Bacillus marinus for reducing salt and alkali and application thereof

Abstract

The invention discloses a bacillus marinus strain which is named as bacillus marinus sp., the strain is preserved in the China General Microbiological Culture Collection Center on March 28, 2023, the preservation number is CGMCC NO.26955, and the preservation number is CGMCC NO.26955. The bacillus marinus strain is named as bacillus marinus sp. The 16S rRNA nucleotide sequence of the strain is as shown in SEQ ID NO: 1. The strain disclosed by the invention has saline-alkaline tolerance and saline-alkaline reduction capabilities, also has multiple functions of fixing nitrogen, producing IAA (Indoleacetic Acid), promoting plant growth and the like, and can promote the growth of plants under saline-alkaline stress conditions by generating volatile acidic metabolites. The invention provides application of the bacillus marinus strain. The strain can be used for reducing salt and alkali in saline-alkali soil, increasing the content of available nitrogen elements in soil and promoting plant growth. The strain provided by the invention has wide application prospects in the aspects of improvement of saline-alkali stress resistance and production capacity of plants, improvement of saline-alkali soil, ecological environment protection and the like, and can be prepared into microbial fertilizers and microbial soil conditioners with plant growth promotion and saline-alkali soil improvement effects.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial application, and specifically relates to a salt-alkali reducing Oceanobacillus sp. strain and its application in the fields of plant growth promotion, research and development of microbial fertilizers and microbial soil conditioners and the like. Background Art

[0002] Due to some natural and human factors, the phenomenon of soluble salts accumulating on the soil surface in some areas is called soil salinization. Soil salinization is a factor that seriously harms land quality, ecological environment and agricultural production. Excessive salt and alkali in saline-alkali soil exceeds the range required for normal plant growth, which will have a negative impact on plant growth and sustainable land use. On the one hand, soil salinization seriously reduces agricultural productivity and soil fertility, thereby affecting crop yields; on the other hand, the expansion of saline-alkali land may also lead to soil desertification and further deteriorate the ecological environment. Therefore, how to improve saline-alkali soil, enhance the salt-alkali resistance of crops, and promote the growth of plants in saline-alkali land has become a major issue in improving agricultural productivity, ensuring sustainable and efficient agricultural development, and building ecological agriculture.

[0003] Xinjiang is the region with the most widespread distribution of saline-alkali land in my country, with an area of ​​14.6 million hectares, accounting for 40.6% of the country's saline-alkali land area and 37.7% of Xinjiang's total cultivated land area. Xinjiang is located in the inland of my country, with many deserts and Gobi. The formation of saline-alkali land is mainly due to factors such as dry climate, low rainfall, high evaporation, and salt accumulation. Soil salinization not only inhibits the normal growth of crops and leads to reduced yields, but also affects the physical and chemical properties of the soil, making it difficult for microorganisms in the soil to survive. In Xinjiang, traditional agricultural practices often focus on increasing crop yields while ignoring the sustainable use of soil. Large-scale reclamation, unreasonable irrigation and fertilization have accelerated the process of soil salinization. Due to the high salt-alkali content of Xinjiang soil, the soil is prone to dryness and compaction, making it difficult for plants to take root and unable to absorb enough nutrients and water. Therefore, how to make plants grow normally in saline-alkali soil and improve yield is a problem that we urgently need to solve.

[0004] Plant Growth-Promoting Rhizobacteria (PGPR) refers to a class of microorganisms that exist in the rhizosphere of plants and can produce plant growth-promoting substances or promote plant growth by inhibiting harmful microorganisms. PGPR can promote plant growth through direct and indirect effects. Direct effects refer to the synthesis and secretion of nutrients required by plants or the change of the valence state of certain elements in the root soil to facilitate plant absorption. Indirect effects mainly use the function of PGPR to prevent plant diseases to reduce the adverse effects of soil-borne pathogenic microorganisms on plant growth and development, and induce plants to enhance systemic resistance and improve the plant's defense capabilities against various diseases.

[0005] Inoculation of salt-alkali tolerant growth-promoting microorganisms is an effective biological method for improving saline-alkali land. This method is simple to operate, highly sustainable and low-cost. At present, some microorganisms have been proven to have the function of promoting plant growth and improving soil physical and chemical properties in different saline-alkali soil environments. However, in the process of using salt-alkali tolerant growth-promoting microorganisms to promote plant growth and improve saline-alkali land, there are still the following shortcomings: few strains; existing strains have poor adaptability to different environments, especially special ecological environments such as saline-alkali land, and poor universality of growth-promoting effects on different plants. Therefore, it is particularly important to screen and develop highly targeted functional microbial agents.

[0006] In existing research, Oceanobacillus has been used to produce anti-tumor active peptides (Sun Mi, 2014), and its fermentation products have been used to prepare anti-aging cosmetics (Chen Peiyan, 2023), and used in pickling kimchi to increase the variety and content of volatile substances, reduce the content of irritating substances, and improve flavor (Su Xiaoyun, 2024), etc. However, there are few related reports on salt-alkali tolerance, salt-alkali reduction, conditioning and improving soil while promoting plant growth. Summary of the invention

[0007] At present, some microorganisms have been proven to have the function of promoting plant growth in saline-alkali soil, but there are also technical problems such as the small number of salt-alkali-reducing and growth-promoting strains and the difficulty of cultivating saline-alkali land. In order to solve the above problems, the technical solution adopted by the present invention is to provide a salt-alkali-reducing marine Bacillus strain and its application. The salt-alkali-reducing marine Bacillus strain is named Oceanobacillus sp., which is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit date of March 28, 2023 and a deposit number of CGMCC NO.26955. The marine Bacillus

[0008] The 16S rRNA nucleotide sequence of Oceanobacillus sp. is shown in SEQ ID NO: 1. The present invention also provides the use of the above-mentioned salt-alkali reducing marine bacillus strain, wherein the marine bacillus Oceanobacillus sp. has multiple functions such as reducing salt and alkali in saline-alkali land, increasing soil nitrogen content, producing IAA, promoting plant growth, and can promote the growth of plants under salt-alkali stress conditions by producing volatile acidic metabolites.

[0009] Preferably, the plants include corn and Arabidopsis thaliana.

[0010] Preferably, when the plant is corn, the Oceanobacillus sp. is used to promote the aboveground fresh weight, underground fresh weight, root length and plant height of corn; when the plant is Arabidopsis thaliana, the Oceanobacillus sp. is used to promote the taproot length, lateral root number, fresh weight and leaf number of Arabidopsis thaliana.

[0011] Preferably, the Oceanobacillus sp. is used for the preparation of microbial fertilizers, microbial agents and microbial soil conditioners having plant growth promotion and soil conditioning and improvement effects.

[0012] Preferably, when the Oceanobacillus sp. is used as a microbial fertilizer, a microbial agent and a microbial soil conditioner, it is used for at least one of salt-alkali tolerance, salt-alkali reduction, IAA production, nitrogen fixation and production of volatile acidic metabolites.

[0013] Compared with the prior art, the present invention has the following advantages:

[0014] The present invention provides a salt-alkali reducing marine bacillus sp. strain, which has a deposit number of CGMCCNO.26955. The strain is isolated from the rhizosphere soil of Xinjiang salt-tolerant plant Ephedra, has strong salt-alkali tolerance and salt-alkali reducing ability, especially the alkali reducing ability can reach 21.10%, can effectively colonize in the plant rhizosphere and improve the soil environment, reduce the stress of salt and alkali on plants, and promote plant growth; the strain has nitrogen fixation ability, can improve soil fertility, promote plant growth and development; the strain can also produce IAA and volatile acidic metabolites, can reduce the damage of salt-alkali stress to plants and promote plant growth. In summary, the strain can be used to improve plant resistance to salt-alkali stress, soil salt reduction, soil alkali reduction, increase soil fertility, increase soil available nitrogen elements and promote plant growth, and can be prepared into microbial fertilizers, microbial agents and microbial soil conditioners with plant growth promotion, soil conditioning and improvement effects.

[0015] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a phylogenetic tree of 16S rRNA of the Oceanobacillus sp. strain of the present invention;

[0017] Figure 2 is a Gram staining result diagram of the Oceanobacillus sp. strain of the present invention;

[0018] Figure 3 is a growth curve diagram of the Oceanobacillus sp. strain of the present invention;

[0019] Figure 4 This is a graph showing the results of a nitrogen fixation experiment of the Oceanobacillus sp. strain of the present invention;

[0020] Figure 5 This is a diagram showing the growth-promoting effect of the Oceanobacillus sp. strain of the present invention on Arabidopsis thaliana under isolated culture conditions;

[0021] Figure 6 It is a bar graph showing the growth-promoting effect of the Oceanobacillus sp. strain of the present invention on Arabidopsis thaliana under the isolation and culture conditions;

[0022] (Significant difference analysis: * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001.)

[0023] Figure 7 This is a graph showing the experimental results of the production of volatile acidic metabolites by the Oceanobacillus sp. strain of the present invention;

[0024] Figure 8 This is a diagram showing the growth-promoting effect of the Oceanobacillus sp. strain of the present invention on corn;

[0025] Fig. 9 It is a bar graph showing the growth-promoting effect of the Oceanobacillus sp. strain of the present invention on corn;

[0026] (Significant difference analysis: * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001.)

[0027] Fig.10This is a diagram showing the growth-promoting effect of the Oceanobacillus sp. strain of the present invention on Arabidopsis thaliana;

[0028] Fig.11 It is a bar graph showing the growth-promoting effect of the Oceanobacillus sp. strain of the present invention on Arabidopsis thaliana.

[0029] (Significant difference analysis: * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001.) DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0031] The present invention provides a salt-alkali tolerant growth-promoting marine bacillus Oceanobacillus sp. Bachu7 strain, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit date of March 28, 2023 and a deposit number of CGMCC NO.26955.

[0032] Example 1

[0033] Isolation, Purification and Identification of Oceanobacillus sp. Bachu7

[0034] 1. Isolation and purification of Oceanobacillus sp. Bachu7 strain

[0035] The rhizosphere soil of the salt-tolerant plant Ephedra sinica in Bachu County, Kashgar Prefecture, Xinjiang Uygur Autonomous Region was collected, and the surface soil, large lumps of soil, and rotten leaves were removed and placed in sterile bags and transported back to the laboratory. More than 5 g of the test soil sample was added to a centrifuge tube containing 45 mL of sterile deionized water, and vortexed for 2 minutes to fully disperse the microorganisms in the test soil in the sterile deionized water to obtain a soil mixture, which was allowed to stand at room temperature for 2-5 minutes to form a soil suspension; 1 mL of the above soil suspension was taken and added to 9 mL of sterile deionized water to obtain 10 -1 g / mL concentration suspension, and so on for gradient dilution to obtain 10 -2 g / mL, 10 -3 g / mL, 10 -4 g / mL, 10 -5 g / mL, 10 -6g / mL concentration gradient soil suspension, vortex oscillate to mix, and then let it stand; take 100 μL of each concentration gradient soil suspension, spread it on LB (NaCl: 7.5%, pH = 9) solid culture medium with a sterilized coating rod, set 3 replicates for one concentration gradient, and invert the culture in a 30°C constant temperature incubator. After colonies are formed, select bacterial single colonies with different colony morphology, color and size, pick single colonies and inoculate them into corresponding LB liquid culture medium for culture, and then perform streak culture; repeat the above streak operation at least 3 times to obtain a pure cultured single colony of Bachu7 strain; take 500 μL of the purified strain and mix it with 500 μL of 50% glycerol, and store it in a -80°C refrigerator for later use.

[0036] 2. Identification of Oceanobacillus sp. Bachu7 strain

[0037] (I) Systematic identification of Oceanobacillus sp. Bachu7 strain

[0038] The universal upstream primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and the universal downstream primer 1492R (5'-GGCTACCTTGTTACGACTT-3') of the bacterial 16S rRNA gene were used, and the genome of the marine Bacillus strain was used as a template for PCR amplification. The PCR reaction system was a 25 μL system: 1 μL of primer 27F, 1 μL of primer 1492R, 12.5 μL of 2×phanta MaxMix, 10.4 μL of ddH2O, and 0.1 μL of DNA template. The PCR reaction conditions were: 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 2 min, a total of 35 cycles, and 72°C pre-extension for 5 min. The amplified PCR product was tested by 1% agarose gel electrophoresis. After the electrophoresis band was detected correctly, the PCR product was sent to Sangon Biotech Co., Ltd. for sequencing, and a sequence of about 1500 bp was obtained. The sequencing results were compared by BLAST in NC BI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and the strain phylogenetic tree was constructed using MEGA X64 software. Figure 1 As shown, the strain is similar to the model strain Oceanobacillus manasiensis YD3-56 T The sequence similarity was the highest, at 98.91%. Therefore, the strain was a member of the genus Oceanobacillus and was named Oceanobacillus sp.Bachu7.

[0039] (II) Physiological and biochemical characteristics of Oceanobacillus sp. Bachu7 strain

[0040] The Bachu7 strain was inoculated on a solid LB (NaCl: 7.5%, pH = 9) medium and the colony morphology was observed. The results are shown in Table 1. The colony characteristics of the Bachu7 strain on the LB solid medium are: the colony shape is round, white with transparent edges, moist surface, smooth and neat edges with protrusions. The strain was stained with Gram stain and showed a blue-purple color, which is a Gram-positive bacterium. Figure 2 shown.

[0041] The physiological and biochemical characteristics of the Bachu7 strain were identified using the biochemical tube of Qingdao Haibo Biotechnology Co., Ltd. The identification results are shown in Table 2: The strain can grow with a variety of substances such as glucose, xylose, and arabinose as the only carbon source, providing reference data for the subsequent application of the strain. At the same time, the catalase reagent of Qingdao Haibo Biotechnology Co., Ltd. was used to determine the ability of the Bachu7 strain to produce catalase. Pick a single colony on a slide rinsed with distilled water, absorb the catalase reagent, and drop 1-2 drops on the colony on the slide. If bubbles appear, it is positive, and if no bubbles appear, it is negative. The results show that a single colony of the Bachu7 strain was picked, and bubbles were generated after the catalase reagent was added, proving that the Bachu7 strain has the ability to produce catalase.

[0042] Table 1 Colony morphology characteristics

[0043]

[0044] h

[0045] Table 2 Experiment on the sole carbon source of Bachu7 strain

[0046]

[0047] Note: “+”: positive; “-”: negative

[0048] Example 2

[0049] Study on the Growth Characteristics of Oceanobacillus sp. Bachu7

[0050] 1. Growth curve determination of Oceanobacillus sp. Bachu7 strain

[0051] A single colony of the Bachu7 strain was selected and cultured in LB (NaCl: 7.5%, pH = 9) liquid medium, placed in a constant temperature shaking incubator at 30°C, 180 r / min. The absorbance of the bacterial solution at 600 nm was measured using an ultraviolet spectrophotometer.600 When the value is 0.6-0.8, the bacterial solution is inoculated into LB liquid culture medium at a 2% inoculum volume and placed in a constant temperature shaking incubator at 30°C and 180r / min for culture. The absorbance of the bacterial solution at 600nm is measured every 4h and the value is recorded. The measurement is repeated 3 times at each time point, and the average value is taken to draw a growth curve. The results are shown in Figure 3 As shown, the logarithmic growth period is 16-36 hours after inoculation of Bachu7, and it enters the stationary phase after 36 hours, which provides theoretical data for the actual production of Bachu7 strain.

[0052] 2. Determination of salt tolerance of Oceanobacillus sp. Bachu7 strain

[0053] A single colony of strain Bachu7 was selected and inoculated into liquid LB (NaCl: 7.5%, pH = 9) medium, and cultured in a constant temperature shaking incubator at 30°C, 180 r / min, and the absorbance of the microorganism at 600 nm was measured with a UV spectrophotometer until the OD of the bacterial solution reached 0. 600 The value is 0.6-0.8. Then the bacterial solution was inoculated in a solid culture medium with pH = 9, salt concentrations of 0g / L, 20g / L, 40g / L, 60g / L, 80g / L, 100g / L, 120g / L, 140g / L, 160g / L, 180g / L, 200g / L and no pH adjustment, salt concentration of 0g / L, and cultured in a 30°C constant temperature incubator; the growth state of the strain was observed every 24 hours, and the diameter of the single colony was counted. The measurement was repeated 3 times at each time point, and the average value was taken. The results are shown in Table 3: Bachu7 strain can grow normally under the conditions of salt concentration of 0g / L to 200g / L, which proves that the strain has excellent salt tolerance and provides a basis for the colonization and application of the strain in saline-alkali land.

[0054] Table 3 Salt tolerance results of Bachu7 strain

[0055]

[0056] Note: “0(-)” means no NaCl, pH is neutral; “0” means no NaCl, pH=9; “+”: positive; “-”: negative.

[0057] 3. Determination of alkali resistance of Oceanobacillus sp. Bachu7 strain

[0058] A single colony of strain Bachu7 was selected and inoculated into liquid LB (NaCl: 7.5%, pH = 9) medium, and cultured in a constant temperature shaking incubator at 30°C, 180 r / min, and the absorbance of the microorganism at 600 nm was measured with a UV spectrophotometer until the OD of the bacterial solution reached 0. 600The bacterial liquid was inoculated into LB liquid culture medium with pH=7, pH=8, pH=9, pH=10, pH=11, pH=12 and pH=13 at an inoculation rate of 2%, and cultured in a constant temperature shaking incubator at 30°C and 180r / min; the absorbance of the bacterial liquid at 600nm was measured by an ultraviolet spectrophotometer every 24 hours, and the liquid culture medium without bacteria was used as the control group. The measurement was repeated 3 times at each time point, and the average value was taken. The results are shown in Table 4. It shows that the strain Bachu7 has excellent alkali resistance and can grow normally under the condition of pH value of 7-13, which provides a basis for the colonization and application of the strain in saline-alkali land.

[0059] Table 4 Alkali resistance results of Bachu7 strain

[0060]

[0061] Note: “+”: positive; “-”: negative.

[0062] 4. Determination of the salt-reducing ability of Oceanobacillus sp. Bachu7 strain

[0063] In this experiment, the Mohr precipitation titration method was used to determine the salt reduction ability of the Bachu7 strain. K2CrO4 (potassium chromate) solution was used as an indicator and AgNO3 (silver nitrate) standard solution was used to titrate the Cl in the fermentation broth. － , with Cl － The concentration reflects the salt concentration. In the liquid culture medium of pH=8, pH=9, and pH=10, OD 600 The value is 0.6-0.8 bacterial solution, placed in a constant temperature shaking incubator at 30°C and 180r / min for cultivation, 0.5mL of bacterial solution is taken every 12h, 2.5μL of 10% K2CrO4 solution is added as an indicator, and titrated with 0.1mol / LAgNO3 solution. When a brick-red precipitate appears and does not fade, it is the titration end point. The results are shown in Table 5, which proves that the Bachu7 strain has the ability to reduce salt, indicating that the Bachu7 strain can effectively improve the soil properties of saline-alkali land and help plants adapt to the saline-alkali land environment.

[0064] Record the amount of AgNO3 solution used and calculate Cl according to the following formula: － The concentration of C2.

[0065] Calculation formula: C2 = (C1 × V1) / V2

[0066] Where: C1 is the concentration of AgNO3 solution, mol / L;

[0067] V1 is the volume of AgNO3 solution consumed during titration, mL;

[0068] V2 is the volume of the bacterial solution during titration, mL;

[0069] According to Cl － The salt reduction rate is calculated according to the following formula:

[0070] Calculation formula: η 盐 =(C 2最高 -C 2最低 ) / C 2最高 ×100%

[0071] Where: η 盐 The salt-reducing ability of the strain;

[0072] C 2最高 is Cl in the culture medium － Concentration before degradation, mol / L;

[0073] C 2最低 is Cl in the culture medium － Concentration after degradation, mol / L.

[0074] Table 5 Salt reduction results of Bachu7 strain

[0075]

[0076] V. Determination of the Alkali-Reducing Ability of Oceanobacillus sp. Bachu7

[0077] OD 600 The Bachu7 bacterial solution with a value of 0.6-0.8 was inoculated into LB liquid culture medium with pH = 8, pH = 9, and pH = 10, respectively, and placed in a constant temperature shaking incubator at 30°C and 180r / min for culture. 4mL of bacterial solution was taken every 12h, centrifuged at 4000rpm for 5min, the supernatant was taken and its pH value was measured with a pH meter, and the alkalinity reduction rate was calculated according to the following formula. The alkalinity reduction results of the Bachu7 strain are shown in Table 6, which proves that the strain has excellent alkalinity reduction function, can effectively improve soil properties, and alleviate the stress caused by alkali to plants, so it can be effectively used in saline-alkali land improvement and plant growth promotion.

[0078] The alkali reduction rate was calculated according to the following formula:

[0079] η 碱 =(pH 前 -pH 后 ) / pH 前 ×100%

[0080] Where: η 碱 is the alkali reduction rate of the strain;

[0081] pH 前 is the pH value of the culture medium before inoculation;

[0082] pH 后 is the pH value of the culture medium after inoculation.

[0083] Table 6 Alkali reduction results of Bachu7 strain

[0084]

[0085] Example 3

[0086] Study on the Growth-Promoting Function of Oceanobacillus sp. Bachu7

[0087] 1. Nitrogen fixation function of Oceanobacillus sp. Bachu7 strain

[0088] Configuration of Ashubei nitrogen-free culture medium (g / L): 0.2g potassium dihydrogen phosphate, 10g glucose, 0.2g magnesium sulfate heptahydrate, 0.1g calcium sulfate, 75g sodium chloride, 5g calcium carbonate, 16g agar, add 1000mL deionized water, adjust pH=7.0-7.2, and sterilize with high temperature and high pressure steam at 115°C for 20min.

[0089] The Bachu7 bacterial solution was inoculated on the nitrogen-free solid medium of Axubei, and each medium was inoculated three times, with 15μL of bacterial solution spotted at each location. The culture was inverted and cultured in a 30℃ constant temperature incubator for 7-9 days to observe the growth of the Bachu7 strain and determine whether the strain has the ability to fix nitrogen. Figure 4 As shown, the Bachu7 strain can grow normally on the Axubei nitrogen-free culture medium, indicating that the Bachu7 strain has the ability to fix nitrogen. The Bachu7 strain can increase the available nitrogen content in the soil through the nitrogen fixation function, promote plant growth and development, reduce dependence on soil nitrogen fertilizers, and help reduce environmental problems such as soil pollution and eutrophication of water bodies caused by excessive use of chemical fertilizers.

[0090] II. IAA production capacity of Oceanobacillus sp. Bachu7 strain

[0091] The Bachu7 strain was inoculated into LB liquid (NaCl: 7.5%, pH = 9) medium and cultured in a constant temperature shaking incubator at 30°C and 180 r / min. Every 2 days, 2 ml of the bacterial solution was taken from the bacterial solution into a centrifuge tube and centrifuged at 14000 rcf for 10 min. Then, the supernatant was taken and added with an equal volume of Salkowski colorimetric solution. The solution was placed in a dark place for reaction for 30 min. A spectrophotometer was used to measure the A 530 The absorbance is measured until 13 days, and the data is recorded. Select A 530The maximum absorbance was calculated by the standard curve equation y=74.595x-2.5256 for the maximum IAA concentration in the bacterial solution. The results are shown in Table 7. The IAA concentration produced by the Bachu7 strain was 30±2 μg / mL, which could effectively promote the root development of plants, thereby improving the plant's ability to absorb nutrients and water and promoting better plant growth.

[0092] Table 7 Quantitative determination of IAA secretion by Bachu7 strain

[0093]

[0094] 3. Detection of volatile acidic metabolites produced by Oceanobacillus sp. Bachu7 strain

[0095] (I) Treatment of Arabidopsis seeds: Place Arabidopsis seeds in a conical flask, add 75% ethanol to cover the seeds, soak for 7 minutes, shake, let stand for 15 seconds, and absorb the upper layer of 75% ethanol; then add 10% NaClO to the seeds and soak for 7 minutes, shake, let stand for 15 seconds, and absorb the upper layer of NaClO; finally, rinse the seeds with sterile deionized water for 5 times, then add sterile deionized water to cover the seeds, and place in a 4°C refrigerator for vernalization for 3 days.

[0096] (II) Growth-promoting effect of Oceanobacillus sp. Bachu7 on Arabidopsis thaliana in isolated culture

[0097] In this study, 1 / 2MS medium with pH=5.8 was used as the control group, and 1 / 2MS medium with different saline-alkali stresses (pH=8, pH=8+2mMNaHCO3, pH=8+100mM NaCl, and pH=8+2mM NaHCO3+100mM NaCl) was used as the experimental group. First, 1 / 2MS solid medium with five conditions (a: pH=5.8, b: pH=8, c: pH=8+2mM NaHCO3, d: pH=8+100mM NaCl, e: pH=8+2mMNaHCO3+100mM NaCl) was prepared and poured into one side of a two-grid culture dish, and LB solid medium was added to the other side. Arabidopsis seeds that had been vernalized were spotted on the above 1 / 2MS solid medium, and the Bachu7 strain was inoculated with LB solid medium on the other side. Three biological replicates were set for each treatment. Transfer to a light incubator for vertical culture, culture conditions: temperature 22 ° C, light intensity 12000 Lux, photoperiod 16h / 8h, relative humidity 50%. During the growth process, the fresh weight, root length and other physiological indicators of Arabidopsis seedlings after 10 days of growth were counted. Arabidopsis root length was measured using ImageJ software. The results are shown in Figure 5 , Figure 6As shown: Under saline-alkali stress, the isolation and cultivation of Bachu7 strain and Arabidopsis thaliana significantly increased the number of lateral roots and leaves of Arabidopsis thaliana, indicating that the Bachu7 strain produced volatile metabolites, which can alleviate saline-alkali stress and promote the growth of Arabidopsis thaliana.

[0098] (III) Verification of the production of volatile acidic metabolites by Oceanobacillus sp. Bachu7 strain

[0099] It is known that the indication range of neutral red pH indicator is 6.8-8.0, alkaline shows orange, acidic shows red. Neutral red medium with pH = 8.0 was added to the isolation medium, the medium turned orange, one side was inoculated with Bachu7 strain, and the other side was not inoculated with strain. After culturing at 30℃ for 3 days, it was found that the color of the medium on the inoculated side changed from orange to red, and the medium on the side not inoculated with strain also turned red. The results are as follows Figure 7 As shown, it indicates that the culture medium changes from alkaline to acidic, and the Bachu7 strain produces volatile acidic metabolites, which enhances the plant's tolerance to salt-alkali stress and is more conducive to plant growth.

[0100] Example 4

[0101] Effects of Oceanobacillus sp. Bachu7 on the Growth of Maize

[0102] 1. Preparation of bacterial suspension

[0103] The Bachu7 strain was inoculated into LB (NaCl: 7.5%, pH = 9) liquid medium and cultured in a constant temperature shaking incubator at 30°C and 180 r / min for 2 days. The culture solution was centrifuged at 5000 g for 10 min, the supernatant was removed, and the cells were resuspended in sterile deionized water to a concentration of 10 8 CFU / mL was used as bacterial suspension.

[0104] 2. Corn Seed Treatment

[0105] Select complete, plump and consistent corn seeds, wash off the red coat of the seeds with sterile deionized water, disinfect with 95% alcohol for 0.5-1 min, and then soak in 0.1% NaClO solution for 15 min; finally, wash the seeds with sterile deionized water for 5 times, and finally put them into a conical flask, add sterile deionized water to immerse the corn seeds, and germinate them overnight at room temperature in the dark.

[0106] 3. Effects of Oceanobacillus sp. Bachu7 on the Growth of Maize Seedlings

[0107] The nutrient soil and vermiculite were mixed in a ratio of 1:1, and sterilized with high-temperature and high-pressure steam at 121°C for 20 min; NaHCO3 and neutral salt were mixed in a ratio of 1:1 to prepare 30g / kg and 45g / kg salt solutions, the sterilized soil and the prepared salt solution were mixed and divided into small black pots, and 3 Xinyu No. 62 corn seeds were sown in each small black pot, with a sowing depth of about 1 cm, and 10 replicates were set for each condition; when the corn grew two leaves, 5mL of the prepared bacterial suspension was applied to each experimental group, and 5mL of sterile deionized water was applied to the control group; watering was carried out in a timely manner during the growth of the corn, and 5mL of the prepared bacterial suspension was applied to the experimental group every other week, and 5mL of sterile deionized water was applied to the control group. After culturing for 14 days, the corn roots were cleaned with water, and the aboveground fresh weight and underground fresh weight, root length and plant height were measured at the same time, and data statistics were performed. The results are shown in Tables 8, 9, Figure 8 , Fig. 9 shown.

[0108] The results showed that under the same salt concentration conditions, the inoculation of Bachu7 strain had a good growth-promoting effect; under the soil conditions treated with 0g / kg salt solution, the average aboveground fresh weight increased by 27.31%, and the average plant height increased by 25.17%; under the soil conditions treated with 30g / kg salt solution, the average aboveground fresh weight increased by 15.35%, the average underground fresh weight increased by 9.80%, and the average plant height increased by 17.39%; under the soil conditions treated with 45g / kg salt solution, the average aboveground fresh weight increased by 34.52%, the average plant height increased by 45.68%, and the average root length increased by 8.43%. In summary, the strain of the present invention has a significant growth-promoting effect on corn under different saline-alkali stress conditions.

[0109] Table 8 Effect of Bachu7 strain on corn growth

[0110]

[0111] Table 9 Growth rate of physiological indicators of corn after treatment with strain Bachu7 suspension

[0112]

[0113] Example 5

[0114] Effects of Oceanobacillus sp. Bachu7 on the Growth of Arabidopsis thaliana

[0115] In this study, 1 / 2MS medium with pH=5.8 was used as the control group, and 1 / 2MS medium with different saline-alkali stresses (pH=8, pH=8+2mMNaHCO3, pH=8+100mM NaCl, and pH=8+2mM NaHCO3+100mM NaCl) was used as the experimental group. First, 1 / 2MS solid culture medium with five conditions (a: pH=5.8, b: pH=8, c: pH=8+2mM NaHCO3, d: pH=8+100mM NaCl, e: pH=8+2mM NaHCO3+100mM NaCl) was prepared, and the plate was turned upside down to cool. After the culture medium was completely cooled, a line was drawn at 1.5cm and 6.0cm from the bottom of the culture dish, and the 1 / 2MS solid culture medium at 1.5cm from the bottom of the culture dish was cut off, and 2mL LB solid culture medium was added. The Arabidopsis seeds that had been vernalized at 4°C were sown on 1 / 2MS medium at 6.0 cm from the bottom of the culture dish. Three biological replicates were set for each condition. At the same time, the Bachu7 strain was inoculated on the LB solid medium at 1.5 cm from the bottom of the culture dish and transferred to the light incubator for vertical culture. The culture conditions were: temperature 22°C, light intensity 12000Lux, photoperiod 16h / 8h, relative humidity 50%. During the growth of Arabidopsis in the light incubator, the physiological indicators such as the root length of Arabidopsis seedlings grown for 10 days were counted. The main root length, number of lateral roots, fresh weight and number of leaves of Arabidopsis were measured respectively. The results are shown in Table 10. Fig.10 , Fig.11As shown in the figure. It can be seen from the figure that under 5 different conditions, the growth of Arabidopsis thaliana in the culture medium without inoculation of strains was significantly inhibited compared with the pH = 5.8 control group, among which NaCl stress had a more obvious inhibitory effect on the growth of Arabidopsis thaliana than NaHCO3 stress. When the strain was inoculated in the culture medium of the salt-alkali stress group, the growth of Arabidopsis thaliana showed different promotion effects compared with the control group. Under pH=5.8 conditions, compared with the control group, the number of lateral roots increased by 429.28% after the application of Bachu7 strain; under pH=8 conditions, compared with the control group, the fresh weight increased by 129.63% and the number of lateral roots increased by 204.90% after the application of Bachu7 strain; when 2mM NaHCO3 stress was applied in the culture medium at pH=8, compared with the control group, the fresh weight increased by 531.63% and the main root length increased by 215.83% after the application of Bachu7 strain; when 100mM NaCl stress was applied in the culture medium at pH=8, compared with the control group, the fresh weight increased by 399.97% after the application of Bachu7 strain; when 100mM NaCl and 2mM NaHCO3 co-stress was applied in the culture medium at pH=8, compared with the control group, the fresh weight increased by 1099.39% after the application of Bachu7 strain. In summary, the Bachu7 strain significantly promoted the growth of Arabidopsis thaliana under different saline-alkali stress conditions, with obvious effects on increasing fresh weight, taproot growth, lateral root growth and biomass.

[0116] Table 10 Growth rates of physiological indicators of Arabidopsis thaliana after treatment with strain Bachu7 suspension under different conditions

[0117]

[0118] “ / ” means that the physiological index was not measured or observed under this condition and could not be calculated.

[0119] Example 6

[0120] The Oceanobacillus sp. Bachu7 strain provided by the present invention has multiple functions such as salt-alkali resistance, salt-alkali reduction, increasing soil nitrogen, producing IAA, producing volatile acidic metabolites and promoting plant growth, and can effectively alleviate the stress effect of salt and alkali on plants, promote plant growth and development, and has important application value in improving plant salt-alkali stress resistance and production capacity, soil conditioning and improvement, ecological environment protection, etc.

[0121] The oceanic bacillus sp. Bachu7 of the present invention can also be used for the preparation of microbial fertilizers, microbial agents and microbial soil conditioners with plant growth promotion and soil conditioning and improvement functions.

[0122] The marine bacillus sp. Bachu7 is prepared into a fertilizer. The specific ingredients may include rice straw, stalks, animal feces, chitin, yeast extract, phosphate, potash fertilizer, potassium humate, vermiculite and the marine bacillus sp. Bachu7.

[0123] The above examples are intended to illustrate the embodiments disclosed by the present invention, which are only preferred embodiments of the present invention and are not to be construed as limitations of the present invention. In addition, the various modifications listed herein and the variations of the methods in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of the present invention.

Claims

1. A marine Bacillus strain having the ability to reduce salt and alkali, characterized in that: The strain is named Oceanobacillus sp., and is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee on March 28, 2023, with a deposit number of CGMCC NO. 26955. The 16SrRNA nucleotide sequence of the Oceanobacillus sp. is shown in SEQ ID NO:

1.

2. An application of the salt-reducing and alkali-reducing marine Bacillus strain according to claim 1, characterized in that: The oceanic bacillus sp. is used for reducing salt and alkali in saline-alkali land, increasing the nitrogen content in soil, promoting plant growth and improving the salt-alkali resistance of plants.

3. The use according to claim 2, characterized in that: The plants include corn and Arabidopsis thaliana.

4. The use according to claim 3, characterized in that: When the plant is corn, the Oceanobacillus sp. can promote the aboveground fresh weight, underground fresh weight, root length and plant height of corn; when the plant is Arabidopsis thaliana, the Oceanobacillus sp. can increase the taproot length, lateral root number, fresh weight and leaf number of Arabidopsis thaliana.

5. A use of the salt-reducing and alkali-reducing marine Bacillus strain according to claim 1, characterized in that: The oceanic bacillus sp. is used for preparing microbial fertilizers, microbial agents and microbial soil conditioners with plant growth promotion and soil conditioning effects.

6. A use according to claim 5, characterized in that When the Oceanobacillus sp. is used as a microbial fertilizer, a microbial agent and a microbial soil conditioner, it is used for at least one of salt-alkali resistance, salt-alkali reduction, nitrogen fixation, IAA production and volatile acidic metabolite production.

Images