Saline-alkaline tolerant strain and culture method and application thereof

Abstract

The invention discloses a saline-alkaline tolerant strain as well as a culture method and application thereof, and relates to the technical field of agricultural biology. Comprising the following steps: S1, screening and culturing strains; s11, carrying out salt tolerance detection on primary strains stored in a laboratory, then carrying out first screening, and screening out strains with OD600 values greater than 1.0 as candidate strains; s12, inoculating the candidate strains into a culture medium, carrying out secondary screening, and screening out strains with the OD600 value greater than 1.0 as saline-alkaline tolerant strains; and S13, inoculating the saline-alkaline tolerant strain into the EPS-producing strain fermentation medium. The application of the saline-alkaline tolerant strain provided by the invention can obviously improve the yield and quality of crops; experiments show that after the HB-02 strain is inoculated, the prevention and treatment effect on wheat root rot reaches 80.2%, the yield of wheat in saline-alkali soil is increased to 703.13 g / m < 2 > from 516.61 g / m < 2 >, the yield is increased by 36.10%, and good disease prevention and growth promotion effects are shown.

Description

Technical Field

[0001] The invention relates to the field of agricultural biotechnology, and in particular to a salt-alkali tolerant bacterial strain and a cultivation method and application thereof. Background Art

[0002] At present, the treatment methods for saline-alkali land are mainly divided into three categories: physical improvement, chemical improvement and biological improvement. Among them, biological improvement is further divided into plant improvement and microbial improvement. Plant improvement mainly improves the physical and chemical properties of the soil by planting some salt-tolerant plants, such as Suaeda salsa, alfalfa, Tamarix, etc., to increase the diversity of soil microorganisms and reduce the salt content of saline-alkali soil. Microbial improvement refers to inoculating salt-alkali-tolerant microbial agents into the soil or plants to improve the plant rhizosphere environment and the physical and chemical properties of the soil, thereby achieving the purpose of improving saline-alkali land. Microbial improvement is pollution-free, environmentally friendly, and can improve the physical and chemical properties of the soil, etc., and has broad application prospects.

[0003] Plant rhizosphere growth-promoting bacteria refer to a type of beneficial bacteria that can colonize the rhizosphere of plants and promote plant growth. They can promote the growth of plants in saline-alkali land by fixing nitrogen, dissolving phosphorus, producing 1-aminocyclopropane-1-carb oxylate (ACC) deaminase, iron carriers and plant hormones. Some salt-alkali tolerant PGPR can help plants establish resistance mechanisms to salt-alkali stress, such as improving plant salt tolerance by changing the absorption and release of ions by plant roots and regulating the expression of specific plant genes, enhancing the survival ability of plants in saline-alkali land, and thus improving the growth state of plants in saline-alkali environments. In addition, inoculating PGPR in saline-alkali soil can promote the decomposition of soil organic matter, improve soil fertility, improve the physical and chemical properties of soil, and thus reduce soil salinization. Therefore, exploring PGPR with salt-alkali tolerant growth-promoting effects and making them into microbial agents will help improve saline-alkali land and increase agricultural productivity.

[0004] At present, there is little research at home and abroad on microorganisms that can improve saline-alkali soil and have the effect of preventing plant diseases and promoting growth.

[0005] After searching, the application scheme of Chinese patent application number CN202310681956.1 discloses a microbial agent produced by liquid fermentation of sugarcane filter mud. Fresh sugarcane filter mud is diluted to 13-17°BX with alcohol waste mash of sugar factory, and a composite enzyme is added for hydrolysis for 1-2h, and then auxiliary materials and the composite agent after expansion are added for liquid fermentation for 3-7 days. After the fermentation is completed, the solid and liquid are separated, and the liquid part is concentrated and a stabilizer is added as a liquid microbial agent. The solid part is dried at low temperature to make a solid microbial agent. The microbial agent in the above patent has the following shortcomings: Although it has certain functionality, it is still insufficient in promoting growth and production. Summary of the invention

[0006] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a salt-alkali tolerant strain and a cultivation method and application thereof.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A salt-alkali tolerant strain, Paenibacillus mucilaginosus, with a preservation number of CGMCC No.24636; the preservation center is: General Microbiology Center of China Culture Collection Administration; the preservation address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the preservation date is April 6, 2022.

[0009] A method for culturing a salt-alkali tolerant bacterial strain comprises the following steps:

[0010] S1: strain screening and cultivation;

[0011] S11: The 219 primary strains stored in the laboratory were tested for salt tolerance and then screened for OD 600 Strains with values ​​greater than 1.0 were selected as candidate strains;

[0012] S12: Inoculate the candidate strains into the culture medium for a second screening to select the OD 600 The strains with values ​​greater than 1.0 were regarded as salt- and alkali-tolerant strains;

[0013] S13: inoculating the strain into a fermentation medium of an EPS-producing strain, measuring the EPS yield, and selecting a strain with a yield of 0.3 g / g as a target strain;

[0014] S14: After screening, Bacillus subtilis YH-3 strain, Bacillus licheniformis ZL-357 strain and Paenibacillus colloids HB-02 strain were obtained. Among them, Paenibacillus colloids HB-02 strain had the strongest EPS secretion ability. Paenibacillus colloids HB-02 strain was selected for subsequent disease prevention and growth promotion effect detection;

[0015] S2: Expansion culture of strains;

[0016] S21: Inoculate the activated HB-02 strain into a fresh NB medium plate and culture at a constant temperature overnight to obtain a sufficient number of colonies;

[0017] S22: Pick up activated single colonies and inoculate them into NB liquid culture medium, shake and culture overnight to allow the strain to grow fully;

[0018] S23: Inoculate the culture into fresh NB medium at 10% inoculum and culture with shaking to obtain a large number of bacterial cells;

[0019] S24: centrifuging the fermentation broth, recovering the bacterial cells and diluting them with sterile water;

[0020] S3: Preparation of microbial inoculants;

[0021] S31: Stir evenly to ensure that the bacterial agent is evenly distributed;

[0022] S32: Packed under mechanical equipment to produce the final microbial agent product.

[0023] Preferably: in S11, the method for selecting the candidate strain specifically includes:

[0024] S11: The growth of the strains in NB medium containing 10% NaCl was tested and the OD 600 The strains with values ​​greater than 1.0 were selected as candidate strains.

[0025] Preferably: in said S12, the method for selecting salt-alkali tolerant strains specifically comprises:

[0026] S12: Test the alkali resistance of the selected salt-tolerant strains. Inoculate the candidate strains into NB medium at pH 10.0 and screen the OD 600 The strains with a value greater than 1.0 were regarded as salt- and alkali-tolerant strains.

[0027] Preferably, in S21, the temperature of the overnight constant temperature culture is controlled at 30°C, in S22, the temperature of the overnight shaking culture is controlled at 30°C, and in S23, the temperature of the shaking culture is controlled at 30°C and the time is controlled at 48h.

[0028] Preferably, in S24, the fermentation broth is centrifuged at 8000 rpm for 10 min, the cells are recovered and diluted with sterile water to a viable cell content of 1×10 8 cfu / mL or 1×10 10 cfu / mL.

[0029] Preferably, in S32, the carrier material is a mixture of one or more of peat and vermiculite.

[0030] Preferably: In S1, PDA medium is used for activation culture of the strain:

[0031] Select 200g potatoes, 20g glucose, 15-20g agar, and 1000mL water; peel the potatoes, cut into small pieces, add water and boil for 0.5h, filter with double-layer gauze, add sugar to the filtrate, and add water to make up to 1000mL.

[0032] Preferably: in S1, the fermentation medium for the EPS-producing strain is specifically:

[0033] Sucrose 20g, K2HPO4 0.2g, KH2PO4 0.5g, NaCl 100g, MgSO4·7H2O 0.5g, yeast extract 3g, add distilled water to make up to 1000mL.

[0034] Preferably: in said S1, the NA medium is specifically:

[0035] 5g beef extract, 10g peptone, 5g NaCl, 15-20g agar, 1000mL water, pH 7.0-7.2. NB medium is NA medium without agar.

[0036] The beneficial effects of the present invention are:

[0037] 1. The application of the salt-alkali tolerant strain provided by the present invention can significantly improve the yield and quality of crops; experiments show that after inoculation with the HB-02 strain, the control effect on wheat root rot reaches 80.2%, and the wheat yield in saline-alkali land increases from 516.61 g / m 2 Increased to 703.13g / m 2 , increasing production by 36.10%, showing a good growth-promoting effect.

[0038] 2. The microbial agent of the present invention can increase the air permeability of the soil and reduce the toxic effects of salt ions on crops by secreting extracellular polymers (EPS); it helps to improve the physical and chemical properties of the soil and enhance soil fertility and microbial diversity.

[0039] 3. Compared with the traditional chemical improvement method, the microbial improvement method adopted in the present invention is pollution-free and has good environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a growth curve diagram of each strain in NB culture medium in a salt-alkali tolerant strain culture method proposed in the present invention. DETAILED DESCRIPTION

[0041] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0042] Embodiment 1:

[0043] A Bacillus type composite microbial agent is prepared, tested and evaluated by the following scheme, which specifically includes:

[0044] 1 Materials

[0045] 1.1 Culture medium

[0046] PDA medium: 200g potato, 20g glucose, 15-20g agar, 1000mL water. Peel the potato, cut into small pieces, add water and boil for 0.5h, filter with double-layer gauze, take the filtrate, add sugar, and add water to make up to 1000mL.

[0047] Fermentation medium for EPS-producing strains: sucrose 20 g, K2HPO4 0.2 g, KH2PO4 0.5 g, NaCl 100 g, MgSO4·7H2O 0.5 g, yeast extract 3 g, add distilled water to prepare to 1000 mL.

[0048] NA medium: 5 g beef extract, 10 g peptone, 5 g NaCl, 15-20 g agar, 1000 mL water, pH 7.0-7.2.

[0049] NB medium is NA medium without agar.

[0050] 1.2 Pathogen culture

[0051] The wheat root rot pathogen Cochliobolus sativus was stored by the Agricultural Microbiology Laboratory of the College of Life Sciences of Hebei Agricultural University. Sterile PDA medium was melted and poured into a plate, and streptomycin sulfate was added to make the final concentration 40 μg / mL. The Cochliobolus sativus piece was inoculated in the center of the PDA plate and cultured at 26°C until the mycelium filled the plate for later use.

[0052] 1.3 Bacterial culture

[0053] A small amount of bacterial moss was picked from the NA slant with a sterilized bamboo stick and inoculated onto a fresh NA medium plate by streaking. The plate was cultured at 30°C overnight. An activated single colony was picked and inoculated into NB medium. The plate was cultured at 30°C overnight with shaking. The culture was inoculated into fresh NB medium at 10% inoculum and cultured at 30°C with shaking for 48 h. The fermentation broth was centrifuged at 8000 rpm for 10 min. The recovered bacteria were diluted with sterile water to prepare a plate with a viable bacterial content of about 1×10 8 cfu / mL or 1×10 10 cfu / mL of bacterial suspension, and detect the viable bacteria content using a bacterial counting plate.

[0054] 1.4 Soil

[0055] The test soil was collected from coastal saline-alkali soil in Nanpaihe Town, Huanghua City, Hebei Province in June 2023. The saline-alkali soil has a soluble salt content of 0.8%, a pH of 8.97, and an electrical conductivity of 3.59 mS / cm.

[0056] 1.5 Wheat seeds

[0057] The wheat seeds used in the experiment were Jimai 418, uncoated.

[0058] 2 Methods

[0059] 2.1 Preliminary screening of bacterial salt tolerance

[0060] The test strain was inoculated into NB medium and cultured overnight at 30°C. The concentration of the bacterial suspension was adjusted to 1×10 8 CFU / mL, transferred to NB medium containing 10% NaCl at a 1% inoculum, cultured at 30°C for 3 days, and measured OD by spectrophotometer 600 Each strain was replicated 3 times and the average value was taken. 600 The value indicates the growth and reproduction of bacteria.

[0061] 2.2 Initial screening of bacterial alkali resistance

[0062] The selected salt-tolerant strains were inoculated into NB medium and cultured overnight at 30°C. The concentration of the bacterial suspension was adjusted to 1×10 8 CFU / mL, transferred to NB medium with pH 10.0 at a 1% inoculum volume, cultured at 30°C for 3 days, and measured OD by spectrophotometer 600 Each strain was replicated 3 times and the average value was taken. 600 The value indicates the growth and reproduction of bacteria.

[0063] 2.3 Determination of the ability of bacteria to produce EPS

[0064] The salt-alkali tolerant strain obtained in the initial screening was inoculated into the EPS-producing strain fermentation medium, with an inoculation amount of 5%, and cultured at 30℃ for 3 days. The fermentation broth was centrifuged at 8000rpm for 10min, and the recovered bacteria were dried at 105℃ and weighed. Three volumes of 95% ethanol were added to the supernatant, and after standing at 4℃ overnight, the supernatant was centrifuged at 8000rpm for 10min, and the recovered precipitate was dried at 105℃ and weighed. The EPS yield per unit weight of the strain was obtained by the ratio of EPS weight to strain dry weight.

[0065] 2.4 Growth curve determination of salt-alkali tolerant strains

[0066] The selected salt-alkali tolerant strains were inoculated into NB medium and cultured at 30°C overnight. The concentration of the bacterial suspension was adjusted to 1×10 8 CFU / mL, the inoculum was transferred to NB medium with pH 10.0 and 10% NaCl at 1% inoculation volume, and cultured at 30°C with shaking. Samples were taken at 0, 4, 8, 12, 24, 48, and 72 h of culture, and the OD of the bacterial solution was measured using a spectrophotometer. 600 The corresponding culture medium without bacterial solution was used as blank control. Each treatment was repeated 3 times and the average value was taken. The culture time was used as the horizontal axis and the OD 600 The value is the vertical axis and the growth curve is drawn.

[0067] 2.5 Detection of the control effect of salt-alkali tolerant strains on wheat root rot

[0068] The pot experiment set up three treatments, namely the blank control group CK0, the pathogen group CK1, and the microbial agent group TR, and each treatment was set up with 5 replicates. Take a flower pot with a diameter of 12.9 cm and a height of 12.4 cm, and sow 12 wheat seeds evenly in each pot. The blank control group was irrigated with water, and no pathogens and salt-alkali tolerant strains were inoculated. The pathogen group was inoculated with wheat root rot bacteria, and the wheat seeds were placed on pathogen bacterial sheets (diameter 7 mm), and 1 wheat seed was placed on each bacterial sheet. The method of inoculating pathogens in the microbial agent group was the same as that of the pathogen group, and the concentration of live bacteria was about 1×10 8 20mL of salt-alkali tolerant bacterial suspension with a cfu / mL was cultured at room temperature, and the bacterial control effect was investigated 20 days after planting. The plant growth status was tested by plant, with a sampling volume of 20 plants, and then the average value was calculated. The severity grading standard of wheat root rot: Level 0: No disease; Level 1: The outer leaf sheath at the base of the wheat plant stem turns dark brown, and the length of the lesion is less than 1 / 2 of the leaf sheath; Level 2: The outer leaf sheath at the base of the wheat plant stem turns dark brown, and the length of the lesion is greater than 1 / 2 of the leaf sheath; Level 3: The inner leaf sheath at the base of the wheat plant stem turns dark brown, and the length of the lesion is less than 1 / 2 of the leaf sheath; Level 4: The inner leaf sheath at the base of the wheat plant stem turns dark brown, and the length of the lesion is greater than 1 / 2 of the leaf sheath; Level 5: Root rot and plant death.

[0069] Disease index = [∑ (number of diseased plants at each level × representative value) / total number of plants investigated × representative value of the most severe disease level] × 100. Control effect = [(disease index of control group - disease index of treatment group) / disease index of control group] × 100.

[0070] 2.6 Field test on growth-promoting effect of salt-alkali tolerant strains

[0071] A wheat planting plot was selected in the coastal saline-alkali land of Nanpaihe Town, Huanghua City, Hebei Province, with an area of ​​about 666.7m 2 , divided into 3 small pieces, each piece is about 222.2m 2 , respectively, as the control group, treatment group 1 and treatment group 2. The control group was not inoculated with bacterial agents; both treatment groups 1 and 2 were inoculated with live bacteria containing approximately 1.0×10 10 cfu / mL of the inoculant, the inoculation amount was 20mL / kg of seeds; in the green period, the two treatment groups were inoculated with salt-alkali tolerant bacteria by irrigation, the concentration of the inoculant was 1.0×10 8 cfu / mL, the inoculation dose is 1000mL / m 2. No fertilizer was applied to the control group and the treatment group, mechanical sowing was used, and the management method was the same as that of general fields. At the jointing stage of wheat, a 5-point sampling method was adopted, 10 plants were selected continuously at each point, 50 plants were selected for each treatment, and the same position in the middle of the uppermost fully extended leaf was selected. A portable chlorophyll content meter was used to measure the relative chlorophyll content (SPAD value) and calculate the average value. Measure the plant height and calculate the average value. The aboveground part of the wheat plant was brought back to the laboratory for drying and weighing, and the aboveground dry weight was determined. At the milky stage of wheat, a 5-point sampling method was adopted for sampling. Three consecutive rows were taken at each point, and 0.4m was taken for each row. The plant height and average number of grains per ear were investigated and counted (30 plants at each random sampling point, and 150 plants were sampled in each group). After the seeds were dried, the thousand-grain weight was weighed, and the yield per square meter was determined by single threshing and harvesting.

[0072] 2.7 Data Processing

[0073] Microsoft Excel 2010 and SPSS 18.0 software were used for statistical analysis of the data. One-way ANOVA and Duncan method were used for variance analysis and multiple comparisons (α=0.05). The data in the table are mean ± standard deviation.

[0074] 3 Results and analysis

[0075] 3.1 Screening of salt-alkali tolerant EPS-producing strains

[0076] The salt tolerance of 219 plant rhizosphere growth-promoting strains preserved in the laboratory was tested. The results showed that 12 strains had significant salt tolerance and could grow significantly in NB medium containing 10% NaCl. 600 The alkali resistance of the 12 strains was further tested. In the NB medium with pH 10.0, 5 strains showed significant alkali resistance. 600 Value>1.0, the growth condition is good. Since saline-alkali land is prone to soil compaction, resulting in poor soil permeability, the extracellular polymers (EPS) secreted by microorganisms can form soil aggregates with soil particles through van der Waals force and electrostatic attraction, increase soil permeability, and reduce the toxic effects of salt ions on crops. This study used the five strains screened to test the EPS secretion ability. The results showed that three of the strains had significant EPS secretion ability, and the yield reached 0.3g / g. These three strains were Bacillus subtilis YH-3, Bacillus licheniformis ZL-357 and Bacillus subtilis HB-02.

[0077] 3.2 Growth curve drawing of salt-alkali tolerant EPS-producing strains

[0078] The growth curves of the selected Bacillus subtilis YH-3 strain, Bacillus licheniformis ZL-357 strain and Paenibacillus colloids HB-02 strain with strong salt-alkali tolerance and significant EPS secretion ability were drawn in NB medium with pH 10.0 and 10% NaCl ( Figure 1 The results showed that the absorbance of the Bacillus HB-02 strain was the highest, and the OD 600 The absorbance value was 1.458, indicating that it had the highest content of viable bacteria; followed by Bacillus licheniformis ZL-357 strain, with an OD of 72 h. 600 The absorbance value was 1.193; the Bacillus subtilis YH-3 strain had the lowest OD600 absorbance value of 1.074 after 72 hours of cultivation. Therefore, the HB-02 strain was selected for disease prevention and growth promotion experiments in the later stage.

[0079] Among them, the preservation number of Bacillus mucilaginosus is CGMCC No.24636; the preservation center is: General Microbiology Center of China Culture Collection Administration; the preservation address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; the preservation date is April 6, 2022.

[0080] 3.3 Analysis of the disease prevention effect of Bacillus subtilis HB-02 strain

[0081] The results of the potted test on the control effect of the HB-02 strain of Bacillus subtilis on wheat root rot are shown in the following table. The root length, aboveground height and aboveground weight of the TR group with the bacterial agent were significantly different from those of the blank control group, and the root length, root weight, aboveground height and aboveground weight of the TR group with the pathogenic bacteria were significantly different from those of the CK1 group with the pathogenic bacteria. The control effect of the TR group on wheat root rot reached 80.2%, which was significant. This shows that the application of the fermentation liquid of the HB-02 strain can effectively prevent and control the occurrence of wheat root rot. Compared with the blank control group without the addition of pathogens, it has a significant disease prevention and growth promotion effect, and effectively prevents the occurrence of soil-borne diseases.

[0082] Control effect of HB-02 strain on wheat root rot

[0083]

[0084] Note: The data in the table are mean ± standard deviation. Different letters after the data in the same column indicate that the data of the same test index and different groups are significantly different at the P<0.05 level.

[0085] 3.4 Analysis of the growth-promoting effect of Paenibacillus subtilis HB-02 strain

[0086] According to the investigation of wheat growth effect, wheat seedlings began to emerge 8 days after sowing, and basically all seedlings emerged after 11 days. The results of the investigation of seedling emergence in the wheat field of the experimental plot showed that the seedlings in the group treated with the microbial agent were better, the leaves were dark green, and the wheat seedlings were thick and strong. After the wheat returned to green after winter, the wheat plants in the treatment group grew well and the leaves were green; the wheat in the control group grew weakly and the leaf tips were yellow. The investigation of the growth of wheat plants during the jointing stage showed that the wheat seedlings in the group treated with the microbial agent grew well, the plants were strong and dense, the leaves were dark green and thick, and the plant height was significantly higher than that of the control group. The plant height of treatment group 1 was 22.98% higher than that of the control group, and the plant height of treatment group 2 was 31.09% higher than that of the control group. The difference between the two microbial agent treatment groups and the control group was significant. The treatment group 2 was 6.59% higher than that of treatment group 1, but the difference between the two was not significant. The relative content of chlorophyll in treatment group 1 was 20.62% higher than that of the control group, and the treatment group 2 was 33.86% higher than that of the control group. The difference between the two microbial agent treatment groups and the control group was significant. Treatment group 2 was 10.98% higher than treatment group 1, and the difference between the two was not significant. The dry weight of treatment group 1 was 17.33% higher than that of the control group, and treatment group 2 was 28.26% higher than that of the control group. There were significant differences between the two bacterial agent treatment groups and the control group. Treatment group 2 was 9.32% higher than treatment group 1, and the difference between the two was not significant. In the detection of indicators such as plant height, relative chlorophyll content and dry weight, treatment group 2 was higher than treatment group 1, indicating that two inoculations of HB-02 strains are beneficial to promote the growth of wheat (as shown in the table below).

[0087] Detection of the growth-promoting effect of Paenibacillus subtilis HB-02 strain in wheat jointing stage

[0088]

[0089] Note: The data in the table are mean ± standard deviation. Different letters after the data in the same column indicate that the data of the same test index and different groups are significantly different at the P<0.05 level.

[0090] At the milky stage of wheat, the growth-promoting effect of HB-02 strain on wheat was analyzed by investigating the indicators such as plant height, number of spikes, number of grains per spike, 1000-grain weight and yield. Compared with the control group, the above indicators of wheat in treatment groups 1 and 2 increased, among which the increase in number of spikes, number of grains per spike and 1000-grain weight was not significant, while the plant height and yield increased significantly. The wheat yield in treatment group 1 increased from 516.61 g / m 2 Increased to 632.84g / m 2 , the yield increased by 22.50%, and the wheat yield of treatment 2 group increased from 516.61g / m 2 Increased to 703.13g / m 2, increased yield by 36.10%, with significant growth-promoting effect. Treatment 2 increased yield by 11.11% compared with treatment 1, with significant effect. In the detection of indicators such as plant height, number of grains per ear, thousand-grain weight and yield, treatment 2 was better than treatment 1, and the yield difference between the two groups was significant. In addition, there was no significant difference between the two groups in other indicators (as shown in the following table). It shows that inoculation with HB-02 strain can significantly increase wheat yield, and the effect of inoculation twice is better.

[0091] Detection of the growth-promoting effect of Paenibacillus subtilis HB-02 strain on wheat at the milky stage

[0092]

[0093] Note: The data in the table are mean ± standard deviation. Different letters after the data in the same column indicate that the data of the same test index and different groups are significantly different at the P<0.05 level.

[0094] 4 Conclusion

[0095] The HB-02 strain of Bacillus colloids, which is salt-tolerant and high in extracellular polymer production, was screened. The control effect on wheat root rot reached 80.2%, which can effectively prevent and control the occurrence of wheat root rot with significant results. At the jointing stage of wheat, the plant height of the group treated with the inoculant was 31.09% higher than that of the control group, the relative chlorophyll content was 33.86% higher than that of the control group, and the dry weight was 28.26% higher than that of the control group. The effect of two inoculations of the HB-02 strain on promoting wheat growth was more significant. At the milky stage of wheat, the wheat yield of the group treated with the inoculant increased from 516.61 g / m 2 Increased to 703.13g / m 2 , increased by 36.10%. Inoculation with HB-02 strain can significantly increase wheat yield, and the effect of two inoculations is better.

[0096] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A salt-alkali tolerant bacterial strain, characterized in that: It is Paenibacillus mucilaginosus, with the deposit number CGMCC No.24636; the deposit center is: General Microbiology Center of China Culture Collection Administration; the deposit address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the deposit date is April 6, 2022.

2. A method for cultivating a salt-alkali tolerant bacterial strain, characterized in that: The steps include: S1: strain screening and cultivation; S11: Conduct salt tolerance test on primary strains stored in the laboratory, and then conduct the first screening to screen out OD 600 Strains with values ​​greater than 1.0 were selected as candidate strains; S12: Inoculate the candidate strains into the culture medium for a second screening to select the OD 600 Strains with values ​​greater than 1.0 were considered salt- and alkali-tolerant strains; S13: The salt-alkali tolerant strain was inoculated into the fermentation medium of the EPS-producing strain, the EPS yield was measured, and the strain with a yield of 0.3 g / g was selected as the target strain; S14: After screening, Bacillus subtilis YH-3 strain, Bacillus licheniformis ZL-357 strain and Paenibacillus colloids HB-02 strain were obtained. Among them, Paenibacillus colloids HB-02 strain had the strongest EPS secretion ability. Paenibacillus colloids HB-02 strain was selected for subsequent disease prevention and growth promotion effect detection; S2: expansion culture of strains; S21: Inoculate the activated HB-02 strain into a fresh NB medium plate and culture at a constant temperature overnight to obtain a sufficient number of colonies; S22: Pick up activated single colonies and inoculate them into NB liquid culture medium, shake and culture overnight to allow the strain to grow fully; S23: Inoculate the culture into fresh NB medium at a 10% inoculum volume and culture with shaking to obtain a large number of bacterial cells.

3. The method for cultivating a salt-alkali tolerant bacterial strain according to claim 2, characterized in that: The following steps are also included: S24: centrifuging the fermentation broth, recovering the bacterial cells and diluting them with sterile water; S3: Preparation of microbial inoculants; S31: Stir evenly to ensure that the bacterial agent is evenly distributed; S32: Packed under mechanical equipment to produce the final microbial agent product.

4. The method for cultivating a salt-alkali tolerant bacterial strain according to claim 2, wherein: In S11, the method for selecting the candidate strain specifically includes: S11: The growth of the strains in NB medium containing 10% NaCl was tested and the OD 600 Strains with values ​​greater than 1.0 were selected as candidate strains; In S12, the method for selecting salt-alkali tolerant strains specifically includes: S12: Test the alkali resistance of the selected salt-tolerant strains. Inoculate the candidate strains into NB medium at pH 10.0 and screen the OD 600 The strains with a value greater than 1.0 were regarded as salt- and alkali-tolerant strains.

5. The method for cultivating a salt-alkali tolerant bacterial strain according to claim 2, characterized in that: In the S21, the temperature of the constant temperature culture overnight is controlled at 30°C, in the S22, the temperature of the shaking culture overnight is controlled at 30°C, and in the S23, the shaking culture temperature is controlled at 30°C, and the time is controlled at 48h; In S24, the fermentation broth was centrifuged at 8000 rpm for 10 min, the cells were recovered and diluted with sterile water to a viable cell content of 1×10 8 cfu / mL or 1×10 10 cfu / mL.

6. The method for cultivating a salt-alkali tolerant bacterial strain according to claim 2, characterized in that: In S32, the carrier material is selected from a mixture of one or more of peat and vermiculite.

7. The method for cultivating a salt-alkali tolerant bacterial strain according to claim 2, characterized in that: In S1, PDA medium is used for activation culture of the strain: Select 200g potatoes, 20g glucose, 15-20g agar, and 1000mL water; peel the potatoes, cut into small pieces, add water and boil for 0.5h, filter with double-layer gauze, add sugar to the filtrate, and add water to make up to 1000mL.

8. The method for cultivating a salt-alkali tolerant bacterial strain according to claim 2, characterized in that: In S1, the fermentation medium for the EPS-producing strain is specifically: Sucrose 20g, K2HPO4 0.2g, KH2PO4 0.5g, NaCl 100g, MgSO4·7H2O 0.5g, yeast extract 3g, add distilled water to make up to 1000mL.

9. The method for cultivating a salt-alkali tolerant bacterial strain according to claim 2, characterized in that: In the S1, the NA medium is specifically: 5g beef extract, 10g peptone, 5g NaCl, 15-20g agar, 1000mL water, pH 7.0-7.

2. NB medium is NA medium without agar.

10. An application of a salt-alkali tolerant bacterial strain, characterized in that: The salt-alkali tolerant strain according to claim 1 is used for soil improvement or crop cultivation.

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