Halomonas sp. Strain with saline-alkaline tolerance and application thereof

Abstract

The invention provides a Halomonas sp. Strain with saline-alkaline tolerance, the Halomonas sp. Strain is named as Vreelandella sp. Strain, is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC NO.30231, the preservation date is April 1, 2024, and the 16S rRNA nucleotide sequence is as shown in SEQ ID NO: 1. The invention further provides a preparation method of the Halomonas sp. Strain. The invention further provides preparation of a microbial fertilizer which can be used for improving saline-alkali resistance of plants and promoting plant growth and can also be used for promoting plant growth and improving saline-alkali soil. The strain can relieve damage of saline-alkali stress to plants and promote plant growth, can be used for preparing functional fertilizers for soil improvement and plant growth promotion, and has wide application prospects in the aspects of saline-alkali soil improvement, agricultural productivity improvement, agricultural ecological environment protection and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial application, and particularly relates to a salt- and alkali-resistant strain of the Halomonas family, genus Vreelandella, and an application thereof. Background Art

[0002] Xinjiang is the largest saline soil distribution area in China. Soil salinization is the main factor causing food production security and hindering the improvement of land use efficiency, and it poses a serious threat to the long-term sustainable development of irrigation agriculture. Excessive soil salt content will destroy the water balance in the soil, change the soil structure, reduce soil permeability, cause soil compaction, make saline-alkali soil difficult to breathe and permeate, the soil surface temperature rises too slowly, and the soil enzyme activity decreases, inhibiting crop growth and reducing yield and quality, seriously affecting agricultural sustainable production. Salt-alkali land is a potential land resource, so improving saline-alkali soil is of great significance to improving soil productivity and maintaining sustainable agricultural development. In recent years, effective measures for saline-alkali land management mainly include physical improvement, chemical improvement and biological improvement. The physical improvement method has significant effects in a short period of time, but there are still problems such as high cost, large engineering volume, and inability to fundamentally improve the physical and chemical properties of the soil. The chemical improvement method can work in a short period of time, but it is costly and environmentally damaging. These methods have limitations and cannot fundamentally improve soil quality and increase crop yields. On the one hand, the biological improvement method can be improved by planting salt-tolerant or salt-resistant plants on saline-alkali land, and on the other hand, it can be improved by microorganisms, which has the advantages of sustainability and environmental friendliness. Microbial fertilizers use microorganisms with salt-tolerant, salt-solubilizing and growth-promoting abilities to improve the soil environment of saline-alkali land and improve the conditions for crop growth. The Xinjiang Uygur Autonomous Region has the characteristics of large temperature differences, sufficient sunshine hours, low precipitation, and dry climate. The local microorganisms have formed unique physiological functions and metabolites in the long-term evolution process, and have strong environmental adaptability.

[0003] Rhizosphere soil generally refers to the thin layer of soil 1-5mm around the rhizosphere. In the soil, plant roots secrete a large amount of secretions (mainly including sugars, amino acids, hormones and vitamins, etc.), making the rhizosphere microbial resources very rich, providing a strain basis for isolating growth-promoting bacteria from the rhizosphere. Plant Growth Promoting Rhizobacteria (PGPR) refers to a type of beneficial bacteria that attaches to the root system of plants and can promote the absorption of nutrients and minerals by plants through autogenous physiological metabolic activity, or improve the stress resistance of plants through some metabolic pathways, thereby promoting plant growth, or inhibiting infection by some pathogenic microorganisms.

[0004] The interaction between PGPR and plants is widely used in agricultural production, and the growth-promoting mechanism between the two has also been widely studied by scholars at home and abroad. Their mechanism of action mainly includes direct effects: phosphorus solubilization, nitrogen fixation, potassium solubilization, iron carrier production, production of plant hormones such as auxin, cytokinin and gibberellin, production of ACC deaminase (regulation of plant ethylene), etc.; indirect effects include: production of antibiotics to inhibit plant pathogenic microorganisms, change of plant rhizosphere soil environment, indirect regulation of plant autogenous metabolic pathways, and improvement of resistance to form a competitive relationship with pathogens. Inoculation of PGPR can make ineffective nutrients in the soil effective and promote the effective absorption and utilization of nutrients by plants.

[0005] According to existing literature reports, Halomonas has a wide range of applications and can be used to produce tetrahydropyrimidine (Tao Yujie, 2024), remove nitrogen (Wang Yuguang, 2023), purify high-salt nitrogen-containing wastewater (Li Yujing, 2022), and degrade phenol (Mu Xianjie, 2020). In 2023, de la Haba et al. reclassified some analyzed species of Halomonas into new genera belonging to the Halomonadaceae family based on the publication priority rule. These genera include Vreelandella, Bisbaumannia, Billgrantia, Franzmannia, Litchfieldella, and Onishia. The genus Vreelandella includes species such as H.aquamarina (synonyms H.meridiana and H.axialensis), H.arcis, and H.titanicae. There is currently no application research on the genus Vreelandella. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a salt-alkali resistant strain of the genus Vreelandella of the family Halomonas and its application in view of the deficiencies in the above-mentioned prior art. The strain has the functions of reducing salt and alkali, tolerating salt and alkali, fixing nitrogen, producing iron carriers, dissolving inorganic phosphorus, producing IAA, producing volatile acidic substances and promoting plant growth. It can be used to improve the salt-alkali tolerance of plants and promote plant growth, and is used for the preparation of microbial fertilizers with plant growth promotion and saline-alkali land improvement effects.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is: a salt-alkali resistant strain of the genus Vreelandella of the family Halomonas, the salt-alkali resistant strain of the genus Vreelandella of the family Halomonas is named Vreelandella titanicae, and is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC NO.30231 and a deposit date of April 1, 2024; the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; the 16S rRNA nucleotide sequence of the Vreelandella titanicae is shown in SEQ ID NO:1.

[0008] The invention provides application of the salt-alkali tolerant Vreelandella titanicae strain, wherein the Vreelandella titanicae strain is used for improving the salt-alkali tolerance of plants and promoting plant growth.

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

[0010] Preferably, when the plant is Arabidopsis thaliana, the Vreelandella titanicae is used to promote the lateral root number, fresh weight and leaf number of Arabidopsis thaliana;

[0011] When the plant is corn, the Vreelandella titanicae is used to promote the underground fresh weight of corn, and under non-stress conditions, is used to promote the aboveground fresh weight, underground fresh weight and leaf fresh weight of corn;

[0012] When the plant is tomato, under saline-alkali stress conditions, the Vreelandella titanicae is used to promote the number of lateral roots and fresh weight of tomatoes, and under non-stress conditions, the Vreelandella titanicae is used to promote the number of lateral roots, root length and fresh weight of tomatoes.

[0013] The present invention also provides the use of the above-mentioned salt-alkali resistant Vreelandella strain of the Halomonas family, wherein the Vreelandella titanicae is used for preparing a microbial fertilizer having the functions of promoting plant growth and improving saline-alkali land.

[0014] Preferably, when the Vreelandella titanicae is used as a microbial fertilizer, it is used for at least one of reducing soil salinity, fixing nitrogen, producing iron carriers, dissolving inorganic phosphorus, producing IAA, and producing volatile acidic substances.

[0015] Compared with the prior art, the present invention has the following advantages:

[0016] The Vreelandella titanicae of the present invention has the functions of reducing salt and alkali, tolerating salt and alkali, fixing nitrogen, producing iron carriers, dissolving inorganic phosphorus, producing IAA, producing volatile acidic substances and promoting plant growth. It can be used to improve the salt and alkali tolerance of plants, reduce soil alkali, increase the nitrogen fixation ability of plants and promote plant growth, and can also be prepared into a microbial fertilizer with plant growth promotion and saline-alkali land improvement functions.

[0017] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the 16S rRNA phylogenetic tree of Vreelandella titanicae of Example 1 of the present invention.

[0019] Figure 2 It is a colony morphology diagram of Vreelandella titanicae in Example 2 of the present invention.

[0020] Figure 3 This is a transmission electron microscope result of Vreelandella titanicae in Example 2 of the present invention.

[0021] Figure 4 This is the growth curve of Vreelandella titanicae in Example 2 of the present invention.

[0022] Figure 5 This is a graph showing the siderophore production experimental results of Vreelandella titanicae in Example 2 of the present invention.

[0023] Figure 6 This is the IAA standard curve of Vreelandella titanicae in Example 2 of the present invention.

[0024] Figure 7 This is a diagram showing the nitrogen fixation experiment results of Vreelandella titanicae according to Example 2 of the present invention.

[0025] Figure 8 This is a graph showing the results of an experiment on the dissolution of inorganic phosphorus by Vreelandella titanicae according to Example 2 of the present invention.

[0026] Fig. 9 This is a graph showing the experimental results of Vreelandella titanicae producing volatile acidic gases according to Example 2 of the present invention.

[0027] Fig.10This is a bar graph of the growth promotion experiment of Vreelandella titanicae on Arabidopsis thaliana under different conditions in Example 3 of the present invention. (Significant difference analysis: * indicates P<0.05; ** indicates P<0.01; *** indicates P<0.001; **** indicates P<0.0001)

[0028] Fig.11 This is a graph showing the results of an experiment on promoting the growth of Arabidopsis thaliana by Vreelandella titanicae according to Example 3 of the present invention.

[0029] Fig.12 This is a bar graph of the growth promotion experiment of Vreelandella titanicae on corn under different conditions in Example 4 of the present invention. (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 graph showing the results of an experiment on promoting corn growth by Vreelandella titanicae according to Example 4 of the present invention.

[0031] Fig.14 This is a bar graph of the growth promotion experiment of Vreelandella titanicae on tomatoes under different conditions in Example 5 of the present invention. (Significant difference analysis: * indicates P<0.05; ** indicates P<0.01; *** indicates P<0.001; **** indicates P<0.0001)

[0032] Fig.15 This is a graph showing the growth promotion experiment results of Vreelandella titanicae on tomatoes according to Example 5 of the present invention. DETAILED DESCRIPTION

[0033] Example 1

[0034] This example is the isolation, purification and identification of Vreelandella titanicae zp-514 strain

[0035] 1. Isolation and purification of Vreelandella titanicae zp-514 strain

[0036] The rhizosphere soil of the desert plant Populus euphratica in Kunyu City, 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. 0.1 g of the test soil sample was added to 900 μL of sterile deionized water, vortexed for 2 min to obtain the soil stock solution, and allowed to stand for 2-5 min to form a soil suspension. 100 μL was aspirated and added to 900 μL of sterile deionized water to obtain 10-1 g / mL soil suspension, and so on for gradient dilution to obtain 10 -2 g / mL, 10 -3 g / mL, 10 -4 g / mL and 10 -5 g / mL soil suspension, vortex oscillation to mix and then stand; take 100μL of each concentration gradient soil suspension, apply it on LB solid medium (pH 9, NaCl concentration 7.5%) with a coating rod, mark the date on the culture dish, invert the marked culture dish and place it in a 30℃ constant temperature incubator for 7 days, select single colonies with different colony morphology, color and size after 7 days, inoculate them into liquid culture medium for culture, then perform streaking culture, repeat the above streaking operation at least 3 times until a single colony is obtained, and record the colony morphology. Take 500μL of bacterial solution and 500μL of 50% glycerol from the purified strain, mix them, and store them in a -80℃ refrigerator for later use.

[0037] 2. Molecular biological identification of Vreelandella titanicae zp-514 strain The universal upstream primer 27F and universal downstream primer 1492R of the bacterial 16S rRNA gene sequence were used to perform PCR amplification using the 16S rRNA gene sequence of the strain as a template. The PCR system was 25 μL: 1 μL of primer 27F, 1 μL of primer 1492R, 12.5 μL of 2×phantaMax Mix, 0.1 μL of DNA template, and deionized water was added to make up to 25 μL. The PCR reaction conditions were: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 57°C annealing for 30 s, 72°C extension for 2 min, a total of 30 cycles; 72°C final extension for 5 min. The amplified PCR product was tested by agarose gel electrophoresis, and then the PCR product was sent to Sangon Biotech Co., Ltd. for sequencing, and a sequence of approximately 1500 bp was obtained. The sequencing results were compared by BLAST (Basic Local Alignment Search Tool (nih.gov)) at NCBI, and the strain phylogenetic tree was constructed using MEGA X64 software. Figure 1 As shown in the figure, the strain is similar to the model strain Vreelandella titanicae BH1 TThe sequence homology is the highest, at 99.43%. According to the priority principle published by de la Haba et al. in 2023, some analyzed members of the Halomonadaceae and Halomonas were reclassified into new genera of the Halomonadaceae, including Vreelandella, Bisbaumannia, Onishia, etc. The strain belongs to the genus Vreelandella, which includes members such as H.arcis and H.titanicae. There are currently no reports on the application research of members of the genus Vreelandella.

[0038] The strain was named Vreelandella titanicae, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC NO.30231 and a deposit date of April 1, 2024; the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; its 16S rRNA nucleotide sequence is shown in SEQID NO:1.

[0039] Example 2

[0040] This example is the identification of the physiological and biochemical characteristics and growth-promoting function of Vreelandella titanicae zp-514 strain.

[0041] 1. Physiological and biochemical characteristics of Vreelandella titanicae zp-514 strain

[0042] The zp-514 strain was inoculated on solid LB medium (pH 9, NaCl concentration 7.5%) to observe the colony morphology. The colony shape of the zp-514 strain on the LB solid medium was round, beige, moist, with smooth and neat edges and protrusions. The results are shown in Table 1. Figure 2 The transmission electron microscopy results are shown in Figure 3 The biochemical tube of Qingdao Haibo Biological was used to identify the physiological and biochemical characteristics of the strain. The identification results are shown in Table 2. The strain can grow with fructose, sucrose, maltose and other substances as the only carbon source, which provides reference data for the subsequent application of the strain.

[0043] Table 1 Colony morphology characteristics

[0044] Strain number size color shape edge Surface condition Degree of bulge zp-514 Smaller Beige Round Smooth and neat Wet Raised

[0045] Table 2 Experiment on the sole carbon source of strain zp-514

[0046] Test indicators glucose fructose Galactose Xylose sucrose maltose zp-514 - + - - + +

[0047] 2. Growth curve determination of Vreelandella titanicae zp-514 strain

[0048] The zp-514 strain was inoculated into LB liquid medium for cultivation and placed in a constant temperature shaker at 30°C and 180 r / min. The absorbance of the bacterial solution at 600 nm was measured using a UV spectrophotometer. 600 When the value is 0.6-0.8, the bacterial solution is inoculated with 1% of the inoculum into LB liquid culture medium and then placed in a constant temperature shaker 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 4 As shown, it provides a theoretical basis for the subsequent application of this strain.

[0049] 3. Identification of the growth-promoting function of Vreelandella titanicae zp-514 strain

[0050] 1. Determination of salt tolerance of Vreelandella titanicae zp-514 strain

[0051] The zp-514 strain was inoculated into LB liquid medium for cultivation and placed in a constant temperature shaker at 30°C and 180 r / min. The absorbance of the bacterial solution at 600 nm was measured and the OD of the bacterial solution was 600 When the value is 0.6-0.8, the bacterial solution is streaked on LB solid culture medium with NaCl concentrations of 0g / L, 20g / L, 40g / L, 60g / L, 80g / L, 100g / L, 120g / L, 140g / L, 160g / L, 180g / L and 200g / L (three biological replicates for each salt concentration), inverted and cultured in a 30°C constant temperature incubator, and the diameter of a single colony is observed every 24 hours. The results of the salt tolerance test of the zp-514 strain are shown in Table 3. The results show that the zp-514 strain can grow under the condition of NaCl concentration of 0g / L-200g / L, indicating that it has good salt tolerance.

[0052] Table 3 Salt tolerance results of zp-514 strain

[0053]

[0054] Note: +: strain grows, -: strain does not grow.

[0055] 2. Determination of alkali resistance of Vreelandella titanicae zp-514 strain

[0056] The zp-514 strain was inoculated into LB liquid medium for cultivation and placed in a constant temperature shaker at 30°C and 180 r / min. The absorbance of the bacterial solution at 600 nm was measured and the OD of the bacterial solution was 600 When the value is 0.6-0.8, the bacterial solution is inoculated with 1% inoculation amount into liquid culture medium with pH values ​​of 7, 8, 9, 10, 11, 12, and 13, respectively, and cultured in a constant temperature shaker at 30°C and 180 r / min. The OD of the bacterial solution is measured every 12 hours with a UV spectrophotometer. 600 The results are shown in Table 4. The results show that the zp-514 strain can grow at a pH value of 7-13, indicating that it has good alkali resistance.

[0057] Table 4 Alkali resistance results of strain zp-514

[0058] pH pH7 pH8 pH9 pH10 pH11 pH12 pH13 zp-514 + + + + + + +

[0059] Note: +: strain grows, -: strain does not grow.

[0060] 3. Determination of temperature tolerance range of Vreelandella titanicae zp-514 strain

[0061] The zp-514 strain was inoculated into LB liquid medium for cultivation and placed in a constant temperature shaker at 30°C and 180 r / min. The absorbance of the bacterial solution at 600 nm was measured and the OD of the bacterial solution was 600 When the value is 0.6-0.8, 100 μL of bacterial solution is taken to plate on a culture dish and the culture dish is placed in a constant temperature incubator for inversion culture for 7 days to observe the growth of the strain. The results are shown in Table 5, which show that the temperature tolerance range of the zp-514 strain is 5-45°C.

[0062] Table 5 Temperature tolerance range results of zp-514 strain

[0063] Temperature(℃) 5 10 15 20 25 30 35 40 45 50 zp-514 + + + + + + + + + -

[0064] Note: +: strain grows, -: strain does not grow.

[0065] Based on the above experiments, it can be seen that the zp-514 strain can grow normally under the conditions of NaCl concentration of 0g / L-200g / L, pH value of 7-13 and temperature range of 5-45℃, which shows that the strain has good environmental adaptability and lays the foundation for the strain to colonize in the soil under various stress conditions.

[0066] 3. Determination of the salt-reducing ability of Vreelandella titanicae zp-514 strain

[0067] The NaCl concentration in the bacterial solution was determined by the AgNO3 titration method, and the NaCl concentration was used to reflect the salt concentration. The zp-514 strain was inoculated into LB liquid culture medium for cultivation, and placed in a constant temperature shaker at 30°C and 180r / min for cultivation. The absorbance of the bacterial solution at 600nm was determined, and the OD of the bacterial solution was 600 When the pH value was 0.6-0.8, they were inoculated in liquid culture media with pH values ​​of 8, 9 and 10, respectively, and cultured in a constant temperature shaker at 30°C and 180r / min. 0.5mL of bacterial solution was taken every 12h, 2.5μL of 10% potassium chromate solution was added as an indicator, and titrated with 0.1mol / L AgNO3 solution. When a brick-red precipitate appeared and did not fade, the titration endpoint was reached. The results are shown in Table 6. The salt reduction rate of the zp-514 strain at pH 8 was 13.84%, indicating that the zp-514 strain can effectively improve the soil properties of saline-alkali land, help plants adapt to the saline-alkali land environment, and provide microbial resources for future saline-alkali land improvement.

[0068] Record the amount of AgNO3 solution used and calculate Cl according to the following formula: - The concentration of C2.

[0069] Calculation formula: C2 = (C1 × V1) / V2;

[0070] Where: C1 is the concentration of AgNO3 solution, mol / L;

[0071] V1 is the volume of AgNO3 solution consumed during titration, mL;

[0072] V2 is the volume of the bacterial solution during titration, mL;

[0073] According to Cl - The salt reduction rate is calculated according to the following formula:

[0074] Calculation formula: η salt = (C2 highest - C2 lowest) / C2 highest × 100%;

[0075] Where: ηsalt is the salt-reducing ability of the strain;

[0076] C2 is the highest when Cl - Concentration before degradation, mol / L;

[0077] C1 is the lowest Cl in the culture medium - Concentration after degradation, mol / L.

[0078] Table 6 Salt reduction results of strain zp-514

[0079]

[0080] 4. Determination of the Alkali-Reducing Ability of Vreelandella titanicae zp-514

[0081] The zp-514 strain was inoculated into LB liquid medium for cultivation and placed in a constant temperature shaker at 30°C and 180 r / min. The absorbance of the bacterial solution at 600 nm was measured and the OD of the bacterial solution was 600 When the value was 0.6-0.8, they were inoculated into LB liquid culture medium with pH values ​​of 8, 9 and 10, respectively, and placed in a constant temperature incubator at 30°C and 180r / min for culture. Every 12h, 4mL of bacterial solution was taken, centrifuged at 4000rpm for 5min, and the supernatant was taken and the pH value was measured with a pH meter. The results are shown in Table 7. The alkalinity reduction rate of the zp-514 strain at a pH value of 10 was 18.9%, proving that the zp-514 strain has the ability to reduce alkalinity, 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.

[0082] The alkali reduction rate was calculated according to the following formula:

[0083] η 碱 =(pH before - pH after) / pH before × 100%;

[0084] Where: η 碱 is the alkali reduction rate of the strain;

[0085] pH before is the pH value of the culture medium before inoculation;

[0086] pH is the pH value of the culture medium after inoculation.

[0087] Table 7 Alkali reduction results of strain zp-514

[0088]

[0089] Based on the above two experiments, it can be seen that the zp-514 strain has the best salt-reducing effect at a pH value of 8, with a salt-reducing rate of 13.84%; the zp-514 strain has the best alkali-reducing effect at a pH value of 10, with an alkali-reducing rate of 18.90%. This shows that the zp-514 strain can reduce the salt content and pH value of the soil, enhance the activity of microorganisms in the soil, and promote plant growth.

[0090] 5. Determination of the siderophore production function of Vreelandella titanicae zp-514 strain

[0091] Preparation of CAS medium (g / L): 10g glucose, 0.5g ammonium sulfate, 0.5g yeast extract powder, 0.3g sodium chloride, 0.3g potassium chloride, 0.3g magnesium sulfate, 0.03g ferrous sulfate, 0.03g manganese sulfate, 5g tricalcium phosphate, add 1L of deionized water, adjust pH = 7.0-7.5, and sterilize at 116°C for 30min.

[0092] The zp-514 strain was inoculated into LB liquid medium for cultivation and placed in a constant temperature shaker at 30°C and 180 r / min. The absorbance of the bacterial solution at 600 nm was measured and the OD of the bacterial solution was 600 When the value is 0.6-0.8, inoculate it on CAS medium and culture it upside down in a 30℃ constant temperature incubator for 7 days. 3+ A blue complex is formed, which is the color of CAS culture medium. If the strain produces iron carriers, iron carriers and Fe 3+ The combination is more stable, and Fe 3+ Take away from the CAS test solution, and a transparent color circle appears around the colony. Figure 5 The results showed that the strain zp-514 has the ability to produce siderophores, which can promote the absorption of Fe and other elements by plants in saline-alkali land, because siderophores can react with Zn +2 and Cr +3 The formation of complexes with heavy metal ions affects the existence of heavy metals, which is beneficial to the absorption of elements such as Fe by plant roots and promotes plant growth and development.

[0093] 6. IAA production function assay of Vreelandella titanicae zp-514 strain

[0094] The prepared 100 μg / mL IAA stock solution was diluted in a concentration gradient to obtain 10 μg / mL, 20 μg / mL, 30 μg / mL and 50 μg / mL IAA solutions of different concentration gradients. 2 mL of bacterial solution and an equal volume of Salkowski colorimetric solution were added to the test tubes in sequence. The mixture was placed in a dark place to react for 30 minutes. The A 530 The absorbance of the measured standard concentration A is shown in Table 8. 530 The results were analyzed by measuring the absorbance. 530 The absorbance is taken as the ordinate and the IAA concentration is taken as the abscissa, and the regression equation y=0.0105x+0.1073 (R2=0.9953) is drawn. Figure 6 shown.

[0095] The zp-514 strain was inoculated into LB liquid medium for cultivation and placed in a constant temperature shaker at 30°C and 180 r / min. The absorbance of the bacterial solution at 600 nm was measured and the OD of the bacterial solution was 600 When the value is 0.6-0.8, inoculate into a liquid culture medium containing 2 mg / L tryptophan. Every 48 hours, take 2 ml of the bacterial solution into a test tube, centrifuge at 14000 rpm for 10 minutes, take 2 ml of the supernatant and add an equal volume of Salkowski colorimetric solution, place in a dark place for reaction for 30 minutes, and use a spectrophotometer to measure A 530 The absorbance was measured for 14 days, and the data were recorded. The measured data were substituted into the regression equation y=0.0105x+0.1073 to calculate the maximum IAA concentration in the bacterial solution. The results are shown in Table 9. The concentration was the highest on the 14th day, which was 37.6471 μg / mL, indicating that the zp-514 strain can promote the growth and development of plants. Auxin is an important plant growth hormone that can be synthesized by a variety of microorganisms. Tryptophan, as a secretion of plant roots, is an important precursor for IAA synthesis. Bacteria use tryptophan and other small molecules secreted by plant roots to synthesize and secrete IAA, which is then absorbed by plants, thereby promoting the proliferation and elongation of plant cells.

[0096] Table 8 IAA standard curve determination

[0097] Test tube serial number IAA concentration (μg / mL) <![CDATA[A 530 Absorbance]]> 0 0 0 1 10 0.199 2 20 0.327 3 30 0.432 4 50 0.623

[0098] Table 9 Quantitative determination of IAA secretion by strain zp-514

[0099] Strain number <![CDATA[A 530 Maximum absorbance]]> IAA concentration (μg / mL) zp-514 0.502 37.6471

[0100] 7. Determination of nitrogen fixation function of Vreelandella titanicae zp-514 strain

[0101] Preparation of Axubei nitrogen-free culture medium (g / L): Add 0.2g potassium dihydrogen phosphate, 10g glucose, 0.2g magnesium sulfate heptahydrate, 0.1g calcium sulfate, 75g sodium chloride, 5g calcium carbonate, and 16g agar to 1L deionized water, adjust the pH value to 7.0-7.2, and sterilize with high temperature and high pressure steam at 115°C for 20min.

[0102] The zp-514 strain was inoculated into LB liquid medium for cultivation and placed in a constant temperature shaker at 30°C and 180 r / min. The absorbance of the bacterial solution at 600 nm was measured and the OD of the bacterial solution was 600 When the value is 0.6-0.8, inoculate it on the nitrogen-free solid medium of Axubei. Inoculate three places on each medium, spot 15μL of bacterial solution on each place, place it in a 30℃ constant temperature incubator and invert it for 7-9 days, and observe the growth of the zp-514 strain. The results are as follows Figure 7 As shown, the ZP-514 strain can grow normally on the Axubei culture medium, indicating that the ZP-514 strain has the ability to fix nitrogen, can provide a large amount of nitrogen source for the soil, increase the nitrogen content in the soil, and promote plant growth and development.

[0103] 8. Determination of the inorganic phosphorus solubilization function of Vreelandella titanicae zp-514 strain

[0104] Preparation of inorganic phosphorus culture medium (g / L): 10g glucose, 0.5g ammonium sulfate, 0.3g magnesium sulfate heptahydrate, 0.03g manganese sulfate tetrahydrate, 0.3g potassium chloride, 0.03g ferrous sulfate heptahydrate, 0.3g sodium chloride, 10.0g calcium phosphate, 16g agar powder, 1L deionized water, adjust the pH value to 7.0-7.2, and sterilize at 115℃ high pressure steam for 20min.

[0105] The zp-514 strain was inoculated into LB liquid medium for cultivation and placed in a constant temperature shaker at 30°C and 180 r / min. The absorbance of the bacterial solution at 600 nm was measured using a UV spectrophotometer. 600 When the value is 0.6-0.8, inoculate it into inorganic phosphorus culture medium, inoculate three places in each culture medium, the inoculation volume is 15μL, and place it in a 30℃ constant temperature incubator and invert it for 7-9 days. Figure 8 The results showed that the strain ZP-514 has the ability to dissolve inorganic phosphorus, can dissolve insoluble calcium phosphates, and promote plant growth. Because soil phosphorus is mainly divided into three forms: water-soluble phosphorus, insoluble inorganic phosphorus, and organic phosphorus, the strain can convert insoluble calcium phosphates into highly effective phosphates through the carbonic acid and organic acid produced by the respiration of plant roots and microorganisms and the decomposition of organic fertilizers.

[0106] 9. Determination of volatile acidic substances produced by Vreelandella titanicae zp-514 strain

[0107] Toluene red is mainly used as an acid-base indicator, and its pH value indication range is 6.8-8.0. The indication color is red when it encounters acid and orange-yellow when it encounters alkali. Pour the LB solid culture medium with toluene red added to both sides of the isolation culture medium, inoculate the zp-514 strain on one side, and use the other side as a blank control. Place the culture medium upside down in a 30°C constant temperature incubator and take it out after incubation for 48 hours. Observe the color of both sides of the culture medium and find that the culture medium on the side inoculated with the zp-514 strain turns red while the color of the culture medium in the control group remains unchanged, proving that the zp-514 strain can produce acidic substances. Select the culture medium after the above-mentioned inoculation treatment and place it in the open and non-open place respectively.

[0108] like Fig. 9As shown, the color of the culture medium placed in the open on the right returned to the initial color of the culture medium, while the color of the culture medium placed in the open on the left remained unchanged, proving that the acidic substance produced by the zp-514 strain is volatile, laying a good foundation for the development of the strain in saline-alkali land.

[0109] Example 3

[0110] This example shows the growth-promoting effect of Vreelandella titanicae zp-514 strain on Arabidopsis thaliana.

[0111] In this embodiment, five groups of saline-alkali stress conditions are set: a represents pH 5.8, b represents pH 8.0, c represents pH 8.0+2mM NaHCO3, d represents pH 8.0+100mM NaCl, and e represents pH 8.0+2mM NaHCO3+100mM NaCl. 1 / 2MS solid culture medium with the above different conditions is prepared respectively, and Arabidopsis thaliana is used as the test plant.

[0112] After the 1 / 2MS solid culture medium is completely cooled, cut off the 1 / 2MS solid culture medium at 1.5cm from the bottom of the culture medium and add 2mL of bacterial solution. Inoculate the vernalized Arabidopsis seeds 2.5cm from the top of the culture medium. Inoculate 20 seeds in each culture medium and repeat three times in each group. Place the treated culture medium vertically in a light incubator with the inoculated bacterial solution or sterilized deionized water as the bottom. The culture conditions are: temperature 22℃, light intensity 12000Lux, photoperiod 16h / 8h, relative humidity 50%. After 7 days of culture, the fresh weight, root length, number of lateral roots, number of leaves and other physiological indicators were measured. The results are shown in Table 10. Fig.10 and Fig.11 As shown, after the zp-514 strain was inoculated into the culture medium of the salt-alkali stress group, the growth of Arabidopsis thaliana showed different promotion effects compared with the control group. Under condition a, the growth rates of lateral root number, fresh weight and leaf number of Arabidopsis thaliana inoculated with zp-514 strain were 571%, 141% and 23%, respectively; under condition b, the growth rates of lateral root number, fresh weight and leaf number of Arabidopsis thaliana inoculated with zp-514 strain were 2717%, 115% and 12%, respectively; under condition c, the growth rates of lateral root number, fresh weight and leaf number of Arabidopsis thaliana inoculated with zp-514 strain were 3542%, 250% and 52%, respectively; under condition d, the growth rates of lateral root number, fresh weight and leaf number of Arabidopsis thaliana inoculated with zp-514 strain were 2200%, 46% and 12%, respectively; under condition e, the growth rates of lateral root number, fresh weight and leaf number of Arabidopsis thaliana inoculated with zp-514 strain were 767%, 53% and 24%, respectively. In summary, the zp-514 strain can promote the growth of Arabidopsis thaliana under both non-stress and saline-alkali stress conditions, especially promote the root growth and development of the plant.

[0113] Table 10 Statistical results of physiological indicators of Arabidopsis thaliana after inoculation with strain zp-514

[0114] Stress conditions Lateral root number growth rate Fresh weight growth rate Leaf number growth rate a 571％ 141％ 23％ b 2717％ 115％ 12％ c 3542％ 250％ 52％ d 2200％ 46％ 12％ e 767％ 53％ 24％

[0115] Example 4

[0116] This example shows the growth-promoting effect of Vreelandella titanicae zp-514 strain on corn.

[0117] The nutrient soil and vermiculite were mixed in a ratio of 1:1, and sterilized three times with high temperature and high pressure steam at 121℃ for 20min to kill the microorganisms in the soil. NaCl, NaHCO3, NaCO3, and NaSO4 were mixed in a ratio of 1:1:1:1, and added to the nutrient soil according to the mass ratio. The mass ratio of the added mixed salt to the soil was 0g / kg, 6g / kg, 9g / kg, and 15g / kg respectively.

[0118] Preparation of bacterial suspension: Inoculate the strain into LB liquid culture medium, culture at 30℃180r / min until OD600 is 0.6-0.8, dilute the bacterial solution 10 times with sterile deionized water to prepare bacterial suspension for use. Select corn seeds of uniform size, full and uniform, first disinfect with 75% alcohol for 15s, rinse with sterile water, soak in 10% sodium hypochlorite solution for 30min, and finally rinse with sterile water 3 times. Soak the seeds in zp-514 bacterial suspension for 3h, and soak the control group in sterile deionized water.

[0119] The treated corn seeds (seeds soaked in zp-514 bacterial suspension, seeds soaked in sterilized deionized water) were sown into the soil treated with salt solution to a depth of about 1 cm. Three seeds were sown in each pot, and the seedlings were thinned out after the corn grew two cotyledons. One seedling was retained in each pot, and the seedling size of each treatment component was kept similar. Six repeated experiments were set for each treatment. The culture conditions were: temperature 28°C, light intensity 20000Lux, light cycle 14h, relative humidity 70% and temperature 22°C, light intensity 0Lux, dark cycle 10h, relative humidity 70% alternately. The results of corn growth for 14 days were measured, and the results are shown in Table 11. Fig.12 and Fig.13As shown in the figure, a, b, c, and d represent the stress conditions of 0g / kg, 6g / kg, 9g / kg, and 15g / kg, respectively. Among them, under condition a, after inoculation with the zp-514 strain, the aboveground fresh weight, underground fresh weight, and leaf fresh weight of the plant were all higher than those of the control group, with growth rates of 66%, 16%, and 7%, respectively; under condition b, after inoculation with the zp-514 strain, the aboveground fresh weight growth rate of corn was 68%, the underground fresh weight growth rate was 6%, and the leaf fresh weight growth rate was 44%; under condition c, after inoculation with the zp-514 strain, the growth rate of the underground fresh weight of corn was 14%; under condition d, after inoculation with the zp-514 strain, the underground fresh weight growth rate of corn was 54%. In summary, the zp-514 strain has a growth-promoting effect on corn under different stress conditions, especially promoting the growth and development of corn roots.

[0120] Table 11 Statistical results of maize physiological indicators after inoculation with strain zp-514

[0121] Stress conditions Above ground fresh weight growth rate Underground fresh weight growth rate Leaf fresh weight growth rate a 66％ 16％ 7％ b 68％ 6％ 44％ c -26％ 14％ -32％ d 26％ 54％ 15％

[0122] Example 5

[0123] This example shows the growth-promoting effect of Vreelandella titanicae zp-514 strain on tomatoes.

[0124] 5 groups of saline-alkali stress conditions: a represents pH 5.8, b represents pH 8.0, c represents pH 8.0+2mMNaHCO3, d represents pH 8.0+50mM NaCl and e represents pH 8.0+2mM NaHCO3+50mM NaCl. Prepare 1 / 2MS solid culture medium under the above different conditions respectively, and use tomato as the test plant. Tomato seed treatment: soak in sterile deionized water for 20 minutes; soak in 75% alcohol for 1 minute; soak in NaClO for 15 minutes; finally wash three times with sterile deionized water. The cleaned tomato seeds were inoculated on the culture medium and placed in an incubator for vernalization for 2 days.

[0125] 2mL of zp-514 strain was inoculated on one side of the culture medium at 1.5cm from the edge as the bottom of the plate, and the vernalized tomato seeds were inoculated at 2.5cm from the top of the culture medium. Ten seeds were inoculated in each culture medium, and 6 replicates were made in each group. The treated culture medium was placed vertically in a light incubator with the inoculated bacterial solution or sterilized deionized water as the bottom. The culture conditions were: day and night length and temperature were 14h 23℃ / 10h 18℃, light intensity was 12000Lux, and relative humidity was 50%.

[0126] After 7 days of cultivation, agronomic traits such as lateral root number, root length, and fresh weight were measured. The results are shown in Table 12. Fig.14 and Fig.15 As shown in the figure, under condition a, the number of lateral roots, root length and fresh weight of tomatoes inoculated with zp-514 strain were significantly increased, with growth rates of 505%, 22% and 168% respectively; under condition b, the number of lateral roots and fresh weight of tomatoes inoculated with zp-514 strain were increased, with increase rates of 57% and 59% respectively; under condition c, the number of lateral roots, root length and fresh weight of tomatoes inoculated with zp-514 strain were better than those of the control group, with growth rates of 205%, 29% and 106% respectively; under condition d, the growth rates of the number of lateral roots, root length and fresh weight of tomatoes inoculated with zp-514 strain were 392%, 15% and 36% respectively; under condition e, the growth rates of the number of lateral roots, root length and fresh weight of tomatoes inoculated with zp-514 strain were 47%, 41% and 46% respectively. In summary, zp-514 strain promotes the growth of tomatoes under no stress and different saline-alkali stress conditions.

[0127] Table 12 Statistical results of tomato physiological indicators after inoculation with strain zp-514

[0128] Stress conditions Lateral root growth rate Root length growth rate Fresh weight growth rate a 505％ 22％ 168％ b 57％ -6％ 59％ c 205％ 29％ 106％ d 392％ 15％ 36％ e 47％ 41％ 46％

[0129] The Vreelandella titanicae zp-514 strain provided by the present invention has the functions of salt-alkali tolerance, salt-alkali reduction, siderophore production, IAA production, nitrogen fixation, inorganic phosphorus dissolution, and volatile acidic substance production, and can promote the growth of crops such as grain crops and vegetable crops, and can effectively alleviate the Na in the soil. + , K + , Cl - It can reduce the stress effects on plants and promote plant growth and development. It has important application value in improving plant tolerance to salt-alkali stress, crop production capacity, saline-alkali land improvement, and research and development of microbial fertilizers.

[0130] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A strain of the genus Vreelandella of the family Halomonas having salt-alkali tolerance, characterized in that: The salt- and alkali-resistant strain of the genus Vreelandella of the family Halomonas is named Vreelandella titanicae, and is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC NO.30231 and a deposit date of April 1, 2024; the 16S rRNA nucleotide sequence of the Vreelandella titanicae is shown in SEQ ID NO:

1.

2. A use of the salt- and alkali-tolerant strain of the Halomonas family Vreelandella as claimed in claim 1, characterized in that: The Vreelandella titanicae is used for improving the salt-alkali tolerance of plants and promoting plant growth.

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

4. The use according to claim 3, characterized in that: When the plant is Arabidopsis thaliana, the Vreelandella titanicae is used to promote the lateral root number, fresh weight and leaf number of Arabidopsis thaliana; When the plant is corn, the Vreelandella titanicae is used to promote the underground fresh weight of corn, and under non-stress conditions, is used to promote the aboveground fresh weight, underground fresh weight and leaf fresh weight of corn; When the plant is tomato, under saline-alkali stress conditions, the Vreelandella titanicae is used to promote the number of lateral roots and fresh weight of tomatoes, and under non-stress conditions, the Vreelandella titanicae is used to promote the number of lateral roots, root length and fresh weight of tomatoes.

5. Use of the salt- and alkali-tolerant strain of the Halomonas family Vreelandella as claimed in claim 1, wherein the Vreelandella titanicae is used for preparing a microbial fertilizer having plant growth promotion and saline-alkali land improvement effects.

6. The use according to claim 5, characterized in that: When the Vreelandella titanicae is used as a microbial fertilizer, it is used for at least one of reducing soil salinity, fixing nitrogen, producing iron carriers, dissolving inorganic phosphorus, producing IAA, and producing volatile acidic substances.

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