Acinetobacter picoteri and application thereof

Abstract

The invention discloses acinetobacter picoteri and application thereof, and relates to the field of microorganisms. The problem that an existing single saline-alkaline stress saline-alkaline tolerant strain cannot better adapt to the saline-alkaline environment of the Songnen plain to meet plant growth is solved. The collection number of the Acinetobacter pittii is CCTCC (China Center for Type Culture Collection) NO: M2024440, and the collection unit of the Acinetobacter pittii is the China Center for Type Culture Collection. The strain is classified as Acinetobacter sp. On the basis of physiological and biochemical characteristics and 16S rRNA (ribosomal Ribonucleic Acid) analysis. The acinetobacter picoteri can grow in an LB culture medium with pH of 8.5-12.0 and soda salt of 80-250 mmol / L, and has the capabilities of dissolving inorganic phosphorus, dissolving potassium and producing indoleacetic acid; the saline-alkali environment of the Songnen plain can be better adapted, and rice seed germination and seedling growth under saline-alkali stress are promoted.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to Acinetobacter pitei and applications thereof. Background Art

[0003] Rice (Oryza sativa L.) is an annual aquatic herb. Currently, there are perennial rice plants. It belongs to the genus Oryza of the Poaceae family. The seeds are divided into two subspecies: indica rice and japonica rice. As one of the main food crops, rice accounts for 24.9% of the total cultivated land area in my country, and the total output accounts for 30.4% of the total grain output. At present, rice cultivation in my country is still mainly based on seedling cultivation and transplanting. Seedling cultivation is an important link in rice production. The growth status of rice seedlings has an important impact on its planting and subsequent growth, development and yield. Because it not only has extremely high nutritional value and delicious taste, but also has strong adaptability, it has become one of the main food crops on saline-alkali land. Studies have reported that planting rice on saline-alkali land can improve saline-alkali land. However, the higher the pH of the land, the more difficult it is for rice to grow.

[0004] There is a type of microorganism with growth-promoting function in the rhizosphere of plants, called Plant Growth Promoting Rhizobacteria (PGPR), which can promote plant growth by inducing plant systemic resistance to biotic and abiotic stresses through nitrogen fixation, phosphorus solubilization, potassium solubilization, plant hormone production, and inhibition of pathogenic microorganisms. PGPR selected from saline-alkali soil is more adaptable to saline-alkali environment. Its application in rice seedling cultivation can play a significant role in promoting rice seed germination and seedling growth, thereby increasing the final yield and economic benefits.

[0005] At present, most studies on salt-alkali tolerant strains focus on single salt-alkali stress. For example, Li Zhanglei et al. used sodium bicarbonate as the salt component to screen out 5 salt-alkali tolerant growth-promoting bacteria in the saline-alkali soil of Daqing. After application, the germination and seedling growth of red bean seeds were significantly improved; Li Dongying used sodium chloride as the salt component to screen out multiple salt-alkali tolerant growth-promoting bacteria in the saline-alkali soil of Songnen Plain. After application, the growth of sorghum seeds was significantly promoted. However, the saline-alkali environment of Songnen Plain is complex. Its main salt component is soda salt composed of sodium chloride, ammonium carbonate, sodium bicarbonate and sodium sulfate in a ratio of 1:1:9:9. In order to better adapt to the saline-alkali environment of Songnen Plain, salt-alkali tolerant growth-promoting bacteria should be screened based on this condition. However, the salt-alkali tolerant strains screened based on single salt-alkali stress cannot better adapt to the saline-alkali environment of Songnen Plain, and thus cannot better promote plant growth in such saline-alkali areas. Summary of the invention

[0006] The present invention aims to provide a Pittsburgh Acinetobacter spp. in view of the problem that the existing single salt-alkali stress tolerant strain in the background technology cannot better adapt to the saline-alkali environment of the Songnen Plain to meet the problem of plant growth in the region. The Pittsburgh Acinetobacter spp. has the ability to dissolve inorganic phosphorus, potassium and produce indoleacetic acid, can better adapt to the saline-alkali environment of the Songnen Plain, and significantly promote the germination of rice seeds and the growth of seedlings under saline-alkali stress. The present invention also provides an application of Pittsburgh Acinetobacter spp.

[0007] The present invention can solve the problem by the following technical solution:

[0008] The invention provides an Acinetobacter pittii. The preservation number of the Acinetobacter pittii is CCTCC NO: M2024440. The depositor is Heilongjiang Bayi Agricultural University and the preservation unit is China Center for Type Culture Collection.

[0009] The present invention also provides a microbial agent, which contains the Acinetobacter piteiris.

[0010] The present invention also provides a microbial fertilizer, which contains the Acinetobacter piteiris.

[0011] The present invention also provides the use of the Acinetobacter pituitii in promoting plant seed germination and seedling growth.

[0012] Preferably, the plant comprises rice.

[0013] Preferably, the use of Acinetobacter pitei in promoting rice seed germination and seedling growth under saline-alkali stress;

[0014] The application method is as follows: select rice seeds, immerse them in sterile water for 30 minutes, disinfect them with 75% ethanol for 5 minutes, soak them in 1% sodium hypochlorite for 1 minute, wash them with sterile water for 3-5 times, put them into a beaker, and add sterile water and 1.0×10 8 CFU / mL of bacterial suspension was used for seed soaking.

[0015] Preferably, the preparation method of the bacterial suspension is:

[0016] The strain was inoculated into LB liquid medium, cultured at 37°C and 150 r / min for 24 h, centrifuged at 5000 r / min for 15 min, the supernatant was discarded, and sterile water was added. This was repeated three times to make 1.0×10 8 CFU / mL bacterial suspension.

[0017] Preferably, the LB medium comprises the following components: 10.0 g / L tryptone, 5.0 g / L yeast extract, 10.0 g / L sodium chloride and 20.0 g / L agar, pH 7.0.

[0018] Compared with the above background technology, the present invention has the following beneficial effects:

[0019] The Acinetobacter Piteri NP55 isolated and screened from salinized soil in the present invention can synthesize indoleacetic acid (IAA) and has the function of dissolving inorganic phosphorus and potassium. The rice seeds under saline-alkali stress were treated with Acinetobacter Piteri NP55, and the germination results showed that the bacteria could alleviate the harm of saline-alkali stress to rice seeds under the conditions of pH 9 and 30mmol / L. The seedling cultivation results showed that the bacteria could increase the growth of the main root and lateral root of rice seedlings in a saline-alkali environment of pH 9 and 30mmol / L. Therefore, the present invention provides excellent growth-promoting bacterial species resources for the germination of rice seeds and the growth of their seedlings, and improves the ability of rice seedlings to resist saline-alkali stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The morphological diagram of the strain NP55 of the present invention (in the figure: A: colony morphology; B: Gram staining);

[0021] Figure 2 is the 16S rRNA phylogenetic tree of the strain NP55 of the present invention;

[0022] Figure 3 The photographs show the effect of the strain NP55 treatment on the germination phenotype of rice seeds under saline-alkali stress in the present invention;

[0023] Figure 4 The effect of the strain NP55 on rice seed germination in the present invention (in the figure: A: germination rate; B: embryo length; C: radicle length; D: number of lateral roots);

[0024] Figure 5 The effect of the strain NP55 on the growth phenotype of rice seedlings in the present invention;

[0025] Figure 6 The effect of the strain NP55 treatment on the growth of rice seedlings in the present invention (A: average stem length; B: average root length; C: average number of lateral roots; D: average seedling thickness; E: average root thickness);

[0026] Figure 7 The effect of the strain NP55 treatment on the growth phenotype of rice seedlings under saline-alkali stress in the present invention;

[0027] Figure 8The effect of the strain NP55 treatment on the growth of rice seedlings under saline-alkali stress (A: average stem length; B: average root length; C: average number of lateral roots; D: average seedling thickness; E: average root thickness). DETAILED DESCRIPTION

[0028] To make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0029] The present invention provides an Acinetobacter pittii, whose Latin name is Acinetobacter sp.Y956373, and the preservation unit is China Center for Type Culture Collection; the preservation address is Wuhan University, Wuhan, China, zip code 430072, the preservation number is CCTCC NO: M2024440, and the preservation date is March 11, 2024.

[0030] The present invention also provides a microbial agent, which contains the Acinetobacter piteiris.

[0031] The present invention also provides a microbial fertilizer, which contains the Acinetobacter piteiris.

[0032] The present invention also provides the use of the Acinetobacter pituitii in promoting rice seed germination and seedling growth.

[0033] Application of the Acinetobacter pitei in promoting rice seed germination and seedling growth under saline-alkali stress;

[0034] Select rice seeds, immerse them in sterile water for 30 minutes, disinfect them with 75% ethanol for 5 minutes, soak them in 1% sodium hypochlorite for 1 minute, wash them with sterile water for 3-5 times, put them into a beaker, add sterile water and 1.0×10 8 CFU / mL of bacterial suspension was used for seed soaking.

[0035] The preparation method of the bacterial suspension is:

[0036] The strain was inoculated into LB liquid medium, cultured at 37°C and 150 r / min for 24 h, centrifuged at 5000 r / min for 15 min, the supernatant was discarded, and sterile water was added. This was repeated three times to make 1.0×10 8 CFU / mL bacterial suspension.

[0037] The LB medium includes the following components: 10.0 g / L tryptone, 5.0 g / L yeast extract, 10.0 g / L sodium chloride and 20.0 g / L agar, pH 7.0.

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0040] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0041] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0042] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0043] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[0044] Example 1 Isolation, identification and safety test of strains

[0045] 1. Screening method of strains

[0046] In June 2023, 10 g of saline-alkali soil from the rice rhizosphere was collected from the saline-alkali base of rice cultivation in Sijiqing Town, Lindian. The soil sample was weighed and poured into a triangular flask containing 90 mL of sterile water. The soil was shaken at 30 °C for 30 min. 1 mL of soil suspension was drawn with a sterile pipette and placed in 9 mL of sterile water to obtain 10 -1 Repeat this process to make 10 dilutions. -2 -10-7 dilution, take 10 -5 , 10 -6 , 10 -7 100 μL of the gradient soil suspension was spread on the modified LB medium plate and cultured at 30°C for 2-4 days. Colonies with different colony morphology were selected for purification, and the purified strains were subcultured 3 times. After confirming that the strains had not separated, 40% glycerol and bacterial solution were used to store them in a ratio of 1:1 and stored at -80°C for later use.

[0047] The improved LB medium includes the following components: 10.24 g / L sodium chloride, 5.00 g / L yeast extract, 10.00 g / L tryptone, 0.43 g / L sodium carbonate, 3.02 g / L sodium bicarbonate, 5.12 g / L sodium sulfate and 20.00 g / L agar, pH 8.5.

[0048] 2. Screening method for strains with growth-promoting function

[0049] The isolated strains were inoculated on Axubei nitrogen-free medium, Montkina organic phosphorus medium, PKO inorganic phosphorus medium, potassium-dissolving medium, and CAS medium, respectively, and cultured at 30℃ for 3 days; those that can grow continuously for 3 generations on Axubei nitrogen-free medium are considered to have nitrogen fixation ability; those that can grow on Montkina organic phosphorus medium, PKO inorganic phosphorus medium or potassium-dissolving medium and produce transparent circles are considered to have phosphorus dissolving ability; those that can grow on CAS medium and produce orange-yellow halos are considered to have iron carrier production ability. The strains were inoculated in LB liquid medium containing 0.50g / L tryptophan, cultured at 30℃, 175r / min in the dark for 2 days, and the supernatant was centrifuged and mixed with an equal amount of Salkowski colorimetric solution on a white porcelain plate. After being placed in the dark for 30 minutes, the color development was observed. Each sample was repeated 3 times, and the blank control was a mixture of uninoculated culture medium and colorimetric agent. If a color reaction occurs, indoleacetic acid (IAA) is produced.

[0050] The culture medium used in the above experimental process is:

[0051] (1) Axubei nitrogen-free culture medium, comprising the following components: 0.50 g / L potassium dihydrogen phosphate, 0.12 g / L sodium chloride, 0.20 g / L magnesium sulfate heptahydrate, 1.00 g / L calcium carbonate, 10.00 g / L sucrose and 20.00 g / L agar, pH 7.0.

[0052] (2) Montgina organophosphate medium, comprising the following components: 2.50 g / L calcium phytate, 0.30 g / L sodium chloride, 0.30 g / L magnesium sulfate heptahydrate, 0.03 g / L ferrous sulfate heptahydrate, 10.00 g / L glucose, 0.50 g / L ammonium sulfate, 0.30 g / L potassium chloride, 0.30 g / L manganese sulfate and 20.00 g / L agar, pH 7.0.

[0053] (3) PKO inorganic phosphorus medium, comprising the following components: 10.00 g / L glucose, 0.06 g / L manganese sulfate, 0.60 g / L sodium chloride, 1.00 g / L ammonium sulfate, 0.60 g / L potassium chloride, 0.06 g / L iron sulfate, 10.00 g / L tricalcium phosphate, 0.30 g / L magnesium sulfate heptahydrate and 20.00 g / L agar, pH 7.0.

[0054] (4) Potassium-dissolving medium: sucrose 5.00 g / L, yeast extract powder 0.50 g / L, potassium feldspar powder 10.00 g / L, disodium hydrogen phosphate 2.00 g / L, sodium chloride 0.10 g / L, magnesium sulfate heptahydrate 0.50 g / L, manganese sulfate 1.00 g / L and agar 20.00 g / L, pH 7.0.

[0055] (5) Preparation method of CAS culture medium:

[0056] ① Take 1.2000g chrome azure, 0.0163g ferric chloride, and 1.4560g hexadecyltrimethylammonium bromide (HDTMA) and dissolve them in 1L sterile water to obtain solution a;

[0057] ② Take 0.2470g disodium hydrogen phosphate dodecahydrate, 0.1190g disodium hydrogen phosphate dihydrate, 0.0080g potassium dihydrogen phosphate, 0.0250g ammonium chloride, and 0.0130g sodium chloride and dissolve them in 10mL sterile water, pH 6.8, to obtain solution b, which is diluted 10 times when used;

[0058] ③ Take 1 mL of 20% sucrose solution, 3 mL of 10% acid hydrolyzed casein, 100 μL of 1 mmol / L calcium chloride solution, and 2 mL of 1 mmol / L magnesium sulfate solution, add them to 100 mL of sterile water, mix well, and heat to about 60°C. Slowly add 5 mL each of solution a and solution b to obtain solution c;

[0059] ④ Add 2 g agar powder to solution c to obtain culture medium d;

[0060] ⑤ Place culture medium d in an autoclave for sterilization (121°C, 20 min) to obtain a blue qualitative detection culture medium.

[0061] (6) LB medium: 10.00 g / L tryptone, 5.00 g / L yeast extract powder, 10.00 g / L sodium chloride and 20.00 g / L agar, pH 7.0.

[0062] 3. After the above two screenings, a bacterial strain was finally obtained, named NP55. The surface of the colony of strain NP55 on the modified LB medium plate was smooth and moist, with neat edges, and the whole was a slightly raised, viscous, opaque, light yellow round shape; the bacteria were rod-shaped, Gram-negative bacteria (see Figure 1 ). The results of physicochemical characteristics showed that strain NP55 was positive in VP test, catalase test and citrate utilization test.

[0063] The measured 16S rRNA sequences were compared with the sequences in the EzBioCloud database. The phylogenetic tree analysis constructed from the strain 16S rRNA sequence showed that strain NP55 and Acinetobacter pittii belonged to the same branch, with a sequence similarity of 98.25%. Combined with the morphological and physicochemical characteristics of the strain and the 16S rRNA sequence analysis, strain NP55 was preliminarily identified as Acinetobacter pittii (see Figure 2 ).

[0064] The 16S rRNA sequence is as follows:

[0065] GGGTTAAGGACAACATAGAGTGTGAGTCTGAGCGGAGAGAGGTA

[0066] GCTTGCTACTGATCTTAGCGGCGGACGGGTGAGTAATGCTTAGGAATC

[0067] TGCCTATTAGTGGGGGACAACATTTCGAAAGGAATGCTAATACCGCA

[0068] TACGTCCTACGGGAGAAAGCAGGGGATCTTCGGACCTTGCGCTAATA

[0069] GATGAGCCTAAGTCGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCA

[0070] AGGCGACGATCTGTAGCGGGTCTGAGAGGATGATCCGCCACACTGGG

[0071] ACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATAT

[0072] TGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGA

[0073] AGGCCTTATGGTTGTAAAGCACTTTAAGCGAGGAGGAGGCTACTTTA

[0074] GTTAATACCTAGAGATAGTGGACGTTACTCGCAGAATAAGCACCGGC

[0075] TAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAA

[0076] TCGGATTTACTGGGCGTAAAGCGCGCGTAGGCGGCTAATTAAGTCAA

[0077] ATGTGAAATCCCCGAGCTTAACTTGGGAATTGCATTCGATACTGGTTA

[0078] GCTAGAGTGTGGGAGAGGATGGTAGAATTCCAGGTGTAGCGGTGAAA

[0079] TGCGTAGAGATCTGGAGGAATACCGATGGCGAAGGCAGCCATCTGGC

[0080] CTAACACTGACGCTGAGGTGCGAAAGCATGGGGAGCAAACAGGATTA

[0081] GATACCCTGGTAGTCCATGCCGTAAACGATGTCTACTAGCCGTTGGGG

[0082] CCTTTGAGGCTTTAGTGGCGCAGCTAACGCGATAAGTAGACCGCCTGG

[0083] GGAGTACGGTCGCAAGACTAAAACTCAAATGAATTGACGGGGGCCCG

[0084] CACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACC

[0085] TTACCTGGCCTTGACATAGTAAGAACTTTCCAGAGATGGATTGGTGCC

[0086] TTCGGGAACTTACATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTC

[0087] GTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTTTCCTTAT

[0088] TTGCCAGCGAGTAATGTCGGGAACTTTAAGGATACTGCCAGTGACAA

[0089] ACTGGAGGAAGGCGGGGACGACGTCAAGTCATCATGGCCCTTACGGC

[0090] CAGGGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGCTACCT

[0091] AGCGATAGGATGCTAATCTCAAAAAGCCGATCGTAGTCCGGATTGGA

[0092] GTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCGGATC

[0093] AGAATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTC

[0094] ACACCATGGGAGTTTGTAGCACCAGAAGTAGCTACCCTATATGTAGTA

[0095] GACAAGGTATCCATAGAGTTCCTCAG.

[0096] 4. Safety test of strain NP55: strain NP55 was inoculated into LB liquid culture medium and cultured at 37°C and 150 r / min for 24 h. The bacterial solution concentration was adjusted to 2.0×10 8 CFU / mL-2.0×10 9 CFU / mL.

[0097] A total of 20 healthy adult Kunming mice were selected, half male and half female (10 mice in the control group and 10 mice in the test group, half male and half female), weighing 18-20g, and the test substance was injected intraperitoneally at a dose of 0.1mL / 10g. The control group was injected with normal saline, and the operation was the same. To ensure a healthy and comfortable environment, every 2 mice were placed in the same cage after injection. During the experiment, the daily activities, diet and defecation of mice in each group were normal, and their mental state was good.

[0098] Fourteen days after intraperitoneal injection, the mice showed no signs of poisoning and no deaths. No obvious lesions of the organs were found by macroscopic examination after autopsy. Further HE staining and section observation showed that strain NP55 was not pathogenic to the lung tissue, liver and kidney of mice compared with the control group. The test results showed that strain NP55 was not pathogenic and the test operation met the NYT1109-2017 standard.

[0099] 5. Quantitative analysis of the growth-promoting function of strain NP55

[0100] The strain NP55 was cultured in PKO inorganic phosphorus liquid medium containing tricalcium phosphate for 12 hours, and the maximum effective phosphorus increment was 77.30±0.05 mg / L. The strain NP55 can dissolve the insoluble phosphorus in the soil and make it available to plants, providing nutrients for plants. It may also promote the growth of plant root cells by producing volatile compounds, thereby increasing plant root growth parameters.

[0101] When strain NP55 was cultured in a potassium-dissolving liquid medium containing potassium feldspar powder for 24 hours, its potassium-dissolving capacity was 97.457±0.01 mg / L. Strain NP55 accelerated plant growth by decomposing potassium feldspar minerals, and it may also convert the difficult-to-use potassium in the soil into fast-acting potassium that can be absorbed and utilized by plants by secreting organic acids such as oxalic acid and acetic acid.

[0102] Strain NP55 can produce 15.92±0.07mg / L IAA when cultured in LB liquid medium containing tryptophan for 2 days. Strain NP55 can synthesize the plant growth hormone indoleacetic acid IAA using tryptophan as a precursor, which acts on the whole process of plant growth and development, can be directly used by plants, and may also participate in the regulation of other life activities in plants, thereby allowing plants to absorb more nutrients, promote plant growth and enhance plant sensitivity to pathogens.

[0103] The strain NP55, whose Latin name is Acinetobacter sp.Y956373, has been deposited in the China Type Culture Collection, with the deposit address being Wuhan University, Wuhan, China, postal code 430072, the deposit number being CCTCC NO: M2024440, the depositor being Heilongjiang Bayi Agricultural University, and the deposit date being March 11, 2024.

[0104] Example 2 Effect of strain NP55 on rice seed germination under saline-alkali stress

[0105] The strain was inoculated into LB liquid medium, cultured at 37°C and 150 r / min for 24 h, centrifuged at 5000 r / min for 15 min, the supernatant was discarded, and sterile water was added. This was repeated three times to make 1.0×10 8 CFU / mL bacterial suspension.

[0106] Select full rice seeds, immerse them in sterile water for 30 minutes, and remove empty and barren seeds. Then disinfect the surface with 75% ethanol for 5 minutes, soak them in 1% sodium hypochlorite for 1 minute, wash them with sterile water for 3-5 times, put them into a beaker, and add sterile water and 1.0×10 8 CFU / mL of bacterial suspension was used for seed soaking.

[0107] Sodium chloride, sodium carbonate, sodium bicarbonate and sodium sulfate were mixed in a ratio of 1:1:9:9 to prepare a saline-alkali solution with a final concentration of 30 mmol / L and a pH of 9. Three treatments were set up, namely CK: sterile water + sterile water soaking; T1: saline-alkali solution + sterile water soaking; T2: saline-alkali solution + 1.0×10 8 Seeds were soaked in CFU / mL bacterial suspension and cultured in a constant temperature incubator at 28°C for 7 days to determine the germination rate, and the length of the embryo, the length of the radicle and the number of lateral roots were measured (germination rate = number of germinated seeds / total number of test seeds × 100%).

[0108] Effects of strain NP55 treatment on rice seed germination phenotype under saline-alkali stress (see photos) Figure 3 ; The effect of strain NP55 treatment on rice seed germination under saline-alkali stress is shown in the bar graph Figure 4 (In the figure: A: germination rate; B: embryo length; C: radicle length; D: number of lateral roots).

[0109] from Figure 3 It can be seen that the rice seeds treated with strain NP55 grew radicles and the plumules further developed. Figure 4 The results show that the radicle length and lateral root number of rice seeds treated with strain NP55 under saline-alkali stress were significantly increased compared with T1, indicating that the treatment with strain NP55 promoted seed germination.

[0110] Example 3 Effect of strain NP55 on the growth of rice seedlings

[0111] The strain was inoculated into LB liquid culture medium, cultured at 37°C and 150 rpm for 24 h, then centrifuged at 5000 rpm for 15 min, the supernatant was discarded, and sterile water was added. This was repeated three times to prepare a bacterial suspension.

[0112] Select complete and full rice seeds and place them in a beaker, immerse them in sterile water for 30 minutes, and remove empty and barren seeds. Then disinfect the surface with 75% ethanol for 5 minutes, soak them in 1% sodium hypochlorite for 1 minute, wash them with sterile water for 3-5 times, put them in a beaker, and add sterile water and 1.0×10 8 CFU / mL bacterial suspension was used for seed soaking. The experiment set up two treatments, namely CK: sterile water + sterile water soaking; T3: sterile water + 1.0×10 8 Soak the seeds in CFU / mL bacterial suspension.

[0113] The soaked rice seeds were evenly spread in a 105mm×105mm×58mm square box, with 100 seeds in each box, and repeated 3 times. After 24 days, the root length, stem length, and number of lateral roots of the rice seedlings were measured. 300mL of sterile water was poured into each box. To prevent water loss, the same bottom plate was placed under the square box during the treatment to ensure that the exuded solution would be absorbed from the bottom of the square box again. During the planting period, each box was irrigated 6 times, 200mL each time, with an interval of 4d.

[0114] The effect of strain NP55 treatment on the growth phenotype of rice seedlings is shown in Figure 5 ;

[0115] The effect of the strain NP55 treatment on the growth of rice seedlings is shown in Figure 6 (In the figure, A: average stem length; B: average root length; C: average number of lateral roots; D: average seedling thickness; E: average root thickness).

[0116] from Figure 5 and Figure 6 It can be seen that compared with the control group CK, the average root length, average number of lateral roots, average seedling thickness and average root thickness of rice seedlings treated with T3 were significantly increased by 27.01%, 58.81%, 29.41% and 20.83%, respectively.

[0117] Example 4 Effect of strain NP55 on the growth of rice seedlings under saline-alkali stress

[0118] The strain was inoculated into LB liquid culture medium, cultured at 37°C and 150 rpm for 24 h, then centrifuged at 5000 rpm for 15 min, the supernatant was discarded, and sterile water was added. This was repeated three times to prepare a bacterial suspension.

[0119] Select complete and full rice seeds and place them in a beaker, immerse them in sterile water for 30 minutes, and remove empty and barren seeds. Then disinfect the surface with 75% ethanol for 5 minutes, soak them in 1% sodium hypochlorite for 1 minute, wash them with sterile water for 3-5 times, put them in a beaker, and add sterile water and 1.0×10 8 CFU / mL of bacterial suspension was used for seed soaking.

[0120] The soaked rice seeds were evenly spread in a 105mm×105mm×58mm square box, with 100 seeds in each box, and repeated 3 times. After 24 days, the root length, stem length, and number of lateral roots of the rice seedlings were measured. 300mL of pH 9, 30mmol / L saline-alkali solution was poured into each box, and the same amount of sterile water was poured into the CK treatment. To prevent the loss of salt and water, the same bottom plate was placed under the square box during the treatment to ensure that the exuded solution would be absorbed from the bottom of the square box again. During the planting period, each box was irrigated 6 times, 200mL each time, with an interval of 4d.

[0121] The effect of strain NP55 treatment on the growth phenotype of rice seedlings under saline-alkali stress is shown in Figure 7 The effect of the strain NP55 on the growth of rice seedlings under saline-alkali stress is shown in Figure 8 (In the figure, A: average stem length; B: average root length; C: average number of lateral roots; D: average seedling thickness; E: average root thickness).

[0122] from Figure 7 and Figure 8 It can be seen that the average root length, average number of lateral roots, average seedling thickness and average root thickness of rice seedlings were significantly increased after treatment with strain NP55, which were significantly different from those of T1, with increases of 41.79%, 26.37%, 33.33% and 8.33% respectively; compared with T1, the average stem length of T2 treatment increased by 5.78%, but the results of variance analysis were not significant.

[0123] Example 5 Preparation of microbial inoculants

[0124] Preparation method of microbial inoculant containing Acinetobacter pitei: strain NP55 is cultured in an activation medium for 16 hours, then inoculated into a 500mL Erlenmeyer flask containing 250mL of LB medium and shaken at 37°C and 150r / min for 20 hours to prepare a primary seed for fermentation, and then inoculated into a fermenter (50L) at an inoculum amount of 1%, and after culturing for 20 hours, the strain morphology is observed under a microscope, and after confirming that there is no contamination, it is moved to a 500L fermenter for expansion culture, and the light absorption value (OD) of the fermentation liquid is measured after 20 hours. 600 ) is greater than 1.800 to terminate the fermentation. The culture conditions are: charging factor 0.7, fermentation ventilation volume 0.5 to 1.0 V / V·min, tank pressure: 0.5 Bar, rotation speed: 150 rpm, dissolved oxygen control 70% to 90%, and adding an appropriate amount of wheat bran to the culture medium to improve the stability of the microbial inoculant and make it easier to store. The effective viable count concentration in the obtained microbial liquid inoculant is 3.0×10 8 -3.5×10 8 CFU / mL, pH 6.5-7.0, its technical indicators meet the requirements of GB20287-2006.

[0125] Example 6 Preparation of microbial fertilizer

[0126] Preparation method of microbial fertilizer containing Acinetobacter pitei: strain NP55 is cultured in an activation medium for 16 hours, then inoculated into a 500mL Erlenmeyer flask containing 250mL of LB medium and shaken for 20 hours at 37°C and 150r / min to prepare a first-class seed for fermentation, and then 1% of the inoculum is inoculated into a fermentation tank (50L), and the strain morphology is observed under a microscope after culturing for 20 hours. After confirming that there is no contamination, it is moved to a 500L fermentation tank for expansion culture, and the light absorption value (OD) of the fermentation liquid is measured after 20 hours. 600 ) is greater than 1.800, and the fermentation is terminated. The culture conditions are: charging factor 0.7, fermentation ventilation volume 0.5 to 1.0 V / V·min, tank pressure: 0.5 Bar, rotation speed: 150 rpm, dissolved oxygen control 70% to 90%. The effective viable count concentration in the obtained microbial culture liquid is 3.0×10 8 -3.5×10 8 CFU / mL, pH 6.5-7.0, its technical indicators meet the requirements of GB20287-2006.

[0127] The obtained bacterial liquid is evenly sprayed on particles with organic matter (rotted chicken manure, humic acid and clay) as the main components at an addition rate of 8%-10%, and then dried in the shade until the moisture content is below 30% for storage and use.

[0128] The effective viable bacterial count of the finished granules was measured by plate counting method and was 0.25×10 8 -0.30×10 8 CFU / mL, the organic matter content is between 45%-55%, and its technical indicators reach NY884-2012.

[0129] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

[0130] Those skilled in the art will appreciate that the embodiments described herein are only descriptions of preferred embodiments of the present invention, and are not intended to limit the scope of the present invention; in order to help readers understand the implementation of the present invention, it should be understood that the protection scope of the present invention is not limited to such specific statements and embodiments. Those skilled in the art can make various other specific variations and combinations that do not deviate from the essence of the present invention based on the technical revelations disclosed in the present invention, and these variations and combinations are still within the protection scope of the present invention.

Claims

1. An Acinetobacter pittii, characterized in that: The deposit number of the Acinetobacter pitei is CCTCC NO: M2024440, and the deposit unit is China Center for Type Culture Collection.

2. A microbial agent, characterized in that: The microbial agent comprises the Acinetobacter pituitarius described in claim 1.

3. A microbial fertilizer, characterized in that: The microbial fertilizer comprises the Acinetobacter pitei described in claim 1.

4. Use of the Acinetobacter pituitii as claimed in claim 1 in promoting plant seed germination and seedling growth.

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

6. The use according to claim 5, characterized in that: Application of the Acinetobacter pitei in promoting rice seed germination and seedling growth under saline-alkali stress; The application method is as follows: select rice seeds, immerse them in sterile water for 30 minutes, disinfect them with 75% ethanol for 5 minutes, soak them in 1% sodium hypochlorite for 1 minute, wash them with sterile water for 3-5 times, put them into a beaker, and add sterile water and 1.0×10 8 CFU / mL of bacterial suspension was used for seed soaking.

7. The use according to claim 6, characterized in that: The preparation method of the bacterial suspension is: The strain was inoculated into LB liquid medium and cultured at 37°C and 150 r / min for 24 h. The culture was centrifuged at 5000 r / min for 15 min, the supernatant was discarded, and sterile water was added. This was repeated three times to make 1.0×10 8 CFU / mL bacterial suspension.

8. The use according to claim 7, characterized in that: The LB medium includes the following components: 10.0 g / L tryptone, 5.0 g / L yeast extract, 10.0 g / L sodium chloride and 20.0 g / L agar, pH 7.0.

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