter cloacae JP2022044: Use NaOH and HCl solutions with a concentration of 1 mol / L to adjust the pH of the liquid PKO medium to 4, 5, 6, 7, 8, 9, 10, and 11, respectively. Inoculate Enterobacter cloacae JP2022044 into the above medium at an inoculation rate of 1%, and culture at 27°C and 180 rpm for 3 days. Use the uninoculated liquid PKO medium as a control. After the culture, the molybdenum antimony colorimetric method was used to determine the soluble phosphorus content in the solution. Each treatment was repeated 3 times.

[0049] The results are as follows Fig.10 As shown in the figure, it can be seen that the effective phosphorus content in the culture solution of Enterobacter cloacae JP2022044 shows a trend of first increasing and then decreasing with the increase of pH value. Among them, the soluble phosphorus content in the culture medium with pH=6 is the highest at 435.44 mg / L, followed by the culture medium with pH=7, with a soluble phosphorus content of 434.77 mg / L, and there is no significant difference between the two. The phosphorus solubility ability of Enterobacter cloacae JP2022044 in the culture medium with pH=11 is the lowest, and the soluble phosphorus content is only 112.42 mg / L.

[0050] 4. Effect of inoculation amount on the phosphate solubilization effect of Enterobacter cloacae JP2022044: Enterobacter cloacae JP2022044 was inoculated into liquid PKO phosphate solubilization medium at 0.5%, 1%, 2%, 4%, and 6% inoculation amounts, respectively, and cultured at 27°C and 180rpm for 3 days. The uninoculated culture medium was used as the control. After the culture, the molybdenum antimony colorimetric method was used to determine the soluble phosphorus content in the solution. Each treatment was repeated 3 times.

[0051] The results are as follows Fig.11 As shown in the figure, it can be seen that different inoculation amounts have a significant effect on the phosphate solubilization effect of Enterobacter cloacae JP2022044. When the inoculation amount is 4%, the soluble phosphorus content in the culture fluid of Enterobacter cloacae JP2022044 is the highest, reaching 503.23 mg / L, followed by 6% inoculation amount, with a soluble phosphorus content of 498.85 mg / L. When the inoculation amount is 0.5% and 1%, there is no significant difference in the soluble phosphorus content in the culture fluid of Enterobacter cloacae JP2022044, which are 463.30 mg / L and 469.67 mg / L, respectively.

[0052] Example 6: Exploring the effect of Enterobacter cloacae JP2022044 on the growth of tomato seedlings, as follows:

[0053] 1. The test plant is tomato, and the tomato variety is "Fen Beibei".

[0054] 2. Preparation of fermentation liquid: Enterobacter cloacae JP2022044 was inoculated into liquid NA medium at 1% inoculation rate, cultured in a constant temperature shaking incubator at 27°C and 180 rpm for 3 days, and the concentration of the liquid was adjusted to 1×10 8 CFU, reserve.

[0055] 3. Effect of phosphate-solubilizing growth-promoting bacteria 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, with one plant planted 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 repeat. After 30 days of growth, carefully remove each tomato from the pot, use a washing bottle to clean and dry the root matrix, and then measure growth indicators such as plant height, stem thickness, fresh weight, and leaf area.

[0056] 4. Results: Adding the fermented bacterial liquid of Enterobacter cloacae JP2022044 can effectively promote the growth of tomato seedlings. As shown in Table 1, after adding the fermented bacterial liquid, the plant height of the tomato seedlings reached 24.62 cm, the stem diameter was 4.65 mm, and the leaf area was 69.43 cm 2 , root weight 1.58g, root length 597.92cm, root area 77.76cm 2 , fresh weight was 15.09g, dry weight was 1.92g, the plant height, stem diameter, leaf area, root weight, root length, root area, fresh weight and dry weight of tomato seedlings added with bacterial solution were significantly higher than those of the control, and there was no significant difference in stem diameter between the control and the control.

[0057] Table 1

[0058]

[0059] In summary, the Enterobacter cloacae JP2022044 provided in the embodiment of the present invention has good salt-alkali resistance and phosphate solubilization effect, grows well in a saline-alkali environment (salinity of 6%, pH=12) and has efficient phosphate solubilization ability, which can significantly promote plant growth.

[0060] 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. Use of a salt-alkali tolerant, phosphate-solubilizing, growth-promoting Enterobacter cloacae in promoting tomato growth in saline-alkali soil, characterized in that: Enterobacter cloacae is named Enterobacter cloacae ( Enterobacter cloacea ) JP2022044, preserved in China Center for Type Culture Collection, with the preservation number of CCTCC No: M 20242251, and the preservation time is October 21, 2024; the Enterobacter cloacae can solubilize phosphate ≥150 mg / L under the conditions of salinity 6% and pH=12.

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