Saline-alkaline tolerant Bachu halomonas strain and application thereof

Abstract

The invention provides a saline-alkaline tolerant Bachu halomonas strain, which is characterized in that the saline-alkaline tolerant Bachu halomonas strain is named as halomonas sp. And is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC NO.28831, and the preservation date is November 01, 2023; the nucleotide sequence of the 16S rRNA is as shown in SEQ ID NO: 1. The invention further provides application of the strain, and the strain is used for improving saline-alkaline resistance of plants, reducing soil salinity and alkalinity and promoting plant growth and is used for preparing fertilizer with plant growth promoting and saline-alkali soil improving effects. The strain disclosed by the invention has the functions of resisting salt and alkali, reducing salt and alkali, fixing nitrogen, decomposing organic phosphorus, dissolving inorganic phosphorus, producing IAA, producing oxidase, producing catalase, producing volatile acidic substances and promoting plant growth. The strain obtained by the invention has important application values in the aspects of improving saline-alkali stress resistance of crops, improving physicochemical properties of saline-alkali soil, increasing crop yield and the like, can be prepared into a fertilizer with plant growth promoting and saline-alkali soil improving effects for use, is a great innovation in saline-alkali soil improvement and crop planting, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of microbial application, in particular to a Halomonas Halomonas sp. strain and its application in improving saline-alkali land and promoting the growth of plants against salt and alkali. Background Art

[0002] At present, saline-alkali soils are widely distributed in more than 30 countries around the world, with a total area of ​​about 956 million hectares. China ranks third in the world in terms of salinization area, and 4 / 5 of saline-alkali land in my country has not been developed and utilized, and is in a state of barrenness and continuous degradation. Soil salinization has become a constraint on the sustainable development of irrigation agriculture in the world, seriously affecting food security.

[0003] The management of saline-alkali land can be considered from two aspects: one is to reduce or improve the content of saline-alkali in the soil, and the other is to allow plants to grow normally in soil with high salt content. The methods to reduce or improve the content of saline-alkali in the soil mainly include physical improvement, chemical improvement, and biological improvement. In recent years, the method of using microorganisms to improve saline-alkali land has been continuously valued, and microbial plant inoculants can also be used to help plants grow in high saline-alkali environments, especially when planting crops on high saline-alkali land, applying salt-alkali-tolerant plant growth-promoting rhizobacteria (PGPR) can not only improve crop productivity but also improve soil fertility. However, at present, the salt-alkali-tolerant growth-promoting strains developed at home and abroad still have problems such as few strains and poor adaptability of existing strains to special environments, especially saline-alkali soil environments. Therefore, screening and obtaining a salt monosaccharide that can resist salt-alkali stress and promote plant growth is of great significance to the sustainable agricultural development of saline-alkali land and the improvement and utilization of saline-alkali soil.

[0004] Halomonas belongs to the family Halomonasaceae. The genus was first proposed in 1980 when Vreeland et al. identified Halomonas elongate. Halomonas is Gram-negative, rod-shaped, and its colonies are mostly white, yellow, orange, brown, etc. It is aerobic, but some strains grow anaerobically in the presence of nitrates and can grow under conditions of absolute salinity (NaCl) of 0 to 32%. In recent years, with the research at home and abroad, more detailed characteristics of Halomonas have been proposed: the main fatty acids of this genus are C 16:1 ,C 17:0 cyclo,C 16:0 ,C 18:1 and C 19:0cyclo; the main respiratory quinone is coenzyme Q-9; the GC content of chromosomal DNA varies greatly, generally between 50% and 75%. The reported bacteria of the genus Halomonas all grow in high-salt environments, such as salt fields and salt lakes, saline wetlands, saline soils, seawater, marine organisms, deep-sea hydrothermal vents, and saline-alkali land.

[0005] Although Halomonas is widely studied and applied in industry, there are still few studies on it in agriculture. Halomonas sp. Bachu26, a known Halomonas strain used for plant growth promotion and saline-alkali land improvement, has good effects in producing ACC deaminase and reducing salt and alkali under different conditions, but it is insufficient in producing IAA, decomposing organic phosphorus, dissolving inorganic phosphorus, and promoting the growth of Arabidopsis. It is an important and valuable task to explore more Halomonas, provide sufficient strain resources for fertilizer utilization, and strengthen relevant research on the genus in plant growth promotion, saline-alkali land improvement, and the rational use of microbial resources. This work is expected to achieve major breakthroughs in farmland salinization improvement projects and the transformation and utilization of saline-alkali land in arid areas.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] In view of the current technical difficulties such as the difficulty in cultivating saline-alkali land and the difficulty in treating saline-alkali soil, the present invention provides a Halomonas strain and its application. The strain has the functions of salt-alkali tolerance, salt-alkali reduction, nitrogen fixation, organic phosphorus dissolution, inorganic phosphorus dissolution, IAA production, catalase production, oxidase production, volatile acidic substances production and plant growth promotion.

[0008] To solve the above technical problems, the technical solution adopted by the present invention is: a salt-alkali tolerant Bachu Halomonas strain, the salt-alkali tolerant Bachu Halomonas strain is named Halomonas sp., and is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC NO.28831 and a deposit date of November 1, 2023; the 16S rRNA nucleotide sequence of the Halomonas sp. is shown in SEQ ID NO:1.

[0009] The present invention also provides the use of the above-mentioned salt-alkali tolerant Halomonas sp. strain, wherein the Halomonas sp. is used to improve the salt-alkali tolerance of plants, reduce soil salinity, increase the available nitrogen content in the soil and promote plant growth.

[0010] Preferably, the plants include corn and Arabidopsis thaliana.

[0011] Preferably, when the plant is corn, the Halomonas sp. strain has the characteristics of increasing the aboveground fresh weight, underground fresh weight, and plant height of corn; when the plant is Arabidopsis thaliana, the Halomonas sp. strain has the characteristics of increasing the number of lateral roots, root length, number of leaves, and fresh weight of Arabidopsis thaliana;

[0012] The present invention also provides the use of the above-mentioned salt-alkali tolerant Halomonas sp., and the Halomonas sp. is used for preparing fertilizers having the functions of promoting plant growth and improving saline-alkali land.

[0013] Preferably, when the Halomonas sp. is used as a fertilizer, it is used for at least one of reducing salt and alkali, decomposing organic phosphorus, dissolving inorganic phosphorus, producing IAA, producing catalase, producing oxidase, and producing volatile acidic substances.

[0014] Compared with the prior art, the present invention has the following advantages:

[0015] The Halomonas sp. strain of the present invention has the functions of salt-alkali tolerance, salt-alkali reduction, nitrogen fixation, organic phosphorus dissolution, inorganic phosphorus dissolution, IAA production, catalase production, oxidase production, volatile acidic substances production and plant growth promotion. It can improve the salt-alkali tolerance of plants, improve soil salinity, increase the available nitrogen content in the soil and promote plant growth, and can also be prepared into a fertilizer with plant growth promotion and saline-alkali land improvement functions.

[0016] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is a phylogenetic tree of 16S rRNA of the Halomonas sp. strain of the present invention;

[0018] Figure 2 is a Gram staining result diagram of the Halomonas sp. strain of the present invention;

[0019] Figure 3 This is a transmission electron microscope result of the Halomonas sp. strain of the present invention;

[0020] Figure 4 is a genome circle map of the Halomonas sp. strain of the present invention;

[0021] Figure 5 is the growth curve of the Halomonas sp. strain of the present invention;

[0022] Figure 6This is a diagram showing the nitrogen fixation results of the Halomonas sp. strain of the present invention;

[0023] Figure 7 This is a diagram showing the results of the decomposition of organic phosphorus by the Halomonas sp. strain of the present invention;

[0024] Figure 8 This is a graph showing the results of the Halomonas sp. strain of the present invention dissolving inorganic phosphorus;

[0025] Fig. 9 It is an IAA standard curve diagram for the identification of the IAA production capacity of the Halomonas sp. strain of the present invention;

[0026] Fig.10 This is a result diagram of the production of volatile acidic gas by the Halomonas sp. strain of the present invention;

[0027] Fig.11 This is a diagram showing the growth-promoting effect of the Halomonas sp. strain of the present invention on corn;

[0028] Fig.12 It is a bar graph of the effect of the Halomonas sp. strain of the present invention on promoting corn growth under different conditions;

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

[0030] Fig.13 This is a diagram showing the growth-promoting effect of the Halomonas sp. strain of the present invention on Arabidopsis thaliana;

[0031] Fig.14 is a bar graph showing the growth-promoting effect of the Halomonas sp. strain of the present invention on Arabidopsis thaliana;

[0032] (Significant difference analysis: * indicates P<0.05; ** indicates P<0.01; *** indicates P<0.001; **** indicates P<0.0001)

[0033] Fig.15 This is a diagram showing the growth-promoting effect of Halomonas sp. on Arabidopsis thaliana under isolated culture conditions of the present invention;

[0034] Fig.16 It is a bar graph showing the growth-promoting effect of Halomonas sp. on Arabidopsis thaliana under the isolation culture conditions of the present invention.

[0035] (Significant difference analysis: * indicates P<0.05; ** indicates P<0.01; *** indicates P<0.001; **** indicates P<0.0001) DETAILED DESCRIPTION

[0036] Example 1

[0037] Isolation, Purification and Identification of Halomonas sp. Bachu37

[0038] 1. Isolation and purification of Halomonas sp. Bachu37 strain

[0039] The soil was collected from the rhizosphere of the salt-alkali tolerant plant Phragmites australis in Bachu County, Xinjiang. The surface soil, large soil pieces and rotten leaves around the roots were removed. The rhizosphere soil of the reeds was shaken off and placed in a sterile sample bag and transported back to the laboratory at low temperature. 5g of the test soil sample was added to a centrifuge tube containing 45ml of sterile deionized water. The soil was vortexed to fully disperse the microorganisms in the soil in the sterile deionized water. The soil was left to stand at room temperature for 2-5 minutes to form a soil stock solution. 1ml of the above suspension was taken and added to 9ml of sterile deionized water to obtain 10 -1 concentration suspension, and so on for gradient dilution to obtain 10 -2 , 10 -3 , 10 -4 Concentrated suspension, vortex oscillation to mix, operate in a sterile clean bench. Take 100 μL of each concentration suspension, apply it on LB (7.5% NaCl, pH = 9) solid culture medium with a sterilized coating rod, repeat a gradient three times, and invert the marked culture dish into a 30°C constant temperature incubator for culture. After 7 days of culture, select bacterial colonies with different colony morphology, color and size, inoculate them into the corresponding liquid culture medium for culture, and then perform streaking culture at least 3 times until a monoclonal strain is obtained; take 500 μL of bacterial solution and 500 μL of 50% glycerol from the purified strain, mix them, and store them in a -80°C refrigerator.

[0040] 2. Identification of Halomonas sp. Bachu37

[0041] (I) Identification of 16S rRNA nucleotide sequence of Halomonas sp. Bachu37 strain

[0042] The bacterial universal upstream primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and the bacterial universal downstream primer 1492R (5'-GGCTACCTTGTTACGACTT-3') were used to amplify the 16S rRNA gene sequence of the strain, and the basic conditions of the PCR reaction were set as follows: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30s, 60℃ annealing for 30s, 72℃ extension for 120s, a total of 35 cycles; 72℃ pre-extension for 5min. After the PCR reaction, the PCR product amplified by the strain was tested by 1% agarose gel electrophoresis, and the image was detected by gel imaging, and then the PCR product stock solution was sent to Sangon Biotech Co., Ltd. for sequencing. Sequence information of about 1500 bp in length was obtained, and the sequence information was compared by BLAST at NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). MEGA7 software was used to perform cluster analysis and phylogenetic tree construction by the NJ method. The sampling was repeated 1000 times, and the bootstrap method was used to test the stability of the calculated phylogenetic tree topology. The phylogenetic tree nodes only displayed values ​​with Bootstrap values ​​greater than 50%. The constructed phylogenetic tree is shown in Figure 1. Figure 1 As shown, the Halomonas maris sp. strain is similar to the model strain of Halomonas maris QX-1 T and Halomonastitanicae BH1 T The closest relationship is that the 16S rRNA sequence similarity is 97.56% and 97.49%, respectively. Therefore, it is speculated that the strain may be a new species of the genus Halomonas.

[0043] (II) Morphological identification of Halomonas sp. Bachu37 strain

[0044] The strain Bachu37 was inoculated on Luria-Bertani (LB) solid medium (pH = 9, NaCl 75g / L) to observe the colony morphology. The colony characteristics of the strain Bachu37 on LB (pH = 9, NaCl 75g / L) solid medium are: the colony morphology is irregular, orange-yellow and opaque, the surface is moist and has protrusions, and the edges are irregular. The results are shown in Table 1. The strain Bachu37 was stained with Gram stain and showed red. The results are shown in Table 1. Figure 2 As shown, it was shown that strain Bachu37 belongs to Gram-negative bacteria.

[0045] The strain Bachu37 was observed by transmission electron microscopy using the phosphotungstic acid negative staining method. The results of the electron microscopy showed that the strain Bachu37 was a short rod-shaped bacillus with blunt ends and a cell width of 0.3-0.5μm and a length of 2.5-3.0μm. Figure 3 .

[0046] Table 1 Colony morphology of strain Bachu37

[0047]

[0048] (III) Identification of physiological and biochemical characteristics of Halomonas sp. Bachu37 strain

[0049] Micro-fermentation tubes were purchased from Haibo Biotechnology Co., Ltd., Qingdao High-tech Industrial Park, and strain Bachu37 was inoculated into them or the strain was inoculated into the corresponding functional culture medium to obtain the physiological and biochemical characteristics of strain Bachu37. The identification results are shown in Table 2. Strain Bachu37 can grow with glucose, sucrose, xylose, arabinose, citrate and other substances as the sole carbon source, which provides a basis for the production and application of the strain.

[0050] Table 2 Some physiological and biochemical indicators of strain Bachu37

[0051]

[0052] Note: “+” means positive; “-” means negative

[0053] (IV) Determination of cell components of Halomonas sp. Bachu37 strain

[0054] Polar lipids determination: The polar lipid composition of strain Bachu37 was analyzed by FMIC-QO01-012-2015 microbiological detection and polar lipid thin layer chromatography (TLC) detection methods. The results showed that the polar lipid components of strain Bachu37 included diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), unidentified phospholipids (UPL1-3), unidentified aminophospholipids (UAPL1-2), and unidentified lipids (UL).

[0055] Fatty acid identification: The fatty acid composition of strain Bachu37 was analyzed using the FMIC-QO01-011-2015 microbiological test and fatty acid gas chromatography (GC) detection method. The identification results showed that the main components of the fatty acid composition of strain Bachu37 were Summed Featuer 8 (C 18:1 ω7c or C 18:1ω6c)55.67% and Summed Featuer3(C 16:1 ω7c or C 16:1 ω6c)20.16%.

[0056] Quinone component determination The quinone components of strain Bachu37 were analyzed using FMIC-QO01-008-2015 microbiological detection and high performance liquid chromatography (HPLC) detection methods. The test results showed that the quinone components of strain Bachu37 were all ubiquinone Q-9 (100%).

[0057] Determination of cell wall chemical components: The cell wall components of strain Bachu37 were determined using the FMIC-QO01-008-2015 microbiological detection method and the thin layer chromatography (TLC) detection method for the chemical components of the cell wall of microbial strains. The results showed that strain Bachu37 had no characteristic cell wall diaminopimelic acid (DAP) component, and its main components were ribose, glucose and galactose.

[0058] Based on the above component determination results, strain Bachu37 is closely related to the two model strains of Halomonas (QX-1 T and BH1 T ) have different degrees of differences, and the specific results are shown in Table 3.

[0059] Table 3 Comparison of cell components of strain Bachu37 and model strains QX-1 and BH1

[0060]

[0061] (V) Whole genome sequence identification of Halomonas sp. Bachu37 strain

[0062] The genome of strain Bachu37 was extracted and sequenced. The sequencing results showed that the genome was 3467491 bp long and the G+C content was 58.63%. The genome circle map was constructed as shown in Figure 4 As shown. Figure 4 Among the genome circles, the outermost circle is the genome size marker; the second and third circles are the CDS on the positive and negative strands; the fourth circle is rRNA and tRNA; the fifth circle is the G+C content; and the innermost circle is the G+C tilt value.

[0063] Evaluation of strain Bachu37 and model strain QX-1 using whole genome analysis Tand BH1. The full genome characteristics of strain Bachu37 are as follows: the genome length of strain Bachu37 (CP137552) is 3467491bp, and the G+C content is 59.27%. A total of 3249 genes are predicted. Strain Bachu37 contains 2 plasmids, plasmid 1 (CP137553) is 18112bp in length, and plasmid 2 (CP137554) is 4364bp in length. Among the protein-coding genes, 2561 genes were identified as functional predicted proteins (84.35%), and 475 genes were annotated as hypothetical proteins (15.65%). Among the non-coding RNA genes, there are 6 16S rRNA genes, 6 23S rRNA genes, 6 5S rRNA genes, and 60 tRNA genes. Comparison of strain Bachu37 with BH1 T 、QX-1 T The relationship between strains Bachu37 and BH1 is shown in Table 4. OrthoANI was below the threshold of 95%-96% for differentiation. T The dDDH value was 15.20%-20.40%, which was similar to that of strain QX-1. T The dDDH values ​​of strain Bachu37 and its closest relative, strain QX-1 T and BH1 T The ANI values ​​of the two species were 73.45% and 73.75%, respectively, which were significantly lower than the ANI standard cut-off level (95-96%) recommended by Richter and Rossello-Mora 2009 for interspecies identification.

[0064] Table 4 Comparison of the genome of strain Bachu37 with model strains QX-1 and BH1

[0065]

[0066] In summary, strain Bachu37 showed morphological characteristics similar to those of the type strain of Halomonas, and its biochemical characteristics were different from those of the type strain of Halomonas. It also had low levels of ANI and dDDH values, which were sufficient to prove that strain Bachu37 was a new species of the genus Halomonas.

[0067] Based on the results of morphological, physiological and biochemical characteristics difference analysis and molecular biology, it is shown that the strain of the present invention is a new species of the genus Halomonas, which is named Halomonas Halomonas sp., and has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee with a deposit number of CGMCCNO.28831. The 16SrRNA nucleotide sequence of Halomonas Halomonas sp. is shown in SEQ ID NO: 1. The discovery of this strain further enriches the resources of beneficial bacteria.

[0068] Example 2

[0069] 1. Study on the growth characteristics of Halomonas p.Bachu37 strain

[0070] (I) Growth curve of Halomonas sp. Bachu37 strain

[0071] Pick a single strain and inoculate it into a sterilized test tube, place it in a constant temperature shaking incubator at 30°C and 180r / min, and measure it with a UV spectrophotometer until the OD of the bacterial solution reaches 600 The bacterial solution was then inoculated into the corresponding culture medium at a 2% inoculum and placed in a constant temperature shaking incubator at 30°C and 180 r / min for culture. After that, the absorbance of the bacterial solution at 600 nm was measured every 4 hours using an ultraviolet spectrophotometer, and the liquid culture medium was used as the control group. The measurement was repeated 3 times at each time point, and the average value was taken. A growth curve was drawn based on the measured data. The results are shown in the figure. Figure 5 .

[0072] 2. Salt-alkali tolerance of Halomonas sp.Bachu37

[0073] (I) Salt tolerance of Halomonas sp. Bachu37

[0074] A single strain was selected and inoculated into a sterile test tube containing liquid LB medium (pH = 9, NaCl 7.5%), and placed in a constant temperature shaking incubator at 30°C, 180 r / min, and the absorbance of the bacterial solution at 600 nm was measured with an ultraviolet spectrophotometer until the OD of the bacterial solution was 0. 600The value is 0.6-0.8; dip the bacterial solution at 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 solid culture medium with salt concentration of 0g / L but no pH adjustment, and place it in a 30℃ constant temperature incubator for inverted culture; observe the diameter of the single strain every 24h, repeat the measurement 3 times and take the average value. The results of the salt tolerance experiment of strain Bachu37 are shown in Table 5. The results show that strain Bachu37 can grow under the conditions of salt concentration of 0g / L-200g / L, indicating that it has good salt tolerance.

[0075] Table 5 Determination of salt tolerance of strain Bachu37

[0076]

[0077] Note: +: strain grows, -: strain does not grow.

[0078] (II) Alkali resistance of Halomonas sp. Bachu37

[0079] A single strain was selected and inoculated into a sterile test tube containing liquid LB medium (pH = 9, NaCl 7.5%), and placed in a constant temperature shaking incubator at 30°C, 180 r / min, and the absorbance of the bacterial solution at 600 nm was measured with an ultraviolet spectrophotometer until the OD of the bacterial solution was 0. 600 The value was 0.6-0.8; the 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 inoculum of 2%, and placed in a constant temperature shaking shaker at 30°C and 180r / min for cultivation; the LB liquid culture medium was used as the control group, and the OD of the strain was measured and counted every 24h using a UV spectrophotometer 600 The results of the alkali resistance test of strain Bachu37 are shown in Table 6. The results show that strain Bachu37 can grow at a pH value between 7 and 11, indicating that it has good alkali resistance.

[0080] Table 6 Alkali resistance determination of strain Bachu37

[0081]

[0082] Note: +: strain grows, -: strain does not grow.

[0083] The above salt-alkali resistance experimental results show that strain Bachu37 can grow normally at salt concentrations between 0g / L-200g / L and pH values ​​between 7-11, indicating that it has good salt-alkali resistance, laying a good foundation for its colonization in saline-alkali land.

[0084] 3. Temperature range in which Halomonas sp. Bachu37 can grow

[0085] The strain Bachu37 was inoculated into solid LB medium (pH = 9, NaCl 7.5%), placed in an incubator at 5°C-50°C (with 5°C as an increment of one condition), and then inverted in a 30°C constant temperature incubator for 7 days to observe whether there was colony growth. The results are shown in Table 7. The results showed that the strain Bachu37 could grow in the range of 5-45°C, proving its excellent environmental adaptability and laying a good foundation for the strain Bachu37 to colonize in an environment with a high temperature difference between day and night.

[0086] Table 7 Determination of the growth temperature of strain Bachu37

[0087]

[0088] Note: +: strain grows, -: strain does not grow.

[0089] Example 3

[0090] Identification of the Growth-Promoting Function of Halomonas sp. Bachu37

[0091] 1. Determination of the ability of Halomonas p.Bachu37 to reduce salt and alkali

[0092] (I) Determination of the salt-reducing ability of the Halomonas p.Bachu37 strain

[0093] This test was carried out by Mohr precipitation titration: K2CrO4 (potassium chromate) was used as an indicator and AgNO3 (silver nitrate) standard solution was used to directly titrate chloride ions. 600 The bacterial solution with a pH value of 0.6-0.8 is placed in a constant temperature shaking incubator at 30°C and 180r / min. Every 12 hours, 0.5mL of bacterial solution is taken and 2.5μL of 10% K2CrO4 solution is added as an indicator. The NaCl concentration is determined by 0.1mol / LAgNO3 titration. The titration endpoint is when a brick-red precipitate appears and does not fade.

[0094] Cl － The concentration calculation formula is: C2 = (C1 × V1) / V2

[0095] Note: C1 = AgNO3 solution concentration (mol / L);

[0096] V1 = volume of AgNO3 solution consumed during titration (mL);

[0097] V2 = volume of bacterial solution during titration (mL).

[0098] Obtain Cl - After the content is calculated, the salt reduction rate ηsalt is calculated according to the formula:

[0099] η 盐 =(C 最高 -C 最低 ) / C 最高 ×100%

[0100] Note: C 最高 is the concentration of culture medium before fermentation (mol / L);

[0101] C 最低 is the concentration of culture medium after fermentation (mol / L).

[0102] The results of the salt reduction test are shown in Table 8. The results show that the strain Bachu37 has a salt reduction effect under the three conditions of initial pH values ​​of 8, 9, and 10, and the salt reduction effect is best when the pH value is 8, with a salt reduction rate of 6.25%.

[0103] Table 8 Average results of salt reduction rate of strain Bachu37

[0104]

[0105] (II) Determination of the alkalinity-reducing function of Halomonas sp. Bachu37

[0106] Inoculate OD 600 The bacterial solution with a pH value of 0.6-0.8 was placed in a constant temperature shaking incubator at 30°C and 180r / min. 0.5mL of the bacterial solution was taken every 12h, and the bacterial solution was centrifuged at 4000rpm for 5min. The supernatant was taken to measure the pH value of the liquid culture medium before and during the culture. The alkalinity reduction rate η was calculated according to the following formula 碱 :

[0107] η 碱 =(pH 前 -pH 后 ) / pH 前 ×100%

[0108] Note: pH 前 is the pH value of the culture medium before fermentation;

[0109] pH 后 is the pH value of the medium after fermentation;

[0110] The results are shown in Table 9. The results showed that strain Bachu37 had an alkali reduction effect under the initial pH values ​​of 8, 9, and 10, and the alkali reduction ability of the strain increased with the increase of the initial pH. When the initial pH value was 10, the alkali reduction rate of strain Bachu37 could reach 20.3%.

[0111] Table 9 Average results of alkali reduction rate of strain Bachu37

[0112]

[0113]

[0114] According to the above method, the salt and alkali reduction rate of strain Bachu37 was finally obtained. When the initial pH value was 8, the alkali reduction rate was 9±2%, and the salt reduction rate was 6±2%; when the initial pH value was 9, the alkali reduction rate was 14±2%, and the salt reduction rate was 3±2%; when the initial pH value was 10, the alkali reduction rate was 20±2%, and the salt reduction rate was 4±2%. The farmland salinization improvement project and the transformation and utilization of arid saline-alkali land are an important direction for industrial structure adjustment. The above experimental results show that strain Bachu37 has considerable salt and alkali reduction capabilities, which lays the foundation for improving the soil properties of saline-alkali land and helping plants adapt to the saline-alkali environment.

[0115] 2. Determination of Catalase

[0116] A catalase kit was purchased from Qingdao Haibo Biotechnology Co., Ltd. The strain Bachu37 was inoculated into LB liquid medium and cultured in a constant temperature shaker at 30°C. 600 When the pH is 0.6-0.8, the reagent is dripped into the bacterial solution. If bubbles are generated, it is positive, otherwise it is negative. The results show that strain Bachu37 has the ability to produce catalase. Excessive hydrogen peroxide can oxidize plant cell membrane lipids and cause cell damage, thus causing serious damage to plants. Therefore, catalase can be used as a hydrogen peroxide scavenger and play an important role in plant resistance.

[0117] 3. Determination of oxidase

[0118] Oxidase test strips were purchased from Qingdao Haibo Biotechnology Co., Ltd. The strain Bachu37 was inoculated into LB liquid medium and cultured in a constant temperature shaker at 30°C until the bacterial solution OD 600The pH value is 0.6-0.8. Use a glass rod to dip the bacterial solution and drop it on the test paper. If the test paper turns blue or purple, it is positive. The results show that strain Bachu37 has the ability to produce oxidase. Oxidase can accelerate the respiration of plants, thereby enhancing the metabolic decomposition of toxins by plants and inhibiting the growth of pathogens. Oxidase can also promote the healing of plant wounds, reduce the invasion of pathogens, and ultimately indirectly promote plant growth.

[0119] IV. Nitrogen fixation ability of Halomonas sp. Bachu37

[0120] Prepare the Axubei nitrogen-free medium. Pipette 15 μL of bacterial liquid and drop it on three different positions of the Axubei medium, place it in a 30℃ constant temperature incubator and invert it for 8 days, observe the growth of the strain, and determine whether the strain has the ability to fix nitrogen. The nitrogen fixation results are shown in Figure 6 The results showed that strain Bachu37 has nitrogen fixation function. Nitrogen is one of the important elements required for plant growth and development. Strain Bachu37 can reduce molecular nitrogen in the air to ammonia through nitrogen fixation, so that plants have enough nitrogen to use, thereby promoting plant growth and development.

[0121] 5. Determination of phosphate solubilization ability of Halomonas sp. Bachu37 strain

[0122] (I) Determination of the ability of Halomonas sp. Bachu37 to degrade organic phosphorus

[0123] Prepare Montana medium. Pipette 15 μL of bacterial liquid and drop it on three different positions of Montana medium, place it in a 30℃ constant temperature incubator and invert it for 8 days to observe whether a transparent circle can be formed around the colony. Figure 7 The diameter of the transparent zone and the colony diameter were determined by the cross method, and the ratio of the two was positively correlated with the ability of the strain to degrade organic phosphorus. The diameter of the phosphorus degrading zone of strain Bachu37 on the Montgina medium was measured to be 23.38 mm, the colony diameter was 10.575 mm, and the D / d was 2.604699, as shown in Table 10.

[0124] Table 10 Results of the experimental study on the decomposition of organic phosphorus by strain Bachu37

[0125]

[0126] (II) Determination of the inorganic phosphorus solubilizing ability of Halomonas sp. Bachu37 strain

[0127] Prepare PKO medium. Pipette 15 μL of bacterial solution and drop it on three different locations of PKO medium. Place it in a 30℃ constant temperature incubator and invert it for 8 days. Observe whether a transparent circle can be formed around the colony. Figure 8 The diameter of the transparent zone and the colony diameter were determined by the cross method, and the ratio of the two was positively correlated with the ability of the strain to dissolve inorganic phosphorus. The diameter of the phosphorus dissolving zone D of the strain Bachu37 on the inorganic phosphorus medium was measured to be 8.52 mm, the colony diameter was 6.38 mm, and the D / d was 1.335707, as shown in Table 11.

[0128] Table 11 Results of the experiment on the solubility of inorganic phosphorus by strain Bachu37

[0129]

[0130] The combined results of the above two experiments show that strain Bachu37 has the ability to dissolve organic phosphorus and inorganic phosphorus, and its ability to dissolve organic phosphorus is more impressive. Phosphorus is also one of the important elements required for plant growth, but much of the phosphorus in the soil exists in the form of organic phosphorus compounds or insoluble phosphorus. Strain Bachu37 has the ability to dissolve phosphorus, convert organic phosphorus compounds into phosphates, convert insoluble phosphorus into soluble phosphorus, increase the effective phosphorus content in the soil, and thus promote plant growth.

[0131] VI. Determination of IAA production capacity of Halomonas sp. Bachu37 strain

[0132] Accurately weigh 10 mg of IAA, first dissolve it with a small amount of anhydrous ethanol, then dilute to 100 mL with distilled water to prepare an IAA solution with a concentration of 100 μg / mL as a stock solution, and then dilute the stock solution to prepare standard solutions with concentrations of 10, 20, 30, 40, and 50 μg / mL, respectively. Take 6 clean test tubes and add 2 mL of standard solution and 2 mL of Salkowski colorimetric solution in sequence. Place in a dark place and wait for the color reaction for 30 minutes. Use a spectrophotometer to measure the absorbance at OD530nm, and use the absorbance as the horizontal axis and the concentration of IAA in the fermentation broth as the vertical axis to draw a standard curve, see. Fig. 9 .

[0133] The strain Bachu37 was inoculated into sterile LB liquid culture medium and placed in a constant temperature shaking shaker at 30°C and 180r / min. Every other day, 2mL of bacterial suspension was taken from the prepared bacterial solution in a centrifuge tube, centrifuged at 14000rcf for 10min, and the supernatant was transferred to a clean test tube. An equal volume of Salkowski colorimetric solution was added. The reaction was allowed to proceed for 30min in a dark place and the color reaction was allowed to proceed. Three parallel tests were performed and the absorbance of A530 was measured and recorded using a spectrophotometer. The concentration of IAA produced at 13d was calculated to be 57±2μg / mL by the regression equation of the standard curve (y=74.595x–2.5256). The results are shown in Table 12.

[0134] Table 12 Average IAA concentration of strain Bachu37 after 3 days

[0135]

[0136] The results showed that strain Bachu37 has the ability to produce IAA, and the concentration of IAA produced can reach 57.1504μg / mL. IAA, also known as 3-indoleacetic acid, is the most important component of auxin. An appropriate amount of IAA has a significant promoting effect on plant growth and development. Strain Bachu37 has the ability to produce IAA and can directly produce IAA for plant use, promoting plant growth and development.

[0137] VII. Ability of Halomonas p.Bachu37 strain to produce volatile acidic substances

[0138] This test was conducted by using neutral red acid-base indicator. It is known that the indication range of neutral red indicator is 6.8-8.0, and the indication color is red for acid and orange for alkaline. LB solid culture medium with neutral red added to both sides of the isolation culture dish was added, and one side was inoculated with strain Bachu37, and the other side was not inoculated with bacteria; the culture dish was placed in a 30℃ constant temperature incubator and inverted for 48 hours, then taken out, and the culture dish inoculated with strain Bachu37 turned red ( Fig.10 A) This indicates that strain Bachu37 produces acidic substances. Two culture media were selected, one was left open for 24 hours, and the other was left open for 24 hours. After 24 hours, the open culture dish turned orange again ( Fig.10 B), the color of the culture dish that is not exposed remains unchanged ( Fig.10 C) This indicates that the acidic substances produced by strain Bachu37 are volatile substances.

[0139] From the above examples, it can be seen that the strain Bachu37 has the functions of salt-alkali tolerance, salt-alkali reduction, nitrogen fixation, organic phosphorus solubilization, inorganic phosphorus solubilization, IAA production, catalase production, oxidase production, volatile acidic substances production, etc., which can have beneficial effects on plant growth, and lays a good foundation for the strain to colonize in high-salt-alkali soil, improve the ability of plants to resist salt-alkali stress, improve salinized soil and promote plant growth.

[0140] Example 4

[0141] Effects of Halomonas sp. Bachu37 on the Growth of Maize Seedlings and Arabidopsis

[0142] 1. Effect of Halomonas sp. Bachu37 on the growth of corn seedlings

[0143] In this part, a pot experiment was conducted on the bacterial suspension of strain Bachu37, with corn seedlings as the test plants.

[0144] The strain Bachu37 was inoculated into LB liquid medium and placed in a constant temperature shaking incubator at 30°C and 180 r / min until the bacterial solution concentration reached 1×10 8 CFU / mL, dilute the bacterial solution 10 times with sterile deionized water to make a bacterial suspension; mix the nutrient soil and vermiculite in a 1:1 ratio, sterilize with high-pressure steam at 121℃ for 20min, and stir evenly with 30g / kg and 45g / kg salt mixtures respectively. Divide the mixed soil, sow 5 Xinyu 62 corn seeds of the same size in each experimental condition area, and sow at a depth of 1cm; set 10 replicates for each group; culture conditions are: temperature 28℃ / 22℃, light intensity 20000Lux / 0Lux, photoperiod 14h / 10h, relative humidity 70%, and culture for 14d.

[0145] When the corn germinated, 5 mL of the prepared strain Bachu37 suspension was applied to the two experimental groups under saline-alkali stress conditions every week, and 5 mL of sterile deionized water was applied to the two control groups; the soil state was observed every 24 hours during this period. If the soil was found to be dry, sterile deionized water was added in appropriate amounts to keep the watering amount of all experimental groups consistent. After 14 days, the corn roots were cleaned with water, and the aboveground fresh weight, underground fresh weight and plant height were weighed and recorded, and the results were statistically analyzed. The results are shown in Fig.11 , Fig.12 and as shown in Table 13. Fig.11 Among the two corns A, B, and C, the one on the left is the control group and the one on the right is the experimental group. It can be seen intuitively that the treatment group with the bacterial suspension of strain Bachu37 has a significant improvement on the growth of corn compared with the control group. Fig.12The aboveground fresh weight, underground fresh weight, plant height and other indicators of corn seedlings under different treatment conditions were measured. Under the treatment of 0g / kg without stress, the aboveground fresh weight of corn seedlings in the experimental group was significantly increased compared with the control group; under the treatment of 30g / kg stress, the underground fresh weight and plant height of corn seedlings in the experimental group increased by 3.70% and 15.20% respectively compared with the control group; under the treatment of 45g / kg stress, the plant height of corn seedlings in the experimental group increased by 16.35% compared with the control group. Table 13 shows the growth rate of various indicators of corn after treatment with the bacterial suspension of strain Bachu37 under different salt stress conditions.

[0146] Table 13 Growth rate of physiological indicators of corn treated with strain Bachu37 suspension at different salt concentrations

[0147]

[0148] 2. Effects of Halomonas sp. Bachu37 on the Growth of Arabidopsis

[0149] (I) Experiment on the growth promotion of Arabidopsis thaliana by Halomonas sp. Bachu37

[0150] This embodiment sets 5 groups of saline-alkali stress conditions: pH = 5.8, pH = 8.0, pH = 8.0 + 100mM NaCl, pH = 8.0 + 2mM NaHCO3 and pH = 8.0 + 100mM NaCl + 2mM NaHCO3. Prepare 1 / 2MS solid culture medium with the above different conditions respectively; take Arabidopsis as the test crop; prepare strain Bachu37 bacterial suspension (the preparation method is shown in Example 4); inoculate 2mL of strain Bachu37 bacterial liquid on one side of the culture dish at 1.5cm from the edge as the bottom of the plate, inoculate the Arabidopsis seeds after vernalization at 2.5cm from the top of the culture dish, inoculate 20 seeds in each culture medium, and do three replicates in each group. The treated culture medium is placed vertically in a light incubator with the inoculated bacterial liquid as the bottom for cultivation, and the culture conditions are: temperature 22℃, light intensity 12000Lux, photoperiod 16h / 8h, relative humidity 50%.

[0151] After 10 days of cultivation, the fresh weight, root length, number of lateral roots, number of leaves and other physiological indicators were measured. Fig.13 , Fig.14 As shown in Table 14. Fig.13 It can be seen intuitively that the growth of Arabidopsis thaliana treated with the bacterial suspension of strain Bachu37 is significantly better than that of the control group; Fig.14To measure the physiological indicators of Arabidopsis thaliana such as fresh weight, leaf number, root length and lateral root number under different stress conditions. The results showed that under the condition of pH 5.8, the lateral root number of Arabidopsis thaliana treated with the bacterial suspension of strain Bachu37 reached a significant increase compared with the control group; under the stress condition of pH 8.0, the lateral root number and leaf number of Arabidopsis thaliana treated with the bacterial suspension of strain Bachu37 were significantly improved compared with the control group; under the stress condition of pH = 8.0 + 100mM NaCl, the leaf number of Arabidopsis thaliana treated with the bacterial suspension of strain Bachu37 reached a strong significant improvement compared with the control group; under the stress conditions of pH = 8.0 + 2mM NaHCO3 and pH = 8.0 + 100mM NaCl + 2mM NaHCO3, compared with the control group, the Arabidopsis thaliana treated with the bacterial suspension of strain Bachu37 had a promoting effect on fresh weight, leaf number and lateral root number. Table 14 shows the growth rate of physiological indicators of Arabidopsis thaliana after inoculation with the bacterial suspension of strain Bachu37 at different salt concentrations. The results of the growth promotion experiment of strain Bachu37 on Arabidopsis thaliana show that strain Bachu37 has a significant effect on promoting the growth of the main root and plant biomass of Arabidopsis thaliana, and inoculation of strain Bachu37 can effectively promote the growth and development of Arabidopsis thaliana.

[0152] Fig.13 , Fig.14 In Table 14, a, b, c, d, and e represent different saline-alkali stress conditions, where a represents a pH value of 5.8; b represents a pH value of 8.0; c represents a pH value of 8.0+100mMNaCl; d represents a pH value of 8.0+2mM NaHCO3; and e represents a pH value of 8.0+2mM NaHCO3+100mM NaCl.

[0153] Table 14 Growth rates of physiological indicators of Arabidopsis thaliana treated with strain Bachu37 suspension under different conditions

[0154]

[0155]

[0156] “ / ” means that the physiological index was not measured or observed under this condition and could not be calculated.

[0157] (II) Isolation and culture experiment of Halomonas sp. Bachu37 strain and Arabidopsis thaliana

[0158] The strain was isolated and cultured from Arabidopsis thaliana in a two-compartment culture dish for observation. The uninoculated Arabidopsis thaliana was used as the control group, and the Arabidopsis thaliana inoculated with strain Bachu37 was used as the experimental group. Different saline-alkali stresses were simulated using 1 / 2MS solid culture medium with pH 5.8, pH 8.0, pH 8.0+2mM NaHCO3, pH 8.0+100mM NaCl, and pH 8.0+2mM NaHCO3+100mM NaCl.

[0159] The experimental results are shown in Fig.15 , Fig.16 As shown in Table 15. Fig.15 It can be seen intuitively that the growth of Arabidopsis in the experimental group is better than that in the control group; Fig.16 It measures the physiological indicators of Arabidopsis such as fresh weight, number of leaves and number of lateral roots. The results showed that under the treatment condition of pH 5.8, the growth rates of fresh weight and number of leaves of Arabidopsis in the experimental group were 74.45% and 33.06% respectively compared with those in the control group; under the stress condition of pH 8.0, the growth rates of fresh weight, number of leaves and then number of lateral roots of Arabidopsis in the experimental group were 118.86%, 30.18% and 410.34% respectively compared with those in the control group; under the stress condition of pH 8.0+100mM NaCl, the fresh weight, number of leaves and number of lateral roots of Arabidopsis in the experimental group were increased compared with those in the control group, and the increase of the number of leaves was significant. ; Under the condition of pH 8.0+2mM NaHCO3, the growth rates of fresh weight and lateral root number of Arabidopsis thaliana in the experimental group were 17.65% and 29.42% respectively compared with those in the control group; Under the condition of pH 8.0+2mM NaHCO3+100mM NaCl, the fresh weight, leaf number and lateral root number of Arabidopsis thaliana in the experimental group increased compared with those in the control group, and the increase in leaf number was significant. Table 15 shows the growth rates of physiological indicators of Arabidopsis thaliana treated with the suspension of strain Bachu37 under different conditions.

[0160] Through the correlation analysis of the growth and development traits (fresh weight, number of leaves, number of lateral roots) of Arabidopsis after isolation culture, it was concluded that the inoculated strain Bachu37 had a significant growth-promoting effect on the growth and development of Arabidopsis without direct contact between the strain and Arabidopsis. Since the isolation culture conditions are sealed, we concluded that these strains produced volatile substances to promote the growth of Arabidopsis plants.

[0161] Fig.15 , Fig.16In Table 15, a, b, c, d, and e represent different saline-alkali stress conditions, a represents a pH value of 5.8; b represents a pH value of 8.0; c represents a pH value of 8.0+100mMNaCl; d represents a pH value of 8.0+2mM NaHCO3; and e represents a pH value of 8.0+2mM NaHCO3+100mM NaCl.

[0162] Table 15 Growth rates of physiological indicators of Arabidopsis thaliana after treatment with bacterial suspension of strain Bachu37 under different conditions

[0163]

[0164]

[0165] “None” means that the treatment of the strain under this condition has no effect on the growth index.

[0166] Example 5

[0167] The Halomonas Halomonas sp. Bachu37 strain of the present invention is isolated from the rhizosphere soil of the aquatic halophyte reed in Bachu County, Kashi, Xinjiang Uygur Autonomous Region, and has a strong salt-alkali tolerance, which provides a basis for the colonization of the strain. The Halomonas Halomonas sp. Bachu37 strain has the characteristics of reducing salt and alkali, fixing nitrogen, dissolving phosphorus, producing IAA, producing catalase, etc., and has long-term development prospects and important application value in improving the ability of plants to tolerate salt and alkali stress, improving salinized soil and promoting plant growth.

[0168] The Halomonas sp. Bachu37 strain of the present invention can also be used for preparing fertilizers having plant growth promotion and saline-alkali land improvement effects.

[0169] The Halomonas sp. Bachu37 strain is prepared into a fertilizer, wherein the specific ingredients are rice straw, stalks, animal feces, chitin, yeast extract, phosphate, potash fertilizer, potassium humate, vermiculite and the strain Bachu37.

[0170] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limiting 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.

[0171] Sequence 1

[0172]

[0173] 。

Claims

1. A Halomonas and its application in promoting plant growth in terms of salt-alkali resistance, characterized in that: The strain is named Halomonas sp., deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC NO.28831 and a deposit date of November 1, 2023; the 16SrRNA nucleotide sequence of Halomonas sp. is shown in SEQ ID NO:

1.

2. The use of the salt-alkali tolerant Halomonas sp. strain according to claim 1, characterized in that: The salt mononas can improve the salt-alkali tolerance of plants, improve the salinity and alkalinity of soil, increase the available nitrogen content in soil and promote plant growth.

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 Halomonas sp. is used to promote the aboveground fresh weight, underground fresh weight, plant height and root length of the corn; when the plant is Arabidopsis thaliana, the Halomonas sp. strain is used to promote the lateral root number, root length, fresh weight and leaf number of the Arabidopsis thaliana.

5. A use of the salt-alkali tolerant Halomonas strain according to claim 1, characterized in that: The Halomonas sp. strain is used for preparing a fertilizer with plant growth promotion and saline-alkali land improvement effects.

6. A use as claimed in claim 5, characterized in that The fertilizer added with Halomonas sp. strain has at least one of the following uses: reducing soil salinity, fixing nitrogen, decomposing organic phosphorus, dissolving inorganic phosphorus, producing IAA, producing H2O2 enzyme and oxidase, and producing volatile acidic substances.

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