A growth-promoting halophilic bacterium, *Sutcliffiella halmapala* BWT1.2507, and its applications.

Abstract

This invention discloses a growth-promoting halophilic Sartcliff strain Sutcliffe's halma piece BWT1.2507 and its applications. Sutcliffe's halma piece BWT1.2507, deposited on September 28, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China, accession number GDMCC No: 67048. This invention isolates and screens a halophilic *Satcliffe* strain from saline-alkali soil in the rhizosphere of maize. Sutcliffe piece of cake BWT1.2507 possesses salt-alkali tolerance, nitrogen fixation, organic phosphorus solubilization, IAA secretion, and iron carrier production capabilities. It exhibits significant effects in promoting healthy maize plant growth and reducing alkali and salt content in saline-alkali soils. This strain is expected to be developed as an agricultural microbial agent and has great potential for improving saline-alkali soils.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a growth-promoting halophilic bacterium, Sutcliffiella halmapala BWT1.2507, and its applications. Background Technology

[0002] Soil salinization is one of the ecological and environmental factors restricting the sustainable development of agriculture in my country. Currently, the global area of ​​salinized land is nearly 1 billion hectares. 2 Soil salinization refers to the phenomenon or process of high salinity or alkalinity caused by the continuous erosion and accumulation of soluble salt ions in the soil surface. It leads to soil compaction, low porosity, and poor fertility, making it difficult for mineral nutrients to be released and absorbed. High salinity and alkalinity cause osmotic stress and ion toxicity to plants, severely inhibiting plant growth and posing a serious challenge to agricultural productivity and the ecological environment. Current methods for improving soil salinization mainly include physical, chemical, and biological methods. The first two methods are prone to secondary soil pollution, deterioration of soil structure, large engineering workload, high remediation costs, and difficulty in implementation. Biological methods mainly utilize plant and microbial improvement techniques. Plant methods are easily limited by geographical, climatic, and environmental factors, hindering large-scale promotion. Microbial improvement techniques, however, are highly regarded and favored due to their strong functional sustainability, short cultivation cycle, ease of operation, and strong colonization ability.

[0003] Halotolerant plant growth-promoting rhizobacteria (HT-PGPR) are a class of microorganisms that colonize the rhizosphere of plants in saline-alkali soils and have functions such as promoting growth and resisting stress. They can effectively improve the plant rhizosphere environment and soil physicochemical properties, reduce the inhibitory effect of salt on crop growth, and promote healthy plant growth by regulating the secretion of plant growth hormones (such as the production of indoleacetic acid and gibberellins), enhancing the absorption of nutrients (through nitrogen fixation, phosphorus solubilization, potassium solubilization, and iron carrier production), and tolerating abiotic stresses (salt and alkali tolerance, high temperature tolerance, and radiation resistance). The application of this type of functional microorganism has been particularly effective in improving low- and medium-salinity soils in my country. Summary of the Invention

[0004] The first objective of this invention is to provide a growth-promoting halophilic bacterium, Sutcliffiella halmapala BWT1.2507, as a microbial germplasm resource for the improvement of saline-alkali soils.

[0005] The plant growth-promoting halophilic bacterium *Sutcliffiella halmapala* BWT1.2507 of this invention was isolated from the rhizosphere saline-alkali soil of maize in Nansha District, Guangzhou City. It was deposited on September 28, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, 510070, China, with accession number GDMCC No: 67048.

[0006] The growth-promoting halophilic Sartcliff b1.2507 is a Gram-positive bacterium with an orange-yellow, viscous, opaque body with wrinkled protrusions. It produces spores, is rod-shaped, and is easily cultured.

[0007] The halophilic bacterium *Satcliffe* BWT1.2507 exhibits strong enzyme activities in alkaline phosphatase, β-galactosidase, leucine arylamidase, valine arylamidase, cysteine ​​arylamidase, α-chymotrypsin, urease, and arginine dihydrolase. It also shows positive results for indole production, gelatin liquefaction, starch hydrolysis, and H2O2 enzyme activity. It can utilize carbon sources such as D-mannose, D-mannitol, D-maltose, potassium gluconate, sucrose, adipic acid, malic acid, and phenylacetic acid.

[0008] The growth-promoting halophilic Sartcrylonitrile bWT1.2507 exhibits salt and alkali tolerance, and can tolerate growth at a NaCl concentration of 19% and a pH range of 4-11. It possesses the ability to fix nitrogen, dissolve organic phosphorus, secrete IAA, and produce siderophores, with the secreted IAA content reaching 9.37 mg / L and the siderophore activity unit (su) reaching 70.36%.

[0009] A second objective of this invention is to provide a microbial inoculant containing *Sartcrylonitrile halophilicus* BWT1.2507, or its sludge, fermentation broth, or suspension.

[0010] Preferably, the bacterial sludge, fermentation broth, or bacterial suspension that promotes the growth of halophilic Sutcliffe BWT1.2507 is prepared by the following method:

[0011] The fermentation medium consisted of 10.0 g / L soybean peptone, 5.0 g / L yeast extract, and 20.0 g / L NaCl. Fermentation conditions were: pH 8.0, temperature 30 ℃, fermentation time 60 h, inoculum size 3%, and rotation speed 240 rpm. The fermentation broth was obtained by centrifugation to remove the supernatant, yielding a bacterial sludge. This sludge was then resuspended to obtain a bacterial suspension. The final liquid fermentation OD was... 600 The effective viable count reached 3.76 × 10⁻⁶, with a value of 2.93. 9 CFU / mL.

[0012] A third objective of this invention is to provide the application of the above-mentioned growth-promoting halophilic Sartcrylonitrile BWT1.2507 or the above-mentioned microbial inoculants in promoting plant growth.

[0013] Preferably, the plant is corn.

[0014] The preferred application is in promoting plant growth in saline-alkali soils.

[0015] Preferably, the saline-alkali soil is saline-alkali soil with a salinity of 0.1-0.3%.

[0016] The fourth objective of this invention is to provide the application of the above-mentioned halophilic Sartcrylonitrile BWT1.2507 or the above-mentioned microbial agents in reducing alkalinity and salinity in saline-alkali soils.

[0017] The present invention has the following advantages over the prior art:

[0018] 1) This invention provides a strain of halophilic Sutcliffe bacterium BWT1.2507, which has high salt tolerance (tolerant to growth at 19% NaC concentration) and acid and alkali tolerance (growing under pH 4-11 conditions);

[0019] 2) The above-mentioned halophilic Sartcliff BWT1.2507 has the ability to fix nitrogen, dissolve organophosphates, secrete IAA and produce siderophores, with the secreted IAA content reaching 9.37 mg / L and the siderophore activity units (su) reaching 70.36%.

[0020] 3) The microbial agent prepared from the above-mentioned halophilic Sartcliff BWT1.2507 has a strong ability to promote growth, reduce alkali and salt in corn saline-alkali soil.

[0021] Therefore, this invention has isolated and screened a halophilic Sutcliffiella halmapala strain BWT1.2507 from maize rhizosphere saline-alkali soil. This strain exhibits salt-alkali tolerance, nitrogen fixation, organic phosphorus solubilization, IAA secretion, and iron carrier production. It shows significant effects in promoting healthy maize plant growth and reducing the alkalinity and salinity of saline-alkali soils. This strain will be developed as an agricultural microbial agent and has great potential for application in improving saline-alkali soils.

[0022] Sutcliffiella halmapala BWT1.2507 was deposited on September 28, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC No: 67048. Attached Figure Description

[0023] Figure 1 Cell morphology and Gram staining morphology of strain BWT1.2507;

[0024] Figure 2 Phylogenetic tree of strain BWT1.2507;

[0025] Figure 3 Determination of nitrogen fixation, organophosphate solubilization, IAA secretion, and siderophore production capabilities of strain BWT1.2507;

[0026] Figure 4 IAA concentration standard curve;

[0027] Figure 5 Results of pH tests on solid culture of strain BWT1.2507;

[0028] Figure 6 Results of pH test for liquid fermentation of strain BWT1.2507;

[0029] Figure 7 Results of salt tolerance (NaCl concentration) test for solid culture strain BWT1.2507;

[0030] Figure 8 Results of salt tolerance (NaCl concentration) test for liquid fermentation of strain BWT1.2507;

[0031] Figure 9 Results of fermentation medium optimization for strain BWT1.2507;

[0032] Figure 10 Results of fermentation time optimization for strain BWT1.2507;

[0033] Figure 11 Results of fermentation temperature optimization for strain BWT1.2507;

[0034] Figure 12 Results of optimized fermentation inoculum size for strain BWT1.2507;

[0035] Figure 13 Results of optimized fermentation speed for strain BWT1.2507;

[0036] Figure 14 The effect of strain BWT1.2507 on promoting growth in potted maize (saline-alkali soil). Detailed Implementation

[0037] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] The culture media, reagents, and equipment used in the following examples were sourced from the following sources:

[0039] 1. Experimental reagents

[0040] (1) LB basal medium: Add 10.0 g of tryptone, 5.0 g of yeast extract and 10.0 g of sodium chloride to 1000 mL of distilled water, adjust the pH to 8.0, and add 15.0 g of agar powder if preparing solid medium. Sterilize at 121 °C for 20 min for later use.

[0041] (2) High-salt screening medium: Add 10.0 g of tryptone, 5.0 g of yeast extract and 120.0 g of sodium chloride to 1000 mL of distilled water and adjust the pH to 8.0. If preparing solid plates, add 15.0 g of agar powder.

[0042] (3) Ashby nitrogen-free medium: Add 10.0 g mannitol, 0.2 g K2HPO4, 0.2 g MgSO4·7H2O, 0.2 g NaCl, 0.2 g CaSO4·2H2O, 5.0 g CaCO3 and 15.0 g agar powder to 1000 mL distilled water, adjust the pH to 8.0, and sterilize at 121 °C for 20 min.

[0043] (4) Mongkina Organic Phosphorus Medium: 10.0 g glucose, 0.5 g (NH4)2SO4, 0.3 g KCl, 0.3 g NaCl, 0.03 g FeSO4·7H2O, 0.3 g MgSO4·7H2O, 0.03 g MnSO4·4H2O, 5.0 g CaCO3, 0.2 g lecithin, 15.0 g agar powder, 1000 mL distilled water, pH adjusted to 8.0, steam sterilized at 115 ℃ for 20 min.

[0044] (5) CAS detection medium: Weigh 60.5 mg of Chromium Azurite S (CAS), 72.9 mg of hexadecyltrimethylammonium bromide (HDTMA), 2.645 mg of ferric chloride, 295.25 mg of sodium dihydrogen phosphate, 1213.5 mg of disodium hydrogen phosphate, 125 mg of ammonium chloride, 37.5 mg of potassium dihydrogen phosphate, and 62.5 mg of sodium chloride and add them to 1000 mL of distilled water. Mix thoroughly and autoclave at 116 °C for 30 min. To prepare solid medium, add 15.0 g of agar powder.

[0045] (6) Test media: ①NB medium: Add 10.0 g of peptone, 3.0 g of beef extract powder, and 5.0 g of sodium chloride to 1000 mL of distilled water, adjust the pH to 8.0, and sterilize at 121 ℃ for 20 min. ②TSB medium: Add 17.0 g of tryptone, 3.0 g of soybean peptone, 5.0 g of sodium chloride, 2.5 g of anhydrous dipotassium hydrogen phosphate, and 2.5 g of glucose to 1000 mL of distilled water, adjust the pH to 8.0, and sterilize at 121 ℃ for 20 min. ③YSP medium: Add 10.0 g of tryptone, 5.0 g of yeast powder, and 20.0 g of sucrose to 1000 mL of distilled water, adjust the pH to 8.0, and sterilize at 121 ℃ for 20 min. ④LB1 medium: Add 10.0 g tryptone, 5.0 g yeast extract, and 20.0 g sodium chloride to 1000 mL distilled water, adjust the pH to 8.0, and sterilize at 121 ℃ for 20 min. ⑤LB2 medium: Add 10.0 g acid-hydrolyzed casein peptone, 5.0 g yeast extract, and 20.0 g sodium chloride to 1000 mL distilled water, adjust the pH to 8.0, and sterilize at 121 ℃ for 20 min. ⑥LB3 medium: Add 10.0 g bacteriological peptone, 5.0 g yeast extract, and 20.0 g sodium chloride to 1000 mL distilled water, adjust the pH to 8.0, and sterilize at 121 ℃ for 20 min. ⑦LB4 medium: Add 10.0 g of soybean peptone, 5.0 g of yeast extract, and 20.0 g of sodium chloride to 1000 mL of distilled water, adjust the pH to 8.0, and sterilize at 121 ℃ for 20 min. ⑧MRS medium: Add 10.0 g of casein digest, 10.0 g of beef extract, 4.0 g of yeast extract, 2.0 g of triammonium citrate, 5.0 g of sodium acetate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, 2.0 g of dipotassium hydrogen phosphate, 20.0 g of glucose, and 1.0 g of Tween-80 to 1000 mL of distilled water, adjust the pH to 8.0, and sterilize at 121 ℃ for 20 min.

[0046] All reagents, including sucrose, glucose, and sodium chloride, were domestically produced analytical grade (AR). Peptone, yeast extract, and agar powder were biochemical reagents (BR). Physiological and biochemical tests were performed using the API ZYM kit and the API 20NE kit. The pH of all culture media was adjusted to 8.0.

[0047] 2. Main instruments and equipment

[0048] Incubator (SHP-080), Guangdong Huankai Biotechnology Co., Ltd.; Shaker (HZQ-X300C), Shanghai Yiheng Scientific Instruments Co., Ltd.; Clean bench (BSC-1304ⅡA2), Suzhou Antai Air Technology Co., Ltd.; Electronic vernier caliper (MNT-150T), Shanghai Minate Industrial Co., Ltd.; Electronic balance (BSAZZ4S), Sartorius Scientific Instruments (Beijing) Co., Ltd.; Ultraviolet spectrophotometer (X-8), Shanghai Yuanxi Instruments Co., Ltd.; Benchtop digital pH meter (ST3100), Ohaus Instruments (Changzhou) Co., Ltd.; PCR instrument (BIO-RAD T100), Bio-Rad Biomedical Products, Inc. (USA); High-speed centrifuge (Sigma 3-18KS), Sartorius GmbH (Germany); Chlorophyll meter (SPAD-502), Konica Minolta.

[0049] Example 1

[0050] 1.1 Isolation and preservation of salt-tolerant strains

[0051] Rhizosphere soil from healthy maize plants was collected from saline-alkali soil in Nansha District, Guangzhou. The soil was preserved on dry ice and brought back to the laboratory. 10.0 g of soil was added to an Erlenmeyer flask containing 300 mL of high-salt selection medium (autoclaved at 121 ℃ for 20 min and then cooled). The flask was then cultured at 30 ℃ and 180 rpm for 2 days. 100 μL of the cultured soil suspension was then pipetted into a centrifuge tube containing 900 μL of sterile water, and the mixture was diluted to a final volume of 10 μL. -2 10 -3 and 10 -4 Gradients were used, with 100 μL of soil dilution from each gradient spread onto high-salt screening medium (solid plates) (3 replicates per treatment). The plates were incubated at 30 ℃ and 180 rpm for 2 days. Single colonies were picked for isolation and purification. The purified strains were inoculated onto solid plates or slant tubes (LB basal medium) for storage at 4 ℃ (short-term storage) or into 25% glycerol tubes for storage at -80 ℃ (long-term storage). A salt-tolerant strain (tolerant to growth in 12% NaCl) was obtained through preliminary screening and named BWT1.2507.

[0052] 1.2 Morphological observation of strain BWT1.2507

[0053] Following the methods outlined in Bergey's Manual of Bacterial Identification, the isolated and purified strain BWT1.2507 was subjected to morphological observation, and its morphological characteristics, texture, color, transparency, and Gram staining were described.

[0054] The results show (e.g.) Figure 1 The strain BWT1.2507 grew well after 2 days of culture on LB solid medium. The cells were orange-yellow, viscous, opaque, and wrinkled, and were easy to culture. After Gram staining, the cells appeared purple under a microscope, indicating that it was a Gram-positive bacterium with spores and a rod-shaped structure.

[0055] 1.3 Physiological and Biochemical Tests

[0056] The enzyme production characteristics of strain BWT1.2507 were tested using the API ZYM kit, and routine physiological and biochemical tests of strain BWT1.2507 were performed using the API 20NE kit. Starch hydrolysis, VP test, MR test, and H2O2 enzyme assay were performed according to the guidelines in "Experimental Techniques in Microbiology" (edited by Qin Cuili, Beijing: Chemical Industry Press, 2023.3).

[0057] The results showed (as shown in Tables 1 and 2) that strain BWT1.2507 exhibited strong enzyme activities in alkaline phosphatase, β-galactosidase, leucine arylamidase, valine arylamidase, cysteine ​​arylamidase, α-chymotrypsin, urease, and arginine dihydrolase (Table 1). It also showed positive results for indole production, gelatin liquefaction, starch hydrolysis, and catalase (Table 2). Carbon sources such as D-mannose, D-mannitol, D-maltose, potassium gluconate, sucrose, adipic acid, malic acid, and phenylacetic acid were all utilized (Table 2).

[0058] Table 1 Enzyme activity test

[0059] Enzyme activity number Enzyme activation reagent result Enzyme activity number Enzyme activation reagent result 1 Comparison - 12 Acid phosphatase +++ 2 alkaline phosphatase +++++ 13 Phenol-AS-BI-phosphohydrolase + 3 butyrate esterase ++ 14 α-Galactosidase - 4 Octate esterase +++ 15 β-galactosidase +++++ 5 Tetradecanoate enzyme - 16 β-glucuronidase - 6 Leucinease ++++ 17 α-glucosidase - 7 Valine acylase ++++ 18 β-glucosidase + 8 Cystine arylamidinase ++++ 19 N-acetyl-glucosaminease - 9 trypsin + 20 α-Mannosidase - 10 α-chymotrypsin ++++ 21 α-Fucosidase - 11 Urease ++++ 22 Arginine decarboxylase ++++

[0060] Note: "-" indicates negative, "+" indicates positive, and the more "+" signs there are, the stronger the enzyme activity.

[0061] Table 2 Routine physiological and biochemical tests

[0062] Test number Routine test result Test number Routine test result 1 Nitrate reduction - 11 Potassium gluconate + 2 Indole production + 12 Decanoic acid - 3 Gelatin liquefaction + 13 adipic acid + 4 glucose acidification reaction - 14 malic acid + 5 Arabic sugar - 15 Citric acid - 6 D-mannose + 16 Phenylacetic acid + 7 D-Mannitol + 17 Starch hydrolysis + 8 N-acetylglucosamine - 18 Acetylmethylmethanol test - 9 D-maltose + 19 Methyl red test - 10 sucrose + 20 catalase test +

[0063] Note: "+" indicates positive; "-" indicates negative.

[0064] 1.4 Molecular biological identification

[0065] Strain BWT1.2507 was identified molecularly using 16S rDNA sequencing. DNA was extracted from strain BWT1.2507 using the HiPureBacterial DNA Kit. PCR amplification was performed using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.2) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.3). The PCR reaction mixture (25 μL) consisted of: 12.5 µL of 2×Taq Master Mix, 1 µL each of forward and reverse primers (10 µmol / L), 0.5 µL of DNA template, and 10 µL of ddH2O. PCR conditions were: 95 °C pre-denaturation for 5 min; 95 °C denaturation for 30 s, 60 °C annealing for 30 s, 72 °C extension for 2 min, 35 cycles; and a final extension at 72 °C for 5 min. After successful amplification by 1% agarose gel electrophoresis, the samples were sent to Suzhou Genewise Biotechnology Co., Ltd. for sequencing. The obtained gene sequences were fully assembled, and the gene sequence is shown in SEQ ID NO.1. BLAST alignment was performed in EzBioCloud (https: / / www.ezbiocloud.net / identify), and a phylogenetic tree of the strain's 16S rDNA was constructed using MEGA 12 software (e.g., Figure 2 ).

[0066] 16S rDNA gene sequencing and database comparison showed that strain BWT1.2507 is similar to *Sutcliffiella halmapala* DSM 8723. T With a similarity of 99.10%, and based on the above morphological observations, physiological and biochemical assays, and molecular biological identification results, strain BWT1.2507 was identified as *Sutcliffiella halmapala*. It was named *Sutcliffiella halmapala* BWT1.2507 and deposited on September 28, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC No: 67048.

[0067] 1.5 Determination of nitrogen fixation, organophosphorus solubility, IAA secretion, and siderophore production capacity.

[0068] Nitrogen fixation characteristics: The seed culture after liquid fermentation in LB medium was diluted to OD... 600 =1. Pipette 20 μL of bacterial culture into nitrogen-free Ashby's solid medium (cross-shaped five-point distribution), and incubate at 30 ℃ for 5 days. Observe the growth around the bacterial strain in the plate. (e.g.) Figure 3 )

[0069] Phosphorus solubility characteristics: BWT1.2507 strain was inoculated into Mongkina organic phosphorus solid medium and incubated in a 30℃ constant temperature incubator for 5 days. The clear zone around the strain in the plate was observed.

[0070] IAA secretion characteristics: Construction of an IAA standard curve (e.g.) Figure 4 Weigh 20 mg of indoleacetic acid standard, dissolve it in a small amount of ethanol, and then dilute to 100 mL with deionized water to obtain an indoleacetic acid standard stock solution with a mass concentration of 200 mg / L. Prepare a series of standard solutions with mass concentrations of 0, 10, 20, 40, 60, 80, and 100 mg / L using deionized water, and store them protected from light. (OD...) 600 =1 strain BWT1.2507 bacterial suspension was inoculated at 3% (w / v) into optimized liquid medium (LB4, LB4+Trp) with and without 0.2 g / L tryptophan, respectively. After incubation at 30 ℃ and 240 rpm for 3 days, an equal volume of Salkowski colorimetric solution was added and mixed thoroughly. The mixture was reacted in the dark for 1 h, and the OD was measured. 530 The concentration of IAA produced by the strain was determined by combining the value with the standard curve.

[0071] Siderophore production characteristics: (1) Plate observation method: The strain BWT1.2507 was inoculated into CAS solid medium and placed in a constant temperature incubator at 30 ℃ for 5 days. The transparent zone around the strain in the plate was observed. (2) Test tube test method: A single colony was picked and inoculated into LB4 liquid medium and placed in a constant temperature shaker at 30 ℃ and 240 rpm for 5 days. Then the bacterial suspension was centrifuged, and 3 mL of supernatant was pipetted into a test tube containing 3 mL of CAS detection solution. After thorough shaking and protection from light for 1 h, the absorbance value (As) was measured at a wavelength of 630 nm using a UV spectrophotometer. Double distilled water was used as a control to zero the sample. The absorbance value of the reaction between 3 mL of sterile LB4 liquid medium and 3 mL of CAS detection solution was used as a reference value (Ar). The siderophore production of strain BWT1.2507 after 5 days of fermentation was quantified, and the siderophore activity unit (su) was calculated. su=[(Ar-As) / Ar]×100%.

[0072] Strain BWT1.2507 was inoculated into nitrogen-free (Ashby's), organophosphorus, and siderophore (CAS) solid media and cultured for 5 days, respectively. The results showed that (e.g.) Figure 3 All of these strains possess the functions of nitrogen fixation, organic phosphorus dissolution, and iron production.

[0073] After inoculating strain BWT1.2507 into tryptophan-enriched medium (LB4 + 0.2 g / L Trp) and CAS liquid detection medium for 5 days of fermentation, the results showed that strain BWT1.2507 secreted IAA at a concentration of 9.37 mg / L. Figure 3 (Table 3) The siderophore activity (su) reached 70.36% ( Figure 3 (Table 4).

[0074] Table 3. IAA Concentration Determination

[0075] strain number <![CDATA[Control OD 530 Mean]]> <![CDATA[Processing OD 530 Mean]]> <![CDATA[OD 530 Difference IAA concentration (mg / L) BWT1.2507 0.148 0.353 0.205 9.37

[0076] Table 4 Ferrocarrier Capacity Test

[0077] strain number <![CDATA[Control OD 630 Mean (Ar)]]> <![CDATA[Detect OD 630 Mean (A S )]]> Average activity of siderogenic carriers (su) / % BWT1.2507 0.533 0.158 70.36

[0078] Note: OD 630 The value is represented by the UV spectrophotometer at 630 nm, and the iron-producing activity su = [(Ar-As) / Ar] × 100%.

[0079] 1.6 Salt and Alkali Resistance Test

[0080] Acid and alkali resistance test: Using LB medium as the basal medium, the pH was adjusted to 4, 5, 6, 7, 8, 9, 10, 11, and 12. LB solid and liquid media (99 mL of liquid media was added to each 250 mL Erlenmeyer flask) were prepared for the above treatments. The media were streaked onto solid plates at different pH treatments and incubated at 30 ℃ for 2 days. The growth of the strains on each plate was observed. 1 mL of seed culture of strain BWT1.2507 (dilution concentration OD) was inoculated into each of the liquid fermentation media at different pH treatments. 600 =1), the total volume of the culture medium was 100 mL, and the culture was placed in a constant temperature shaker at 30 ℃ and 180 rpm for 2 days. The OD of each treatment was measured using a UV spectrophotometer. 600 value.

[0081] Salt tolerance test: Using LB medium as the basal medium, the NaCl concentrations in the solid medium were set at 120 g / L (12%), 150 g / L (15%), 180 g / L (18%), 190 g / L (19%), 200 g / L (20%), 210 g / L (21%), and 240 g / L (24%), and the NaCl concentrations in the liquid medium were set at 0 g / L (0%), 10 g / L (1%), 20 g / L (2%), 30 g / L (3%), 60 g / L (6%), 90 g / L (9%), 120 g / L (12%), 150 g / L (15%), 180 g / L (18%), 190 g / L (19%), and 200 g / L (20%). Solid and liquid media for the above treatments were prepared (99 g / L was added to a 250 mL Erlenmeyer flask). The culture medium was streaked onto solid plates treated with different NaCl concentrations (mL liquid culture medium), and incubated at 30 ℃ for 2 days. The growth of the strains on each plate was observed. 1 mL of seed culture of strain BWT1.2507 (dilution concentration OD) was inoculated into each of the liquid fermentation media treated with different NaCl concentrations. 600 =1), the total volume of the culture medium was 100 mL, and the culture was placed in a constant temperature shaker at 30 ℃ and 180 rpm for 2 days. The OD of each treatment was measured using a UV spectrophotometer. 600 value.

[0082] Acid and alkali resistance tests were performed on strain BWT1.2507, and the results showed (e.g.) Figure 5 , Figure 6 It can grow in the pH range of 6-11 on LB solid medium and in the pH range of 4-11 on liquid fermentation medium, with the optimal fermentation pH being 8.

[0083] Salt tolerance tests were performed on strain BWT1.2507, and the results showed (e.g.) Figure 7 , Figure 8 It can tolerate a maximum NaCl concentration of 19% on LB solid medium and a maximum NaCl concentration of 18% on liquid fermentation medium. The optimal NaCl concentration for fermentation is 20 g / L (2%).

[0084] Example 2: Fermentation optimization and preparation of strain BWT1.2507

[0085] 2.1 Optimization of Fermentation Medium

[0086] NB, TSB, YSP, LB1, LB2, LB3, LB4, and MRS were selected as test media (pH adjusted to 8.0) for the optimization experiment of fermentation medium for strain BWT1.2507. The inoculum size was 1 mL of seed culture (dilution concentration OD). 600 =1), the total volume of the culture medium was 100 mL, and all were placed in a constant temperature shaker at 30 ℃ and 180 rpm for 2 days. Then, the OD of each treatment was measured using a UV spectrophotometer. 600 value.

[0087] The results show (e.g.) Figure 9 LB4 medium is best suited for liquid fermentation of strain BWT1.2507, which consists of soybean peptone, 10.0 g / L; yeast extract, 5.0 g / L; sodium chloride, 20.0 g / L; and a fermentation pH of 8.0.

[0088] 2.2 Fermentation time

[0089] Fermentation times were set at 0 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96 h, and 108 h. Based on the optimized culture medium (i.e., LB4 medium) described in section 2.1, 1 mL of the BWT1.2507 seed culture (dilution concentration OD) was added. 600 =1) The culture medium was inoculated into optimized culture media with different fermentation time treatments. The total volume of the culture medium was 100 mL. The media was placed in a constant temperature shaker at 30 ℃ and 180 rpm for fermentation. The cell concentration OD of the fermentation broth was measured at different fermentation time stages. 600 Value test.

[0090] The results show (e.g.) Figure 10 The bacterial cell concentration of strain BWT1.2507 reached a high level in 24 h and peaked in 60 h, which was determined to be the optimal fermentation time.

[0091] 2.3 Fermentation temperature

[0092] Fermentation temperatures were set at 24 ℃, 26 ℃, 28 ℃, 30 ℃, 32 ℃, 34 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, and 40 ℃. Based on the optimized culture medium described in section 2.2 above, 1 mL of the BWT1.2507 seed culture (dilution concentration OD) was added. 600 =1) Inoculate the culture medium into optimized culture media at different fermentation temperatures, with a total culture medium volume of 100 mL. Place the media on a constant-temperature shaker at 180 rpm for each fermentation temperature treatment. After 60 h of fermentation, measure the cell concentration (OD) of each treatment. 600 value).

[0093] The results show (e.g.) Figure 11 The strain BWT1.2507 showed good growth at temperatures ranging from 24 ℃ to 37 ℃, with the optimal fermentation temperature being 30 ℃.

[0094] 2.4 Fermentation inoculum size

[0095] Fermentation inoculum amounts of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 10%, and 15% (mass fraction) were set. Based on the optimized culture medium described in section 2.3 above, the seed culture of strain BWT1.2507 (dilution concentration OD) was added. 600 =1) Inoculate the optimized culture medium with the above inoculation amounts (1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 10 mL, and 15 mL), with a total culture medium volume of 100 mL for each treatment. Incubate at 30 ℃ and 180 rpm on a constant-temperature shaker for fermentation. After 60 h of fermentation, determine the cell concentration (OD) of each treatment. 600 value).

[0096] The results show (e.g.) Figure 12 The inoculum amount did not significantly affect the fermentation of strain BWT1.2507, and the final fermentation level was quite similar. An inoculum amount of 3% is recommended.

[0097] 2.5 Fermentation speed

[0098] Fermentation speeds were set at 120 rpm, 150 rpm, 180 rpm, 210 rpm, 240 rpm, 270 rpm, and 300 rpm. Based on the optimized culture medium described in section 2.4 above, 3 mL of the BWT1.2507 seed culture (dilution concentration OD) was added. 600 =1) The inoculum was injected into the optimized culture medium for different fermentation speed treatments (seed culture inoculum amount was 3%), with a total culture medium volume of 100 mL. The media were placed in constant temperature shakers at 30 ℃ for fermentation at different fermentation speeds. After 60 h of fermentation, the cell concentration (OD) of each treatment was measured. 600 value).

[0099] The results show (e.g.) Figure 13 Since strain BWT1.2507 is an aerobic bacterium, the cell concentration is higher at higher fermentation speeds. The optimal fermentation speed is 240 rpm, and the final optimized cell concentration OD is determined by this factor. 600 The value reached 2.93, and the effective viable bacteria count reached 3.76 × 10⁻⁶. 9 CFU / mL.

[0100] Example 3: Effects of strain BWT1.2507 on promoting growth and stress resistance in maize

[0101] In a greenhouse, a saline-alkali soil environment was simulated. Normal soil and sand were mixed at a 1:1 mass ratio (organic matter 13.92 g / kg, available nitrogen 51.07 mg / kg, available phosphorus 17.82 mg / kg, available potassium 58.34 mg / kg, pH 8.26, salinity 0.05%, electrical conductivity 168 μS / cm). 0%, 0.1% (low salinity), and 0.3% (medium salinity) NaCl were added and thoroughly mixed. The mixture was then placed in pots corresponding to the treatments, and corn seedlings were transplanted. After 7 days of growth, the pots were watered with water and a BWT1.2507 bacterial suspension (OD200). 600 =1) 50 mL of each of the following treatments were applied: 0%, 0.1%, and 0.3% NaCl treatments were applied to potted maize plants and then watered with water, designated as CK1, CK2, and CK3 respectively; 0%, 0.1%, and 0.3% NaCl treatments were applied to potted maize plants and then watered with BWT1.2507 bacterial suspension (OD100). 600 =1), set as T1, T2 and T3, and water with clean water and BWT1.2507 bacterial suspension once every 5 days (50 mL per pot). After 35 days, harvest the corn plants, measure the stem diameter and maximum leaf width with electronic vernier calipers, and measure the plant height, root length and maximum leaf length with a ruler. Weigh the fresh weight and dry weight of the aboveground parts and the fresh weight and dry weight of the underground parts with electronic balance (the dry weight was measured after drying in an oven). Use a chlorophyll meter to determine the chlorophyll content.

[0102] Experimental results show that:

[0103] In a greenhouse simulating a saline-alkali soil environment, using corn plants as potted crops, an experiment was conducted to promote the growth and salt tolerance of strain BWT1.2507. The results are as follows: Figure 14 As shown in Table 5, application of BWT1.2507 bacterial suspension to potted maize plants in medium-low saline-alkali soils (0.3% NaCl and 0.1% NaCl concentrations) significantly promoted plant growth. Compared to the control (CK), maize plants treated with BWT1.2507 bacterial suspension showed increases in stem diameter of 1.95%-8.56%, plant height of 7.95%-15.11%, root length of 8.30%-8.90%, aboveground fresh weight of 16.85%-29.96%, dry weight of 36.11%-50.00%, underground fresh weight of 21.82%-33.12%, dry weight of 26.67%-58.33%, maximum leaf width of 6.12%-18.40%, maximum leaf length of 10.65%-17.13%, and chlorophyll content of 6.98%-13.81%. This was particularly evident in moderately saline-alkali soils (0.3% NaCl and 0.1% NaCl concentrations). The growth-promoting effect is relatively obvious under NaCl concentration conditions.

[0104] Table 5. Agronomic traits of maize pot plants with strain BWT1.2507 under different NaCl concentrations.

[0105] Agronomic traits CK10%NaCl + water T10%NaCl + BWT 1.2507 CK20.1% NaCl + water T20.1%NaCl + BWT 1.2507 CK30.3% NaCl + water T30.3%NaCl++BWT1.2507 Stem diameter (mm) 3.84±0.43a 3.92±0.34a 4.10±0.38a 4.18±0.53a 3.97±0.50a 4.31±0.17a Plant height (cm) 21.37±1.67bc 22.43±2.34bc 23.03±4.44bc 24.86±4.15a 20.92±2.02c 24.08±0.60ab Root length (cm) 35.83±8.07a 39.11±4.25a 35.29±3.23a 38.43±3.89a 32.67±4.88a 35.38±4.86a Fresh weight of above-ground parts (g / plant) 2.72±0.03c 3.21±0.05b 2.79±0.06c 3.26±0.06b 2.67±0.06c 3.47±0.09a Fresh weight of underground parts (g / tree) 1.69±0.05c 1.86±0.05b 1.65±0.06cd 2.01±0.08a 1.54±0.05d 2.05±0.07a Dry weight of aboveground parts (g / tree) 0.35±0.03bc 0.43±0.03ab 0.36±0.04bc 0.49±0.05a 0.32±0.03c 0.48±0.06a Dry weight of underground parts (g / tree) 0.13±0.02d 0.17±0.02ab 0.15±0.02bc 0.19±0.02a 0.12±0.01d 0.19±0.03a Maximum leaf width (cm) 19.21±2.33b 21.69±2.43ab 20.26±2.34ab 21.50±2.69ab 19.02±3.18b 22.52±2.23a Maximum leaf length (cm) 35.65±2.97ab 38.99±3.30a 34.17±4.25ab 37.81±6.20a 30.82±5.43b 36.10±4.07ab Chlorophyll (μg / g) 20.85±2.13b 21.33±1.96ab 21.64±2.47ab 23.15±0.52ab 20.92±1.99b 23.81±1.40a

[0106] Note: For the same agronomic trait, a significant difference analysis was performed (same row). Different letters indicate significant differences (P < 0.05).

[0107] Example 4: Application of strain BWT1.2507 in reducing alkalinity and salinity in saline-alkali soils

[0108] The potted soil from each treatment in Example 3 was naturally air-dried, crushed, sieved, and a certain amount was dissolved in deionized water. The conductivity of each soil treatment was measured using a conductivity meter. The pH value of each soil treatment was measured using a benchtop digital pH meter.

[0109] Table 6 shows the changes in soil electrical conductivity and pH after treatment with BWT1.2507 bacterial suspension. Compared with the control soil, the electrical conductivity and pH of maize planting soil under moderate and low salinity and alkalinity conditions were significantly reduced (P<0.05). The overall electrical conductivity decreased by 8.75%-9.98%, and the overall pH decreased by 2.38%-7.37%, indicating that strain BWT1.2507 has a good ability to reduce alkali and salt in saline-alkali soil.

[0110] Table 6. Changes in soil electrical conductivity and pH after treatment with strain BWT1.2507 in maize.

[0111] Processing Number Soil electrical conductivity (μS / cm) Soil pH CK1 168±11.06e 8.26±0.19abc T1 153±7.21e 8.07±0.17bcd CK2 751±17.79c 8.33±0.18ab T2 665±13.65d 7.96±0.14cd CK3 1823±53.08a 8.41±0.16a T3 1641±48.34b 7.79±0.11e

[0112] The above detailed description is a specific description of the embodiments of the present invention. These embodiments are not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included in the patent scope of this case.

Claims

1. Promotes the growth of Halophilic Sutcliffe ( Sutcliffiella halmapala BWT1.2507, accession number GDMCCNo: 67048.

2. A microbial inoculant, characterized in that, The microbial agent contains the growth-promoting halophilic Sartcrylonitrile BWT1.2507 as described in claim 1, or its bacterial sludge, fermentation broth, or bacterial suspension.

3. The microbial agent according to claim 2, characterized in that, The bacterial sludge, fermentation broth, or bacterial suspension of *Satcliffe bacillus* BWT1.2507 that promotes growth is prepared by the following method: The fermentation medium consisted of 10.0 g / L soybean peptone, 5.0 g / L yeast extract, and 20.0 g / L NaCl. The fermentation conditions were pH 8.0, temperature 30 ℃, fermentation time 60 h, inoculum size 3%, and rotation speed 240 rpm. The fermentation broth was obtained by centrifugation to remove the supernatant and obtain bacterial sludge. The bacterial sludge was then resuspended to obtain a bacterial suspension.

4. The application of the growth-promoting halophilic Sartcrylonitrile BWT1.2507 as described in claim 1 or the microbial agent as described in claim 2 in promoting plant growth, wherein the plant is corn.

5. The application according to claim 4, characterized in that, This is an application to promote plant growth in saline-alkali soil, where the saline-alkali soil is saline-alkali soil with a salinity of 0.1-0.3%.

6. The application of the growth-promoting halophilic Sartcrylonitrile BWT1.2507 as described in claim 1 or the microbial agent as described in claim 2 in reducing alkali and salinity in saline-alkali soil, wherein the saline-alkali soil is saline-alkali soil with a salinity of 0.1-0.3%.

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