Salt-tolerant bacillus XYK4-2 and application thereof

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses salt-tolerant bacillus XYK4-2 and application thereof. The salt-tolerant bacillus XYK4-2 is preserved in the China General Microbiological Culture Collection Center on May 28, 2024, the address is Institute of Microbiology, Chinese Academy of Sciences, No.3, Beichen West Road, Chaoyang District, Beijing, the preservation number is CGMCC No.30804, and the salt-tolerant bacillus XYK4-2 is classified and named as the salt-tolerant bacillus; the salt-tolerant bacillus XYK4-2 disclosed by the invention has the capabilities of dissolving phosphorus, fixing nitrogen, dissolving potassium, complexing iron and generating auxin, has the capability of generating the auxin (IAA) of 5.71 mg / L, has an effective growth promoting effect on crops such as tomatoes and hot peppers, is wide in application and has certain application potential.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and more specifically, to a salt-tolerant Bacillus XYK4-2 and its application. Background Art

[0002] As a major agricultural producer in the world, my country has long relied on chemical fertilizers and pesticides to increase crop yields and resist pests and diseases. However, this high-input, high-consumption production model has caused serious environmental problems, including the deterioration of the soil environment, soil compaction, eutrophication, decreased soil fertility, and weakened resistance to salt, alkali, pests and diseases, and heavy metals. These problems not only affect the sustainable development of agricultural production, but also have a profound negative impact on the ecological environment.

[0003] As farmers gain more knowledge about agricultural production, the traditional concept of using chemical fertilizers is constantly changing, and more and more farmers are beginning to realize the importance of biofertilizers in soil and crop management. Biofertilizers, especially microbial fertilizers, are gradually becoming an ideal choice to replace traditional chemical fertilizers due to their low energy consumption, high resource conversion rate and eco-friendliness. Compared with ordinary fertilizers, microbial fertilizers can effectively regulate plant growth, improve crop product quality, reduce environmental pollution, and ultimately achieve a low-input, high-return production and management model.

[0004] After application, microbial fertilizers can produce bioactive substances such as sugars, amino acids, nucleotides, etc., and combine with soil organic colloids and mineral embryos to form and optimize soil aggregate structure and improve soil physical and chemical properties. This process helps solve problems such as soil hardening, compaction, and water and fertilizer leakage caused by long-term application of chemical fertilizers. At the same time, microbial fertilizers can also enhance plant resistance, produce disease resistance, compete with pathogens for nutritional environment, and limit their growth and reproduction; in addition, microbial fertilizers can also induce plants to produce systemic resistance, and use their unique functions to decompose heavy metals and other harmful substances in the soil, thereby purifying and repairing damaged soil and reducing the impact of diseases.

[0005] Although microbial fertilizers have shown great potential in agricultural applications, the current research on growth-promoting bacteria still faces some technical problems. First of all, there are few existing growth-promoting bacteria resources. Although a variety of strains have been discovered, their application scope and functions still need to be further explored and expanded. The promotion and utilization of growth-promoting bacteria is relatively low, which limits its widespread application in agricultural production. Based on the above statements, the present application provides a salt-tolerant Bacillus XYK4-2 and its application. Summary of the invention

[0006] In order to solve the problems that the existing resources of growth-promoting bacteria are limited, the application scope and functions of the discovered growth-promoting bacteria still need to be further explored and expanded, and the promotion and utilization of growth-promoting bacteria are relatively low, the present application provides a salt-tolerant Bacillus XYK4-2 and its application.

[0007] In the first aspect, the present application provides a halotolerant Bacillus strain XYK4-2, which adopts the following technical solution:

[0008] A halotolerant Bacillus XYK4-2, wherein the halotolerant Bacillus XYK4-2 was deposited on May 28, 2024 at the General Microbiology Center of China Culture Collection Administration, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No.30804 and a classification name of Bacillus shalotolerans.

[0009] Preferably, the 16S rRNA nucleotide sequence of the halotolerant Bacillus sp. XYK4-2 is as shown in SEQ ID NO.1.

[0010] In a second aspect, the present application provides a method for isolating and culturing halotolerant Bacillus XYK4-2, using the following technical solution:

[0011] A method for isolating and culturing halotolerant Bacillus XYK4-2 comprises the following steps:

[0012] S1. Soil samples were collected from Xiayukou Mine in Shaanxi Province, and bacteria and actinomycetes were isolated by the stepwise dilution method. The isolated bacteria and actinomycetes were screened by using Montkin Organophosphate Medium, Montkin Inorganic Phosphorus Medium, Ashby Nitrogen Fixation Medium, PKO Potassium-Resolving Medium, and CAS Iron-Resolving Medium to obtain strain XYK4-2.

[0013] S2. Select a single colony of strain XYK4-2 and inoculate it into LB liquid culture medium, shake and culture it on a shaker at a temperature of 30-34°C and a rotation speed of 180-200 r / min for 16-24 h to obtain seed solution, inoculate the seed solution into LB liquid culture medium at a volume fraction of 3-5%, place it on a shaker at a temperature of 30-34°C and a rotation speed of 180-200 r / min for 16-24 h to obtain fermentation liquid.

[0014] Preferably, the culture temperature is 32° C., the culture speed is 190 r / min, and the culture time is 24 h.

[0015] Preferably, the seed solution inoculation amount is 4% of the LB liquid culture medium by volume.

[0016] In a third aspect, the present application provides an application of halotolerant Bacillus XYK4-2, using the following technical solution:

[0017] The use of the halotolerant Bacillus XYK4-2 described in the first aspect above in the preparation of a growth-promoting bacterial agent.

[0018] The use of the halotolerant Bacillus XYK4-2 described in the first aspect in products promoting the growth of tomatoes or peppers.

[0019] In a fourth aspect, the present application provides a growth-promoting bacterial agent, which adopts the following technical solution:

[0020] A growth-promoting bacterial agent comprises the halotolerant Bacillus XYK4-2 described in the first aspect above.

[0021] Preferably, the active ingredient of the growth-promoting bacterial agent is live bacteria, fermentation broth or bacterial suspension of halotolerant Bacillus subtilis XYK4-2.

[0022] Preferably, the growth-promoting bacteria agent is in the form of a wettable powder, a water dispersible agent, a water suspension or a dispersible oil suspension.

[0023] In summary, this application has the following beneficial effects:

[0024] The salt-tolerant Bacillus XYK4-2 disclosed in the present application has the ability to solubilize phosphate, fix nitrogen, solubilize potassium, chelate iron and produce auxin. Its ability to produce auxin (IAA) is 5.71 mg / L, and it has an effective growth-promoting effect on crops such as tomatoes and peppers. It is widely used and has certain application potential.

[0025] This application screened out plant rhizosphere growth-promoting bacteria strains with multiple growth-promoting functions from soil samples from Shaanxi mines. The obtained strains have strong resistance to harsh growth conditions, are widely adaptable to various environments, and have good activity in different types of soils. Therefore, the selected strains were combined with dominant horticultural crops such as tomatoes and peppers for potted plant experiments to further verify the growth-promoting effect of the strains, screen out strains with excellent performance, enrich the market strain resources, and provide data support for the construction of efficient and widely applicable growth-promoting agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a diagram showing the results of the in-dish screening of the strain XYK4-2 in Example 1 of the present application for phosphorus solubilization, nitrogen fixation, potassium solubilization, iron complexation and auxin production;

[0027] Figure 2 This is a diagram showing the growth-promoting effect of the strain XYK4-2 in Example 2 of the present application on potted tomatoes and peppers;

[0028] Figure 3The plate culture characteristics and scanning electron microscope morphology of strain XYK4-2 in Example 3 of the present application;

[0029] Figure 4 Constructing a phylogenetic tree for the 16S rDNA sequence of strain XYK4-2 in Example 3 of the present application;

[0030] Figure 5 This is a graph showing the growth of strain XYK4-2 in Example 4 of the present application at different pH and salt concentrations;

[0031] Figure 6 This is a growth curve diagram of strain XYK4-2 in Example 5 of the present application;

[0032] Figure 7 This is a diagram for optimizing the single-factor fermentation conditions of strain XYK4-2 in Example 5 of the present application;

[0033] Figure 8 This is a response surface diagram of the effects of the interaction of various factors on the OD value of strain XYK4-2 in Example 5 of the present application. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0035] The culture medium and reagents involved in the examples of this application are as follows:

[0036] (1) LB liquid medium: 10.0 g of peptone, 5.0 g of yeast powder, 2.0 g of sodium chloride, 1000 mL of distilled water, pH 7.0 (solid culture medium is made by adding 18.0 g of agar powder to the liquid culture medium).

[0037] (2) Montgina organophosphate medium (1 L): glucose 10.0 g, NaCl 0.3 g, KCl 0.3 g, FeSO4·7H2O0.03 g, (NH4)2SO4 0.5 g, MnSO4·7H2O 0.03 g, MgSO4·7H2O 0.3 g, lecithin 0.2 g, CaCO3 5 g, yeast extract 0.4 g, distilled water 1000 ml, pH 7.0-7.5.

[0038] (3) Montgina inorganic phosphate medium (1 L): glucose 10.0 g, (NH4)2SO4 0.5 g, NaCl 0.3 g, KCl 0.3 g, MgSO4·7H2O 0.3 g, FeSO4·7H2O 0.03 g, MnSO4·7H2O 0.03 g, Ca(PO4)2 2.5 g, yeast extract 0.4 g, distilled water 1000 mL, pH 7.0-7.5.

[0039] (4) Ashby nitrogen-fixing medium (1 L): KH2PO4 0.2 g, NaCl 0.2 g, MgSO4·7H2O 0.2 g, K2SO4·2H2O 0.2 g, CaCO3 5.0 g, glucose 5.0 g, mannitol 5.0 g, pH 7.0.

[0040] (5) PKO potassium-lysing medium (1 L): 10.0 g glucose, 5.0 g tricalcium phosphate, 5.0 g magnesium chloride, 0.1 g ammonium sulfate, 0.2 g potassium chloride, 0.25 g magnesium sulfate, 17.0 g agar, pH 7.0.

[0041] (6) CAS iron-dissolving medium (mg / L): 68.5% chrome azurol S (CAS), 72.9% hexadecyltrimethylammonium bromide (HDTMA), 2.645% ferric chloride hexahydrate, 295.25% sodium dihydrogen phosphate dihydrate, 1213.5% disodium hydrogen phosphate dodecahydrate, 125% ammonium chloride, 37.5% potassium dihydrogen phosphate, 62.5% sodium chloride, 9000% agar, pH 6.8±0.1. Weigh 10.87 g, heat to boiling and completely dissolve in 1L distilled water, sterilize at 120°C for 30 min and set aside.

[0042] (7) Salkowski colorimetric solution for IAA production determination: 15 mL 0.5 mol / L FeCl3, 300 mL concentrated sulfuric acid, 500 mL distilled water. Mix before use and store in the dark. Add 4 mL of Salkowski colorimetric solution to 1 mL of sample.

[0043] Example 1

[0044] Isolation of strain XYK4-2 and determination of its growth-promoting ability

[0045] Soil samples were collected from Xiayukou Mine in Shaanxi Province, and 41 bacterial strains and 24 actinomycete strains were isolated by serial dilution method. The isolated bacteria and actinomycete strains were screened by using Montana organic phosphorus medium, Montana inorganic phosphorus medium, Ashby nitrogen fixation medium, PKO potassium-solubilizing medium, and CAS iron-solubilizing medium.

[0046] The ability of the strain to demineralize elements was evaluated by measuring the transparent zone (D), colony diameter (d), and calculating D / d;

[0047] At the same time, the strain's ability to produce tryptophan (IAA) was determined: the isolated strain was inoculated in LB liquid culture medium containing 100 mg / L L-tryptophan, and cultured at 28°C and 180 r / min for 2 days. Then, 200 μL of the bacterial suspension was transferred to a 48-well plate, and 800 μL of Salkowski colorimetric solution was added. 200 μL of uninoculated LB liquid culture medium and 800 μL of Salkowski colorimetric solution were used as controls. After being placed at room temperature and away from light for 20 minutes, the color turned pink for positive, indicating that it can secrete IAA. The darker the color, the greater the secretion intensity.

[0048] Quantitative determination of the IAA production capacity of the strain: the strain was inoculated in LB liquid medium containing 500 mg / L L-tryptophan, and its OD was measured by L-tryptophan. 600 nm OD value, take 5 mL in a 10 mL centrifuge tube, centrifuge at 10000 r / min for 10 min, take 1 mL of the supernatant, add 4 mL of Salkowski colorimetric solution, place in the dark at room temperature for 20 min, and measure the OD value. 530 The OD value at nm was used to find the corresponding IAA content on the standard curve.

[0049] Table 1 The results of in-dish screening of strain XYK4-2 for phosphorus solubilization, nitrogen fixation, potassium solubilization and iron complexation

[0050] Organophosphate Inorganic phosphorus Nitrogen fixation Potassium Complex Iron Colony diameter d (mm) 5.14±0.51 9.51±1.91 5.50±0.39 7.26±0.37 1.97±0.32 Transparent ring diameter D (mm) 12.60±1.71 12.22±1.98 9.07±1.08 10.86±007 16.57±1.23 Transparent circle diameter / colony diameter (D / d) 2.45 1.28 1.65 1.5 8.41

[0051] Combination Figure 1 The results in Table 1 show that the strain XYK4-2 with the ability to solubilize phosphorus, fix nitrogen, solubilize potassium, chelate iron and produce galactin was isolated from the rhizosphere soil of the mine, and its IAA production capacity was 5.71 mg / L. Figure 1 A), inorganic phosphorus (such as Figure 1 B) Nitrogen fixation (such as Figure 1 C), potassium solution (such as Figure 1 D) Complex iron (such as Figure 1 E) and IAA production (e.g. Figure 1 F).

[0052] Example 2

[0053] Detection of the growth-promoting ability of strain XYK4-2 on tomato and pepper

[0054] (1) Preparation of bacterial suspension: XYK4-2 was inoculated into LB liquid medium and cultured on a shaker at 28°C and 180 r / min for 48 h. After the strain was fully grown, the OD of the suspension of each strain was measured using a spectrophotometer. 600 and adjust OD with sterile water 600 =1.0.

[0055] (2) Potted treatment: Select tomato and pepper varieties with excellent performance and sow them in seedling pots mixed with fine sand and vermiculite in a ratio of 1:2. After the seeds break through the soil and germinate, irrigate each pot with 50 mL of bacterial solution. The control group is irrigated with an equal amount of LB liquid culture medium. The second irrigation is carried out 14 days after the first irrigation. The plants are harvested after 55 days of growth.

[0056] (3) Determination of indicators: After the plants are harvested, the plant height, stem diameter and root length are measured, and the plants are washed, rinsed and naturally dried. The fresh weight is measured, and the plants are then placed in bags and dried in an oven at 105°C for 10 min. They are then dried at 60°C until the weight no longer changes, and the dry weight of the plants is measured.

[0057] Table 2 Effects of XYK4-2 treatment on the growth of potted tomatoes and peppers

[0058] deal with Plant height(cm) Stem diameter (cm) Root length (cm) Fresh weight(g) Dry weight (g) Number of blades tomato CK <![CDATA[25.00±4.36 b ]]> <![CDATA[4.83±0.43 a ]]> <![CDATA[11.60±3.65 b ]]> <![CDATA[17.01±4.49 b ]]> <![CDATA[1.57±0.82 b ]]> XYK4-2 <![CDATA[31.20±6.20 a ]]> <![CDATA[5.57±0.86 a ]]> <![CDATA[17.80±1.48 a ]]> <![CDATA[25.75±4.29 a ]]> <![CDATA[2.95±0.68 a ]]> chili CK <![CDATA[21.3±1.4 a ]]> <![CDATA[3.47±0.24 a ]]> <![CDATA[10.31±1.90 b ]]> <![CDATA[6.46±1.61 b ]]> <![CDATA[0.71±0.22 b ]]> <![CDATA[12.33±2.25 b ]]> XYK4-2 <![CDATA[21.87+2.16 a ]]> <![CDATA[3.46±0.17 a ]]> <![CDATA[14.48±1.02 a ]]> <![CDATA[9.63±1.16 a ]]> <![CDATA[0.95±0.05 a ]]> <![CDATA[18.5±3.21 a ]]>

[0059] Note: Different letters indicate significant differences (p < 0.05)

[0060] Combination Figure 2 The results in Table 2 show that strain XYK4-2 can significantly improve the growth of tomatoes and peppers. The plant height, stem diameter, root length, fresh weight and dry weight of tomatoes increased by 21%, 15%, 53%, 51% and 88% respectively compared with the control group, and the root growth was significantly higher than that of the control group. Although the plant height and stem diameter of peppers treated with strain XYK4-2 were not significantly different from those of the control group, the number of leaves, root length, fresh weight and dry weight increased by 50%, 40.4%, 49.1% and 34.0% respectively compared with the control group. This shows that strain XYK4-2 has a significant growth-promoting effect on the growth of tomatoes and peppers. Among them, the tomato control group (such as Figure 2 A and Figure 2 C), tomato strain XYK4-2 treated group (e.g. Figure 2 B and Figure 2 D); pepper control group (such as Figure 2 E and Figure 2 G), pepper strain XYK4-2 treatment group (such as Figure 2 F and Figure 2 H).

[0061] Example 3

[0062] Identification of strain XYK4-2

[0063] (1) Morphological identification

[0064] The purified strain XYK4-2 was inoculated into LB solid medium, and the morphological characteristics of the colonies in the dish were observed. The colonies were picked and observed under a scanning electron microscope. The strain XYK4-2 appeared as ivory white to yellowish white colonies on the LB solid medium. The colony surface was flat and moist, and the colony edge was smooth and neat, which was a typical bacterial colony characteristic. The bacteria were rod-shaped and rod-shaped under a scanning electron microscope, with a length of 1.3~2.4 um and a width of 0.4~0.6 um. Figure 3 As shown; Among them, the back colony characteristics of strain XYK4-2 on LB solid medium (such as Figure 3 A), positive colony characteristics of strain XYK4-2 on LB solid medium (such as Figure 3 B); SEM observation of bacterial morphology of strain XYK4-2 (e.g. Figure 3 C).

[0065] (2) Molecular identification

[0066] The genomic DNA of strain XYK4-2 was extracted and 16S rRNA was amplified by PCR. The sequencing results of the amplified products were submitted to NCBI, and similarity analysis was performed with the known 16S rRNA gene sequences in Gen Bank by BLAST to find similar species; the corresponding model strains were downloaded from the GGDC website (http: / / ggdc.dsmz.de / .), and the evolutionary tree was constructed using the neighbor method of MEGA11.

[0067] The 16S rRNA nucleotide sequence of halotolerant Bacillus XYK4-2 is shown in SEQ ID NO.1:

[0068]

[0069] Combination Figure 4 The results showed that strain XYK4-2 was clustered with Bacillus halotoleran DSM8802 (MN483266) with a confidence rate of 96%. Combined with morphological characteristics, XYK4-2 was determined to be Bacillus halotoleran.

[0070] This application will obtain the halotolerant Bacillus XYK4-2 deposited on May 28, 2024 at the General Microbiology Center of the China Culture Collection Administration, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 30804 and the classification name Bacillus shalotolerans.

[0071] Example 4

[0072] Detection of acid, alkali and salt tolerance of strain XYK4-2

[0073] Prepare LB culture medium with different initial pH (1, 3, 5, 7, 9, 11, 13) and NaCl concentration (0.5%, 1%, 2%, 5%, 10%, 12%, 15%) to culture the strain, and measure the OD of the culture medium after 24 hours of cultivation. 600 The growth status of strain XYK4-2 in different pH and salt concentrations was observed.

[0074] Combination Figure 5 The results show that: the acid and alkali resistance test results (such as Figure 5 A), strain XYK4-2 basically cannot grow at pH 1 and pH 13, starts to grow at pH 3, grows rapidly at pH 5-9, and grows a little at pH 11; the results of salt tolerance test (such as Figure 5 B) Strain XYK4-2 can grow rapidly when the NaCl concentration is <10%, and strain XYK4-2 almost stops growing when the NaCl concentration is 10%-15%.

[0075] Example 5

[0076] Optimization of Fermentation Process of Strain XYK4-2

[0077] The present application optimizes the fermentation temperature, pH value and inoculation amount of strain XYK4-2 to obtain the optimal fermentation parameters.

[0078] (1) Growth curve determination of strain XYK4-2

[0079] A single colony of strain XYK4-2 was picked and inoculated into 100 mL of LB liquid medium, and cultured on a shaker at 28°C and 180 r / min for 24 h to obtain seed liquid. The seed liquid was inoculated into the LB liquid medium at a volume fraction of 1%, and the LB liquid medium without seed liquid was used as a control. Three replicates were set for each group. The LB liquid medium was placed on a shaker at 28°C and 180 r / min for culture. Samples were taken every 8 h and the OD of the fermentation liquid within 48 h was measured by UV spectrophotometry. 600 Draw the growth curve of the strain.

[0080] Combination Figure 6 The results showed that strain XYK4-2 multiplied rapidly within 16 hours of inoculation, grew slowly in the following 16-24 hours, and basically reached a stable growth period after 24 hours. Therefore, 24 hours was regarded as the optimal growth period of strain XYK4-2.

[0081] (2) Single factor test method to screen fermentation conditions, the results are as follows Figure 7 As shown, A is temperature, B is pH, C is inoculation amount, D is rotation speed, and E is sample addition amount.

[0082] Screening of optimal fermentation temperature:

[0083] Select 28℃, 30℃, 32℃, 34℃, and 36℃, and inoculate the seed liquid into LB liquid medium at a volume fraction of 1%, and perform shaking culture under the same conditions. Set up 3 replicates for each treatment, and measure the growth curve OD 600 The OD value at the time point with the maximum value.

[0084] Combination Figure 7 A The results showed that the bacterial concentration of strain XYK4-2 reached the highest when the growth temperature was 32℃. 32℃ was selected as the optimal fermentation temperature of strain XYK4-2, and temperatures of 30, 32, and 34℃ were selected for response surface optimization experiments.

[0085] Screening of optimal fermentation pH:

[0086] H3PO4 and NaOH were used to prepare LB liquid culture medium with different pH values ​​of 6, 7, 8, 9, and 10, and shaking culture was carried out under the same conditions. Each treatment was repeated 3 times, and the OD under the same time conditions was measured. 600 value.

[0087] Combination Figure 7 Results B showed that the effective viable bacteria value of strain XYK4-2 reached a peak at pH = 6, and then gradually decreased. pH = 6 was selected as the optimal fermentation pH, and pH = 5, pH = 6, and pH = 7 were selected for response surface optimization experiments.

[0088] Screening of optimal fermentation inoculum amount:

[0089] Select 1%, 2%, 3%, 4%, 5%, and 5 inoculum sizes, inoculate into sterilized LB liquid medium, and culture on a shaker under the same conditions. Set up 3 replicates for each treatment, and measure the OD under the same time conditions. 600 value, and screen out the optimal culture inoculum size.

[0090] Combination Figure 7 C The results showed that the OD of the fermentation broth of strain XYK4-2 600 The value gradually increased with the increase of inoculum size and reached a peak value at 4%.4% inoculum size was selected as the optimal fermentation inoculum size, and 3%, 4% and 5% inoculum sizes were selected for response surface optimization experiments.

[0091] Screening of optimal fermentation speed:

[0092] Select 120 r / min, 140 r / min, 160 r / min, 180 r / min, 200 r / min, and 5 other speeds, inoculate the seed solution with a volume fraction of 1% in LB liquid medium, and shake culture under the same conditions. Set up 3 replicates for each treatment, and measure the OD under the same time conditions. 600 value to determine the optimal culture speed.

[0093] Combination Figure 7 D The results show that the OD of strain XYK4-2 in 180 r / min and 200 r / min fermentation broth 600 There is little difference between the two, and both can be used as the optimal fermentation speed for the experiment.

[0094] Screening of optimal liquid volume:

[0095] Select 60mL, 80mL, 100mL, 120mL, and 140mL of 5 liquid volumes, inoculate the seed liquid with a volume fraction of 1% into LB liquid medium, and shake culture under the same conditions. Set up 3 replicates for each treatment, and measure the OD under the same time conditions. 600 value to determine the optimal culture medium volume.

[0096] Combination Figure 7 E The results showed that when the fermentation volume of strain XYK4-2 was 120 mL, the OD 600 The highest value.

[0097] Example 6

[0098] Optimization of fermentation conditions by response surface methodology

[0099] According to the results of the single factor experiment, temperature (A), pH (B), and inoculum size (C) were selected as independent variables, and OD 600 The Box-Behnken central composite test was used to design the response surface optimization of the three parameters of the fermentation conditions. Figure 7 The results in the regression equation were fitted to obtain OD value = 1.38-0.0451A+0.0172B+0.0241C-0.0032AB+0.0579AC-0.0231BC-0.0295A 2 -0.0463B 2 -0.0475C 2 ,Then the variance of the model was analyzed, and the results of variance analysis are shown in Table 3.

[0100] As shown in Table 3, the P value of the constructed model is <0.05, and the model is significantly different; the P value of the lack of fit term is 0.8254>0.05, indicating that the lack of fit term is not significant, the model is not lack of fit, and the choice is reasonable. 2 =0.8782, indicating that the model can explain 87.87% of the response value changes, the adjusted determination coefficient R2Adj=0.724, and the model fits the experiment generally. 2 , C 2 The response surface model significantly reflected the OD 600 The relationship between the value and the inoculation amount, temperature, and pH, the F value judgment, and the three factors on OD 600 The order of influence of the values ​​is: temperature > inoculum size > pH value.

[0101] Table 3 Results of variance analysis of the regression equation of response surface experiment for strain XYK4-2

[0102] Variance source Sum of squares Freedom Mean square F value P value Significance Model 0.0636 9 0.0071 5.66 0.0162 * A-Temperature 0.0163 1 0.0163 13.05 0.0086 ** B-pH 0.0024 1 0.0024 1.90 0.2106 C-Inoculum size 0.0047 1 0.0047 3.74 0.0944 AB 0.0000 1 0.0000 0.0318 0.8635 AC 0.0134 1 0.0134 10.76 0.0135 * BC 0.0021 1 0.0021 1.71 0.2327 A² 0.0037 1 0.0037 2.94 0.1300 B² 0.0090 1 0.0090 7.24 0.0310 * C² 0.0095 1 0.0095 7.61 0.0281 * Residual 0.0087 7 0.0012 Lack of Fit 0.0016 3 0.0005 0.2992 0.8254 Pure Error 0.0071 4 0.0018 sum 0.0723 Coefficient of determination <![CDATA[R 2 =0.8782]]> <![CDATA[R 2 Adj=0.724]]>

[0103] Note: Different letters indicate significant differences (*=p<0.05, **=p<0.01)

[0104] Combination Figure 8 The results in Table 3 show that the interaction between temperature and pH is the strongest, and the interaction between inoculation amount and temperature is the weakest. The regression model was analyzed using Design Expert13 software, and the optimal fermentation conditions were determined to be inoculation amount 3.933%, pH = 6.881, and temperature 34.00℃. Under these conditions, the predicted OD 600The value of OD is 1.337. Considering the feasibility and practicality of the actual experiment, the optimal fermentation parameters were finally determined to be 4% inoculation, pH = 7, and temperature 34°C. Under these conditions, OD 600 The value is 1.391, which is not much different from the model predicted value of 1.3249.

[0105] The salt-tolerant Bacillus XYK4-2 disclosed in the present application has the ability to solubilize phosphate, fix nitrogen, solubilize potassium, chelate iron and produce auxin. Its ability to produce auxin (IAA) is 5.71 mg / L, and it has an effective growth-promoting effect on crops such as tomatoes and peppers. It is widely used and has certain application potential.

[0106] This specific embodiment is only an explanation of the present application, and it is not a limitation of the present application. After reading this specification, those skilled in the art can make non-creative modifications to the present embodiment as needed, but as long as it is within the scope of the claims of the present application, it is protected by the patent law.

Claims

1. A salt-tolerant Bacillus strain XYK4-2, characterized in that: The salt-tolerant Bacillus XYK4-2 was deposited on May 28, 2024 at the General Microbiological Center of the China Culture Collection Administration, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 30804 and a classification name of salt-tolerant Bacillus ( Bacillus halotolerans ).

2. The halotolerant Bacillus XYK4-2 according to claim 1, characterized in that The 16S rRNA nucleotide sequence of the halotolerant Bacillus sp. XYK4-2 is shown in SEQ ID NO.

1.

3. Use of the halotolerant Bacillus XYK4-2 according to claim 1 or 2 in the preparation of a growth-promoting bacterial agent.

4. Use of the halotolerant Bacillus XYK4-2 according to claim 1 or 2 in products for promoting the growth of tomatoes or peppers.

5. A growth-promoting bacterial agent, characterized in that: It includes the halotolerant Bacillus XYK4-2 according to claim 1 or 2.

6. The growth-promoting bacterial agent according to claim 5, characterized in that The active ingredient of the growth-promoting bacterial agent is live bacteria, fermentation liquid or bacterial suspension of halotolerant Bacillus subtilis XYK4-2.

7. The growth-promoting bacterial agent according to claim 5, characterized in that The growth-promoting bacteria agent is in the form of a wettable powder, a water dispersant, a water suspension or a dispersible oil suspension.

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