Acinetobacter johnsonii sip23-7 and application thereof

Abstract

This invention discloses a strain of Acinetobacter shenyi SIP23-7 and its applications. This Acinetobacter shenyi SIP23-7 was deposited at the China Center for Type Culture Collection (CCTCC) on September 9, 2021, with accession number CCTCC NO: M20211152, located at Wuhan University, Wuhan, China. This strain possesses growth-promoting properties such as phosphorus solubilization, IAA secretion, and siderophore production, effectively promoting plant growth. This provides a potential phosphorus-solubilizing bio-fertilizer for crops. Using microbial fertilizers containing this inoculant can improve crop yield and quality, improve the soil environment, and contribute to the sustainable development of agriculture.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to Acinetobacter sheni SIP23-7 and its applications. Background Technology

[0002] Plant growth and development require a large amount of nutrients, and phosphorus is one of the main essential nutrients for plant growth. Usually, fertilization is used to meet the nutritional needs of plants. However, soluble phosphate fertilizers applied to the soil tend to precipitate easily, and plants only recover about 25% of the phosphate fertilizer, making it difficult for plants to absorb phosphorus and leading to deficiency. This severely restricts crop growth and hinders sustainable agricultural development. Phosphorus-solubilizing microorganisms (PSMs) are an important subgroup that can phosphate-solubilize phosphorus in the form of PO42-. 3- Insoluble phosphorus in its original form is converted into HPO4. 2- and H2PO4 - Soluble phosphorus, existing in plant form and readily absorbable, provides plants with more available phosphorus, thereby improving the utilization rate and efficiency of insoluble phosphorus solubilization and promoting plant growth. For example, arbuscular mycorrhizalfungi (AMF) promotes phosphorus absorption by plants. Inoculating crops with phosphate-solubilizing bacteria (PSB) has the potential to reduce phosphate fertilizer usage by 50% without significantly decreasing crop yield. Therefore, research on the utilization of phosphate-solubilizing microorganisms is of great significance.

[0003] Existing microorganisms with phosphate-solubilizing and growth-promoting effects include Acinetobacter calcoaceticus, but there are no reports of Acinetobacter scheni having phosphate-solubilizing and growth-promoting effects. Summary of the Invention

[0004] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide Acinetobacter shenyi SIP23-7 and its applications, wherein Acinetobacter shenyi SIP23-7 possesses phosphate solubilization ability, IAA secretion ability, siderophore production ability, and ACC deaminase production ability.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: an Acinetobacter schindleri, named Acinetobacter schindleri SIP23-7, was deposited at the China Center for Type Culture Collection on September 9, 2021, with accession number CCTCC NO: M20211152, and the deposit address is Wuhan University, Wuhan, China.

[0006] The Acinetobacter sheni SIP23-7 mentioned above can be used to degrade inorganic phosphorus and / or organic phosphorus.

[0007] The aforementioned Acinetobacter sheni SIP23-7 can be used to prepare preparations containing ferrophiles.

[0008] The Acinetobacter shensi SIP23-7 mentioned above can be used to prepare IAA preparations.

[0009] The aforementioned Acinetobacter sheni SIP23-7 can be used to promote plant growth, preferably maize growth.

[0010] The Acinetobacter sheni SIP23-7 mentioned above can be used to prepare phosphorus-solubilizing and growth-promoting biological agents or phosphorus-solubilizing and growth-promoting biological fertilizers.

[0011] An agent for degrading inorganic and / or organic phosphorus, comprising Acinetobacter sheni SIP23-7 as described above.

[0012] A preparation containing ferrophiles, comprising the aforementioned Acinetobacter schenckii SIP23-7.

[0013] An IAA preparation comprising the aforementioned Acinetobacter schenckii SIP23-7.

[0014] An agent for promoting plant growth, comprising Acinetobacter schenckii SIP23-7 as described above.

[0015] A phosphorus-solubilizing and growth-promoting biological agent, comprising the aforementioned Acinetobacter sheni SIP23-7, can also be prepared into a phosphorus-solubilizing and growth-promoting bio-fertilizer.

[0016] In the above applications or preparations, Acinetobacter shensi SIP23-7 can exist in solid form or in fermentation broth, etc., and its form does not affect its efficacy.

[0017] The present invention has the following beneficial effects:

[0018] This invention isolates a phosphate-solubilizing strain from the rhizosphere soil of Sophora flavescens. The strain was identified through morphological, physiological, biochemical, and molecular biological methods. It belongs to the genus Acinetobacter sp. and shows the highest 16S ribosomal RNA sequence similarity (99%) to Acinetobacter schindleri strain LUH5832, and is named SIP23-7. Its phosphate-solubilizing ability, IAA secretion ability, and siderophore production ability, among other growth-promoting characteristics, were measured. Using maize as the research material, the phosphate-solubilizing bacterium SIP23-7 was formulated into an inoculum to study its effects on maize seedling growth. By measuring morphological indicators such as plant height, stem diameter, leaf area, root length, and root diameter of corn seedlings, as well as physiological and biochemical indicators such as above-ground fresh weight, above-ground dry weight, underground fresh weight, underground dry weight, and root activity, and comparing them with the control group (CK), corn treated with the inoculant SIP23-7 showed varying degrees of increase in all indicators except for stem diameter and above-ground dry weight. In particular, the underground dry weight increased by 275% compared to the control group. This provides a potential phosphorus-soluble bio-fertilizer for crops. Using microbial fertilizers containing this inoculant can improve crop yield and quality, improve the soil environment, and contribute to the sustainable development of agriculture. Attached Figure Description

[0019] Figure 1 The results show the colony morphology and electron microscopy observations.

[0020] Figure 2 This is a growth curve of the strain.

[0021] Figure 3 These are the results of the strain's physiological and biochemical identification.

[0022] Figure 4 This is a phylogenetic tree of the strains constructed based on the 16S rDNA gene sequence.

[0023] Figure 5 The results show the phosphorus solubilization ability of strain SIP23-7.

[0024] Figure 6 The results show the changes in soluble phosphorus content in the supernatant of strain SIP23-7 after inoculation into inorganic phosphorus medium.

[0025] Figure 7 The results show the pH changes of the supernatant after strain SIP23-7 was inoculated into inorganic phosphorus medium.

[0026] Figure 8 The results are qualitative determinations of the iron-loving ability of strain SIP23-7.

[0027] Figure 9 This is the TTF standard curve.

[0028] Figure 10 This is a diagram showing the root growth of maize seedlings under different treatments. Detailed Implementation

[0029] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0030] Example 1: Screening and Identification of Strains

[0031] I. Strain Screening

[0032] 5g of Sophora flavescens rhizosphere soil was taken from the Sophora flavescens planting base of Shanxi Zhendong Authentic Medicinal Herbs Development Co., Ltd., added to sterile water, and shaken at 28℃ and 150r / min for 30 minutes to ensure thorough mixing. The mixture was then diluted to a final concentration of 10. -3 10 -4 10 -5 10 -6 Each gradient was selected and spread onto organophosphate solid medium (Monkina medium), and incubated at 28℃ for 4 days. After three generations of streaking, single colonies of phosphate-solubilizing bacteria were obtained. Single colonies were picked up with a sterile toothpick and stored in 25% glycerol at -80℃.

[0033] II. Strain Identification

[0034] 1. Morphological and physiological-biochemical identification of the strain

[0035] (1) Morphological identification of strains

[0036] The selected strains were inoculated into LB solid medium and incubated at 28℃ for 3-4 days. The color, shape, size, and other characteristics of the colonies were observed. Specific results are shown in [link to results]. Figure 1 The growth curve of the strain is shown in the figure. Figure 2 .

[0037] Depend on Figure 1 It can be seen that the colony morphology is spherical, the colony is small, the center is raised, the surface is smooth, and it is opaque.

[0038] (2) Physiological and biochemical identification of strains

[0039] Referring to the physiological and biochemical characteristics of bacteria in the "Manual of Systematic Identification of Common Bacteria" and "Experimental Techniques in Microbiology", Gram staining test, gelatin liquefaction test, malonate test, citrate test, methyl red test, catalase test, VP test, and phenylalanine deaminase test were performed on phosphate-solubilizing bacteria.

[0040] Identification results: The strain was Gram-negative. It was negative for vortex, methyl red, gelatin liquefaction, phenylalanine deaminase, indole, starch hydrolysis, and urease tests; positive for malonate, citrate, and lecithin tests. Some experimental results are shown in […]. Figure 3 .

[0041] 2. Molecular biological identification and phylogenetic analysis of the strain

[0042] Genomic DNA was extracted from the bacterial strain, and 16S rDNA was amplified using universal bacterial primers. The primer sequences are as follows:

[0043] 27-F:5'-AGAGTTTGATCCTGGCTCAG-3',

[0044] 1492-R:5'-GGTTACCTTGTTACGACTT-3'.

[0045] PCR amplification conditions were as follows: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 45 s, 55℃ annealing for 45 s, 72℃ extension for 2 min, for 35 cycles; final extension at 72℃ for 10 min. The purified PCR products were sent to Beijing BGI Genomics Co., Ltd. for sequencing. Sequences were analyzed using NCBI homology sequence BLAST. Results were analyzed using MEGA 7.0 software. Neighbor-joining (NJ) was used with the Kimura 2-parameter as the model, and bootstrap replication was performed 1000 times to test model stability. A phylogenetic tree was then constructed.

[0046] Identification results: After amplification of the 16S rDNA of the strain, the amplification products were analyzed by 1.0% agarose gel electrophoresis, and all bands were single bands of 1.5 kb in size. The sequences obtained after cloning were compared with the NCBI database. Figure 4 Phylogenetically, the strain belongs to the genus *Acinetobacter* and has the highest 16S ribosomal RNA sequence similarity (99%) to *Acinetobacterschindleri* strain LUH5832. This strain was named *Acinetobacter sp. SIP23-7* and deposited on September 9, 2021, at the China Center for Type Culture Collection (CCTCC), accession number M20211152, Wuhan University, Wuhan, China.

[0047] Example 2: Determination of the growth-promoting ability of the strain

[0048] I. Determination of the phosphorus solubilization ability of strain SIP23-7

[0049] The Monkina organic phosphorus solid medium was divided into four equal regions. 1 μL of fresh bacterial suspension of the screened strain SIP23-7 was added to each region for spot inoculation. The plates were then incubated at 28°C. The phosphorus-solubilizing ability of the phosphate-solubilizing bacteria was preliminarily assessed based on the ratio of the diameter of the phosphate-solubilizing zone (D) to the colony diameter (d) (see Table 1). See Table 1 for details. Figure 5 , Figure 5 A and Figure 5 B represents the culture medium without inoculation and the culture medium inoculated with SIP23-7 strain, respectively.

[0050] Depend on Figure 5 It can be seen that when strain SIP23-7 is inoculated into an organic phosphorus solid medium by spot inoculation, a transparent zone can be observed around the colony after 4 days of culture, indicating that strain SIP23-7 has phosphorus solubilization ability.

[0051] II. Phosphorus solubilization kinetics of strain SIP23-7

[0052] The seed culture of strain SIP23-7 was inoculated at a rate of 1% into 50 mL of NBRIP liquid medium with calcium phosphate as the sole phosphorus source. The medium was incubated at 28°C with shaking at 180 rpm for 9 days. Three replicates were taken every 24 hours, with a blank medium (without inoculation) serving as a control. The soluble phosphorus content and pH of the supernatant were determined according to the above procedure. Specific results are shown in [link to results]. Figure 6 and Figure 7 .

[0053] Depend on Figure 6 It can be seen that as the number of days of cultivation increases, the calcium phosphate solid fixed at the bottom of the culture flask becomes smaller and smaller, and eventually disappears.

[0054] Depend on Figure 7 It can be seen that as the number of days of cultivation increases, the concentration of the strain gradually increases, while the pH value first decreases and then remains in a relatively stable state.

[0055] III. Determination of Potassium Solubility

[0056] Select strain SIP23-7 and inoculate it onto a potassium feldspar solid medium plate. After incubating at 28°C upside down for 3-4 days, observe the potassium dissolution zone.

[0057] Meanwhile, the ratio of the diameter of the potassium-solubilized zone to the diameter of the colony (D / d) was recorded and calculated to be 5.33 (see Table 1), which can preliminarily determine that strain SIP23-7 has potassium-solubilizing ability.

[0058] IV. Determination of the heptaphilic production capacity of strain SIP23-7

[0059] Qualitative determination of iron-producing ability: Strain SIP23-7 was inoculated in the center of CAS solid medium and placed in a 30℃ constant temperature incubator for 72 hours. If a yellow-green halo appeared around the strain, it indicated that the strain had the ability to produce iron.

[0060] Quantitative measurement of iron affinity: The strain was inoculated into 30 mL of MKB liquid medium at a ratio of 1:100; cultured at 28℃ and 200 rpm for 24 h on a shaker; centrifuged (10000 rpm, 10℃) for 15 min, and the supernatant was mixed with an equal volume of CAS blue detection solution; using uninoculated MKB medium as a control, after incubation at 25℃ for 1 h, the OD values ​​of strain 23-7 (T) and the uninoculated blank control (CK) were measured. 630 The value of is used to calculate the production of ferrocarriers using the following formula (results are shown in Table 1):

[0061]

[0062] Test results: A yellow-green halo appeared around strain SIP23-7, indicating that strain SIP23-7 has the ability to produce heparin (see details). Figure 8 ).

[0063] V. Determination of IAA secretion capacity of strain SIP23-7

[0064] The method for determining the IAA secretion capacity of strain SIP23-7 was based on the study "Screening and Biodiversity of Endophytic and Rhizosphere Bacteria with ACC Deaminase Activity from Limonium sinense" (Feng Weiwei et al., Acta Microbiologica Sinica, 2016), and the specific procedure is as follows:

[0065] Qualitative detection of tryptophan-containing IAA: Following the Salkowski colorimetric method, after culturing in a medium containing tryptophan for 7 days, mix with the colorimetric reagent and develop at room temperature for 2 minutes. If a pink color appears, it is positive, indicating that IAA can be produced.

[0066] Quantitative detection of IAA production: The strain was inoculated into sterile nitrogenous medium (containing 0.5 mg / ml tryptophan) and cultured in a shaker at 28℃ for 7 days (uninoculated strains served as controls). After centrifugation at 4000 r / min, the supernatant was collected in a test tube, and an equal volume of RI reagent was added. After mixing, the mixture was incubated in the dark for 30 min, and the OD value was measured at a wavelength of 530 nm. The corresponding IAA concentration was calculated using the relationship curve between IAA concentration and OD value.

[0067] Results: Qualitative experiments showed that strain SIP23-7 can produce IAA. A standard curve of IAA production confirmed that strain SIP23-7 has the ability to produce IAA, with a secreted IAA concentration of 14.191 μg / mL. IAA production by this strain can accelerate plant growth and metabolism, thus promoting plant growth.

[0068] The specific data on the growth-promoting ability of strain SIP23-7 are shown in Table 1:

[0069] Table 1 Results of growth-promoting ability assay for strain SIP23-7

[0070]

[0071] Example 3: Pot inoculation experiment of strain SIP23-7

[0072] Experimental procedure: The glycerol-preserved bacterial strain SIP23-7 was inoculated into liquid LB medium at a 1:100 ratio for initial activation, lasting 5–6 hours. Then, 500 μL of the activated bacterial suspension was inoculated into a 150 mL Erlenmeyer flask at a 1:100 ratio and incubated overnight with shaking for 12 hours. The bacterial cells were washed three times with sterile water, and the bacterial suspension concentration was adjusted to 10⁻⁶. 8 The inoculum was prepared at CFU / mL. The inoculum was added to 240g (60g × 4) of seedling substrate and mixed thoroughly. The control group (CK) was replaced with an equal volume of sterile water. Germinated corn seeds were then sown (the seeds were soaked in warm water overnight before sowing). The cultivation substrate was a mixture of decomposed peanut shells, vermiculite, perlite, and eggshells in a mass ratio of 50:25:20:5. The mixture was wrapped in gauze and sterilized twice at 121℃ in an autoclave. The mixture was then divided into seedling pots, with 10 replicates, and 2 corn seeds were planted in each pot. The pots were placed in a greenhouse (25℃) for cultivation. Once the seedlings emerged uniformly, one seedling of uniform size was transplanted from each pot. Watering was done quantitatively every 3 days. After 20 days, morphological indicators such as plant height, stem diameter, above-ground fresh weight, above-ground dry weight, underground fresh weight, underground dry weight, root length, and root diameter were measured.

[0073] Construction of a standard curve for root vigor in corn seedlings:

[0074] Prepare a 1 mg / mL (1%) TTC solution. Take 2 mL of this solution and transfer it to a 100 mL volumetric flask. Add 2 g of Na₂S₂O₄ and shake well. Add 40 mL of ethyl acetate and shake until fully dissolved. Make up to volume with ethyl acetate to prepare a 20 μg / mL TTF standard solution. Construct a TTF standard curve. Measure the OD value at a wavelength of 485 nm and plot the standard curve (see details). Figure 9 The regression equation is as follows:

[0075]

[0076] After the corn seedlings had grown to 20 days, the seedlings in both the SIP23-7 inoculant treatment group and the control group were removed from the soil samples, cleaned, and their OD values ​​were measured according to the method of Zhu Xiuyun et al. (Zhu Xiuyun et al., 2020). 485Substitute the value of TTC into the regression equation to calculate the root activity of maize in the SIP23-7 treatment group and the control group (CK). The amount of TTC reduction is then determined, which represents the root activity of the maize plant.

[0077] Data Analysis: Experimental data were analyzed using Excel and SPSS 13.0 statistical software for ANOVA and multiple comparisons. Root samples were obtained using an Epson scanner, and the WinRHIZO Pro root analysis system software was used to analyze the obtained root samples.

[0078] Experimental Results: Table 2 shows that by comparing the plant height, stem diameter, and other morphological indicators of the group treated with SIP23-7 fungicide with the control group treated with sterile water, the group treated with SIP23-7 showed varying degrees of increase in all indicators except stem diameter and above-ground dry weight, especially the underground dry weight, which increased by 275% compared to the control group. Furthermore, different treatments of plant height have varying degrees of impact on the root morphology and root activity of maize seedlings, such as... Figure 10 As shown, the root system of the SIP23-7 treatment group was more developed than that of the CK group.

[0079] As shown in Table 2, the TTC reduction intensity of the SIP23-7 treatment group was higher than that of the CK group, based on the root morphology of maize seedlings ( Figure 10 The results of the study and the determination of root activity showed that the treatment of corn with SIP23-7 inoculant promoted the growth of corn seedlings.

[0080] Table 2. Results of morphological index determination of maize under different treatments

[0081]

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A type of Acinetobacter schenckii ( Acinetobacter schindleri ), characterized in that, Named Acinetobacter sheni SIP23-7, it was deposited at the China Center for Type Culture Collection on September 9, 2021, with accession number CCTCC NO: M20211152, and the deposit address is Wuhan University, Wuhan, China. Acinetobacter sheni SIP23-7 simultaneously possesses the functions of degrading organic phosphorus, degrading inorganic phosphorus, potassium solubilization, producing heparin, secreting IAA, and producing ACC deaminase.

2. The application of Acinetobacter sheni SIP23-7 as described in claim 1 in the degradation of inorganic phosphorus and / or organic phosphorus.

3. The use of Acinetobacter sheni SIP23-7 as described in claim 1 in the preparation of a formulation containing ferrophile.

4. The use of Acinetobacter sheni SIP23-7 as described in claim 1 in the preparation of IAA formulations.

5. The application of Acinetobacter sheni SIP23-7 as described in claim 1 in promoting maize growth.

6. The application of Acinetobacter sheni SIP23-7 as described in claim 1 in the preparation of phosphorus-solubilizing and growth-promoting biological agents or fertilizers.

7. A formulation for degrading inorganic phosphorus and / or organophosphorus phosphorus, characterized in that, Includes Acinetobacter sheni SIP23-7 as described in claim 1.

8. A preparation containing heptaphilin, characterized in that, Includes Acinetobacter sheni SIP23-7 as described in claim 1.

9. An IAA formulation, characterized in that, Includes Acinetobacter sheni SIP23-7 as described in claim 1.

10. A plant growth-promoting preparation or phosphorus-solubilizing and growth-promoting biological agent or fertilizer, characterized in that, Includes Acinetobacter sheni SIP23-7 as described in claim 1.

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