Bacillus Velez C5, fermentation liquid and its application

Abstract

The invention discloses a salt-tolerant growth-promoting Bacillus Velez C5, a fermented liquid and an application thereof. The Bacillus Velez C5 has a deposit number of CGMCC No 27764. A microbial agent and a microbial fertilizer are also disclosed. The Bacillus Velez C5 of the invention has a significant effect on the growth of plants tolerant to salt-alkali land; the Bacillus Velez C5 of the invention can be used as an active ingredient of microbial agents, microbial fertilizers, etc., to promote plant growth and alleviate the impact of saline-alkali land on plant growth. The Bacillus Velez C5 of the invention is a plant endophyte, and its application in plants is environmentally friendly and effective, and its preparation method is simple, which is suitable for popularization and production application, and overcomes the problems of unstable effect, single function, limited resources, etc. of the strains used in the research and development of microbial fertilizers.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a salt-tolerant growth-promoting Velez Bacillus C5, a bacterial fermentation liquid and applications thereof. Background Art

[0002] At present, the cumulative coverage area of ​​saline-alkali land in the world has reached 950 million hm2. 2 , accounting for about 10% of the world's average surface area, and is still increasing at a rate of 1.25 million hm2 per year. 2 The speed of soil salinization is increasing, so soil salinization has become one of the most serious environmental problems in the world. The distribution of wheat and corn, the main crops grown in my country, overlaps with these saline-alkali areas.

[0003] Excessive soil salinity is an important abiotic stress that limits the growth and yield of crops. Nearly 20% of irrigated soils worldwide are affected by salinity. Yield losses caused by soil salinization account for about 70% of yield losses in crops such as rice, corn and wheat. The improvement and utilization of salinized soil is an urgent problem to be solved in agricultural production.

[0004] As my country's agriculture moves towards green and high-quality development, the strategies of quality-oriented agriculture, green-oriented agriculture and brand-oriented agriculture have gradually become the main theme of my country's agricultural development. As a new green input and a biological product with priority support for development, microbial fertilizers and their industries have been able to develop and grow rapidly. Microbial fertilizers have the advantages of salt-adapted planting, ecological priority, combination of use and maintenance, and quality and efficiency improvement. They are a unique way to scientifically and efficiently utilize salinized land that can be replicated and promoted. Microbial agents are an emerging biological improvement method that has obvious advantages of safety, efficiency and environmental protection compared with traditional methods. Microbial agents directly improve the poor growth environment of the rhizosphere of plants in saline-alkali land by exerting the multiple functions of salt-tolerant soil microorganisms, effectively alleviate the adverse effects of soil salinization stress on crop growth, and greatly improve the fertility of saline-alkali salinized soil. Among them, microbial fertilizer improvement, as an emerging biological improvement measure, has great potential due to its advantages of environmental protection and high ecological benefits. However, the strains currently used in the research and development of microbial fertilizers have problems such as unstable effects, single functions, and limited resources. It is of great significance and application value to screen out efficient and stable multifunctional strains and develop a salt-tolerant and growth-promoting microbial fertilizer.

[0005] However, there are many problems in the current development of microbial fertilizers (microbial agents). The core of the development of microbial fertilizers is excellent growth-promoting or antagonistic strains. The lack of core strains is the main problem. The functionality of the strains is relatively simple, and the stability of the strains is poor. The mechanism of action between the strains and crops is also unclear, etc.

[0006] Therefore, screening new and excellent salt-tolerant and growth-promoting strains is an urgent problem to be solved, and the existing technology needs to be further developed. Summary of the invention

[0007] The present invention aims to solve the above problems and provides a salt-tolerant growth-promoting plant endophyte Bacillus Velez C5, a bacterial fermentation liquid and an application thereof.

[0008] Endophytic growth-promoting bacteria can establish a harmonious symbiotic relationship with plants. They refer to non-pathogenic microorganisms that exist in plants during their life cycle. Endophytes do not have adverse effects on the host and play an important role in plant disease control, secondary metabolite synthesis, plant growth regulation and stress resistance. Endophytes have a long-term symbiotic relationship with host plants. On the one hand, endophytes promote plant growth and improve plant stress resistance and disease resistance through a series of measures such as absorbing water and nutrients, inducing the production of hormones, iron carriers and antibacterial secondary metabolites, regulating proline content, and increasing the activity of antioxidant enzymes; on the other hand, plants affect the development process and diversity of endophytic bacteria and fungi through lignification and change the metabolic function of endophytes, accelerating the colonization of endophytes in host plants.

[0009] Specifically, the technical solution provided by the present invention is as follows:

[0010] In a first aspect, the present invention provides a strain of Bacillus Velez, which is Bacillus Velez C5, and its deposit number is CGMCC No 27764. Bacillus Velez C5 was deposited on July 3, 2023 at: General Microbiological Center of China National Committee for the Preservation of Microorganisms, with a deposit number of CGMCC No 27764, and the address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the depositing unit is Qingdao Agricultural University.

[0011] In a second aspect, the present invention further provides a bacterial fermentation liquid prepared using the Bacillus Velez subtilis C5 described in the first aspect.

[0012] In a third aspect, the present invention further provides an active extract prepared using the Bacillus Velezii C5 described in the first aspect.

[0013] In a fourth aspect, the present invention further provides a fermentation medium for culturing the Bacillus Velez subtilis as claimed in claim 1, comprising the following components: 10 g / L NaCl, 5 g / L peptone, and 10 g / L yeast extract.

[0014] In a fifth aspect, the present invention further provides a method for culturing the Bacillus Velezii as described in the first aspect, wherein the seed solution of strain C5 is inoculated into 20 mL LB medium at a 5% inoculation rate and cultured at 28° C. and 160 r / min for 20 h.

[0015] In a sixth aspect, the present invention provides a microbial agent, which includes the Bacillus Velez C5 described in the first aspect, the bacterial fermentation broth described in the second aspect, and / or the active extract described in the third aspect.

[0016] Furthermore, the microbial agent also includes other bacterial species that have complementary or synergistic effects with Bacillus Velez C5.

[0017] In a seventh aspect, the present invention further provides a microbial fertilizer, comprising the Bacillus Velez C5 described in the first aspect, the bacterial fermentation broth described in the second aspect, and / or the active extract described in the third aspect.

[0018] Furthermore, the microbial fertilizer also includes other bacterial species that complement or synergize with Bacillus Velez C5.

[0019] In an eighth aspect, the present invention further provides the use of the Bacillus Velez C5 as described in the first aspect, the bacterial fermentation liquid as described in the second aspect, the active extract as described in the third aspect, the microbial agent as described in the sixth aspect, and the microbial fertilizer as described in the eighth aspect in plant salt stress tolerance and / or growth promotion. The plant may be corn, pepper, tobacco, tomato, etc.

[0020] The present invention has the following beneficial effects:

[0021] 1. The present invention screened out a salt-tolerant growth-promoting Bacillus Velezii C5, whose preservation number is CGMCC No.27764; it has a significant effect on promoting plant growth and alleviating the effects of saline-alkali land on plant growth;

[0022] 2. The Velezella C5 of the present invention has a significant effect on the growth of plants tolerant to saline-alkali soils;

[0023] 3. The Bacillus Velez C5 of the present invention can be used as an active ingredient of microbial agents, microbial fertilizers, etc., to promote plant growth and alleviate the impact of saline-alkali land on plant growth;

[0024] 4. The Bacillus Velezii C5 of the present invention is a plant endophyte, and its application in plants is environmentally friendly and effective. The preparation method is simple and suitable for popularization and production application. It overcomes the problems of unstable effect, single function, limited resources, etc. of the strains used in the research and development of microbial fertilizers. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only one embodiment of the present invention, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0026] Figure 1 : Identification diagram of the growth-promoting function of strain C5 of the present invention;

[0027] Figure 2 : Schematic diagram of the morphological observation results of the strain C5 of the present invention;

[0028] Figure 3 : Schematic diagram of Gram staining results of strain C5 of the present invention;

[0029] Figure 4 : Schematic diagram of spore staining results of strain C5 of the present invention;

[0030] Figure 5 : Schematic diagram of the gene amplification product of strain C5 of the present invention;

[0031] Figure 6 : Schematic diagram of the 16srDNA evolutionary tree of strain C5 of the present invention;

[0032] Figure 7 : The salt tolerance identification curve of the strain C5 of the present invention;

[0033] Figure 8 : Schematic diagram of the plate experiment for detecting salt tolerance of strain C5 of the present invention;

[0034] Fig. 9 : Schematic diagram of the effect of the fermentation liquid of strain C5 of the present invention on the germination rate and germination potential of corn seeds;

[0035] Fig.10 : Schematic diagram of detecting the salt tolerance and growth-promoting function of C5 fermentation liquid at different dilution multiples of the present invention on tobacco seedlings;

[0036] Fig.11 : Effect of the fermentation liquid of strain C5 of the present invention on the antioxidant enzyme activity of tobacco seedlings under salt stress. DETAILED DESCRIPTION

[0037] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. The following embodiments are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention. The experimental methods used in the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used in the present invention, unless otherwise specified, can be obtained from commercial channels. In addition, other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0038] Example 1: Isolation of endophytic bacteria in plants

[0039] Select crop materials with good growth from saline-alkali land in Dongying, Shandong, store them at low temperature and transport them back to the laboratory for isolation and screening of microorganisms. First, wash the root or stem of the crop, clean the surface with ultrasound, and then disinfect it with alcohol and sodium hypochlorite. Soak it in 75% alcohol for 3-5 minutes, and then soak it in 2% sodium hypochlorite for 30-40 minutes. After the surface disinfection is completed, rinse it with sterile water for 5 times, dry it under sterile conditions, add 6ml of sterile water to each sample, grind it in a sterile mortar, let it stand for 10-15 minutes, and then dilute the grinding supernatant to 10 by multiples. -3 , apply 80-100 μL to LB medium, repeat each treatment sample 3 times, and culture at 30℃ for 2-3 days.

[0040] The last sterile water rinse after surface disinfection was used as a control to test whether it was endophytic bacteria. If no bacteria grew on the control plate, subsequent screening of plant endophytic bacteria was performed. According to the above method, 79 strains were screened for subsequent screening and identification, as shown in Table 1.

[0041] Table 1 Screening of plant endophytes

[0042]

[0043] Example 2: Analysis of the growth-promoting function of endophytic strains

[0044] Prepare a culture medium that dissolves phosphate, potassium and fixes nitrogen, sterilize it under high pressure at 115°C for 20 min, then pour it into a culture dish for later use. Pipette 2 μL of the activated seed solution and spot it in the center of the culture medium. Incubate it upside down at 28°C for 72 h and observe the results.

[0045] The experimental results are shown in Table 2. The 79 endophytic strains isolated and screened were analyzed for their functions of solubilizing organic phosphorus, solubilizing inorganic phosphorus, solubilizing potassium and fixing nitrogen. The experimental results showed that 12 strains had the functions of solubilizing organic phosphorus, inorganic phosphorus, solubilizing potassium and fixing nitrogen. Among them, strains C1, C2, C4, C5, JC1 and JC2 had good functions of solubilizing organic phosphorus and inorganic phosphorus, fixing nitrogen and dissolving mineral potassium, and strain C3 had the functions of solubilizing inorganic phosphorus, solubilizing potassium and fixing nitrogen.

[0046] To analyze the ability of the strain to dissolve inorganic phosphorus, the culture medium was configured as follows: 10 g / L glucose, 0.5 g / L ammonium sulfate, 0.3 g / L sodium chloride, 0.3 g / L magnesium sulfate, 0.03 g / L manganese sulfate, 0.3 g / L potassium sulfate, 0.03 g / L ferrous sulfate, 5 g / L calcium phosphate, and 15 g / L agar.

[0047] The configuration of the culture medium for analyzing the ability of the strain to dissolve organic phosphorus is as follows: 10 g / L glucose, 0.5 g / L ammonium sulfate, 0.5 g / L yeast extract powder, 0.3 g / L sodium chloride, 0.3 g / L potassium chloride, 0.03 g / L magnesium sulfate, 0.03 g / L ferrous sulfate, 5 g / L calcium phosphate, and 15 g / L agar.

[0048] The configuration of analyzing whether the strain has the ability of nitrogen fixation is as follows: 10 g / L glucose, 0.5 g / L ammonium sulfate, 0.5 g / L yeast extract powder, 0.3 g / L sodium chloride, 0.3 g / L potassium chloride, 0.03 g / L magnesium sulfate, 0.03 g / L ferrous sulfate, 5 g / L calcium phosphate, and 15 g / L agar.

[0049] Table 2 Analysis of the growth-promoting function of endophytes

[0050]

[0051]

[0052] Example 3: Identification of the growth-promoting function of endophytic strains

[0053] Prepare a culture medium that dissolves phosphate, potassium and fixes nitrogen, sterilize it under high pressure at 115°C for 20 min, then pour it into a culture dish for later use. Pipette 2 μL of the activated seed solution and spot it in the center of the culture medium. Incubate it upside down at 28°C for 72 h and observe the results.

[0054] The growth-promoting functions of the above 12 strains were identified. As shown in Table 3, the analysis showed that the seven strains C1, C2, C3, C4, C5, JC1 and JC2 had good growth-promoting abilities of solubilizing organic phosphorus, inorganic phosphorus, potassium and nitrogen fixation, and were significantly higher than the other five strains. Therefore, these seven strains were selected for further analysis.

[0055] Evaluation standard for potassium and phosphorus solubility: diameter ratio = transparent circle diameter / plaque diameter * 100%

[0056] Nitrogen fixation evaluation criteria: Determination of colony diameter.

[0057] Figure 1 The ability of strain C5 to dissolve phosphate, potassium and fix nitrogen was shown

[0058] Table 3 Analysis of the growth-promoting function of endophytes

[0059]

[0060]

[0061] Example 4: Endophytic strains secrete auxins and gibberellins

[0062] The ability of the seven strains C1, C2, C3, C4, C5, JC1 and JC2 screened in Example 3 to secrete siderophores, auxins and gibberellins was determined by the methods of the prior art.

[0063] The results of the experiment showed that among the seven strains, only three strains, C2, C5 and JC1, had the ability to secrete both auxin and gibberellin. Auxin can promote plant growth, and gibberellin can promote seed germination.

[0064] The secretion of auxin and gibberellin content of C2, C5 and JC1 was measured. The experimental results showed that among the three strains, C5 could produce more auxin and gibberellin, with the contents of 5.07μg / ml and 5.23μg / ml respectively. Therefore, the comprehensive growth-promoting ability of strain C5 was the strongest.

[0065] Table 4 The ability of different endophytes to secrete auxins and gibberellins

[0066]

[0067] Example 5: Morphological observation of endophytic strain C5

[0068] The strain C5 was streaked and cultured with single colonies by the plate streaking method. Figure 2 It can be seen that a single colony of strain C5 grown on LB medium for 24 hours is oval with irregular edges, moist and sticky on the surface, milky white, opaque and raised. Figure 3 As shown in the figure, the Gram staining of strain C5 and the indicator bacteria Bacillus subtilis are both purple, so strain C5 is identified as a Gram-positive bacterium. Figure 4 Spore staining showed that strain C5 could produce spores.

[0069] Example 6: Physiological and biochemical identification of endophytic strain C5

[0070] Physiological and biochemical identification of strain C5 showed that the sole carbon source utilization experiment showed that strain C5 could produce acid from lactose, sucrose and maltose, hydrolyze starch and liquefy gelatin, and produce auxin and gibberellin. The indole test result was positive, and the oxidase test result was negative. It did not have the ability to produce cellulase and had motility.

[0071] Table 3 Physiological and biochemical test results of strain C5

[0072]

[0073] Example 7: Schematic diagram of 16srDNA gene sequence amplification of endophytic strain C5

[0074] Based on morphological observation and physiological and biochemical experiments, strain C5 was preliminarily identified as a Bacillus strain. DNA of strain C5 was extracted and used as a template to amplify the 16S rDNA nucleotide fragment using the 16S rDNA universal upstream primer 27F and downstream primer 1492R. The amplification system is shown in Table 1. The amplified product was sent to Sangon for sequencing.

[0075] The primer sequences are as follows:

[0076] Upstream primer 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′;

[0077] Downstream primer 1492R: 5′-TACGGTTACCTTGTTACGACTT-3′.

[0078] Table 4 16S rDNA PCR system

[0079]

[0080] The sequencing results showed that the length of 16SrDNA of strain C5 was 1445bp. The sequencing results are shown in SEQ ID No: 1 in the sequence table. The sequence obtained was submitted to NCBI for BLAST comparison, and the strain sequence with higher similarity was selected for download. The phylogenetic tree was constructed using the software MEGA 6.0. For details, see Figure 6 The results of 16S rDNA sequence alignment showed that C5 had the highest similarity with Bacillus velez.

[0081] Therefore, combined with the morphological, physiological and biochemical characteristics of the bacteria and the results of 16S rDNA sequence analysis, it can be determined that the strain is Bacillus velez, and it is named Bacillus velez C5.

[0082] The above-screened Bacillus Velez C5 was deposited on July 3, 2023 at the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC No. 27764, and the address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The requested depository unit is Qingdao Agricultural University.

[0083] Example 8: Analysis of salt tolerance of endophytic strain C5

[0084] Take the strain C5 seed liquid stored at -80℃ and inoculate it into 20ml LB medium. After culturing in a shake flask at 28℃ and 160r / min for 20h, transfer the bacterial liquid of strain C5 to LB liquid culture medium with different salt concentrations at an inoculation rate of 5% in the same amount. In this embodiment, the mass concentration of NaCl in the liquid culture medium is 1%, 2%, 4%, 6%, 8% and 10%, respectively. After culturing in a shaker at a temperature of 27-29℃ and a rotation speed of 120-180r / min for 24h, observe the growth of strain C5 under different salt concentrations, test the biomass of the bacteria, and determine the salt tolerance of the salt-tolerant growth-promoting bacteria strain. After 24h, measure the bacterial concentration (OD600) to determine the salt tolerance of the strain, and obtain the following Figure 7 The salinity tolerance line graph is shown.

[0085] according to Figure 7 and Figure 8 The results showed that the salt-tolerant growth-promoting bacteria strain C5 had a wide adaptability to NaCl and could grow at salt concentrations of 1 to 6%, which indicated that the salt tolerance of strain C5 could reach up to 6%.

[0086] Example 9: Effect of endophytic strain C5 on corn seed germination under salt stress

[0087] Select corn seeds with complete and full grains as experimental materials. Select corn seeds with full grains, no mold, uniform size and no damage. First, place them in 70% alcohol solution for surface disinfection for 20 seconds, then disinfect them with 5% sodium hypochlorite for 2 minutes, wash them with sterile water 3-5 times, and finally soak them for 6 hours.

[0088] Strain C5 was fermented in LB medium at 28°C and 160r / min for 20h and then diluted for subsequent treatment. Normal conditions and salt stress conditions were set, and 3 treatments were used for each condition, namely control, sterilized LB, and C5 bacterial solution, among which C5 bacterial solution and sterilized LB were diluted 100 times with sterile water before treatment.

[0089] After soaking the seeds, use filter paper to absorb the surface moisture and place them in a 9 cm culture dish. Place a double layer of filter paper in the culture dish in advance, and then put the culture dish into a 25°C constant temperature incubator for culture. During this period, add water to keep it moist. After 72 hours, measure the germination rate and germination potential.

[0090] Table 5 Experimental design

[0091]

[0092] Germination rate = (number of germinated seeds / number of test seeds) × 100%.

[0093] Germination potential = total number of germinated grains ÷ total number of test grains × 100%.

[0094] 2. Experimental results and analysis

[0095] Under the condition of 150mM NaCl, seed germination was inhibited, and the germination rate of the control group was only 30.3%. After treatment with C5 fermentation liquid, the germination rate of seeds was increased to 66.7% and the germination potential was increased to 55.1% compared with the control group. Fig. 9 The results in Table 6 show that strain C5 can improve the germination rate of corn seeds.

[0096] Table 6 Effects of C5 on corn seed germination under salt stress

[0097]

[0098] Example 10: Effects of fermentation broth of strain C5 at different dilution multiples on growth of tobacco seedlings under salt stress 1. Experimental method:

[0099] The tobacco pot experiment was carried out in the greenhouse of Qingdao Agricultural University, with a temperature of 28°C, a photoperiod of 14h / 10h, a light intensity of 1000Lux, and nutrient soil.

[0100] Plant 2 tobacco seeds per hole in a hole tray filled with peat soil, cover with 0.5 cm of peat soil, place the hole tray in a tray, add 1 L of water, wait for the seeds to germinate, water twice a week, each time with 1 L of water, wait for the tobacco seedlings to grow to the four-leaf and one-heart stage, transplant them into pots (10 cm high, 10 cm in diameter at the top), and transplant them into large pots (20 cm high, 20 cm in diameter at the top) again after one week for a potted experiment.

[0101] The following four treatment groups were set up for tobacco:

[0102] (1) Blank control group (no treatment);

[0103] (2) Salt stress treatment group (150 mmol / L NaCl saline was used to treat peat soil, and the salinity of nutrient soil was set to 4‰);

[0104] (3) Salt stress treatment group + strain C5 bacterial solution treatment (28°C, 160 r / min culture for 20 h, then diluted 5 times and irrigated with roots)

[0105] (4) Salt stress treatment group + strain C5 bacterial solution treatment (28°C, 160 r / min culture for 20 h, then diluted 10 times and irrigated with roots)

[0106] (5) Salt stress treatment group + strain C5 bacterial solution treatment (28°C, 160 r / min culture for 20 h, then diluted 15 times and irrigated with roots)

[0107] The tobacco seedlings of each treatment were irrigated with a fixed amount of 50 ml, and the tobacco was treated once. During the period, pymetrozine and nitenpyram were sprayed to prevent pests and diseases. After the tobacco grew for 3 weeks, its growth index was measured.

[0108] 2. Experimental results and analysis

[0109] like Fig.10 The growth of tobacco plants was inhibited after salt stress, and their plant height, stem diameter, leaf number and leaf area were reduced by 29.82%, 35.74%, 37.40% and 34.80% compared with normal soil. The roots were irrigated with 5-fold, 10-fold and 15-fold diluted fermentation liquid of strain C5, respectively. After treatment with 5-fold diluted fermentation liquid, the imbalance of ion homeostasis in the soil and plant environment was aggravated due to the high concentration, thus inhibiting plant growth. After root irrigation with 10-fold diluted fermentation liquid, the plant height, stem diameter, leaf number and leaf area of ​​tobacco increased by 25.00%, 17.59%, 30.00% and 31.73% compared with plants under salt stress, which was close to the growth of tobacco seedlings under normal soil conditions. After treatment with 15-fold diluted fermentation liquid, the strain had no significant growth-promoting function on tobacco salt tolerance due to the high dilution multiple, so the 10-fold diluted fermentation liquid of the strain was selected for subsequent research.

[0110] Example 11: Effect of strain C5 fermentation liquid on the growth of pepper seedlings

[0111] 1. Experimental methods:

[0112] Mix nutrient soil and perlite in a ratio of 9:1 to obtain a seedling medium. Take pepper seeds and place them in seedling trays to germinate in the dark at 28°C. After germination for one week, transplant them into flower pots and place them in a greenhouse at 28°C, 4h / 10h photoperiod, and a light intensity of 1200Lux for 2 weeks. Select materials with consistent growth to conduct the following potted plant experiments. Take strain C5 in LB medium and shake flask ferment for 20 hours at 28°C and 160r / min for subsequent treatment

[0113] The following four treatment groups were set up for pepper:

[0114] (1) Blank control group (no treatment);

[0115] (2) Salt stress treatment group (nutrient soil was treated with 150 mmol / L NaCl salt water, and the salinity of the nutrient soil was set to 4‰);

[0116] (3) Treatment with the fermentation liquid of strain C5 (cultured at 28°C, 160 r / min for 20 h and then diluted 10 times for root irrigation)

[0117] (4) Salt stress treatment + strain C5 fermentation broth treatment

[0118] The pepper seedlings in each of the above treatments were irrigated with a fixed amount of 50 ml for two times. During the period, pymetrozine and nitenpyram were sprayed to prevent diseases and insect pests. After the peppers grew for 4 weeks, their growth indicators were measured.

[0119] 2. Experimental results and analysis

[0120] (1) As shown in Table 7, the fermentation liquid of salt-tolerant strain C5 can effectively promote the growth of pepper under salt stress conditions; as shown in Table 7, the plant height, leaf number and leaf area of ​​pepper in the group treated with the fermentation liquid of strain C5 under salt stress increased significantly compared with the group treated with salt stress, increasing by 16.15%, 19.59% and 15.40% respectively. It was also found that the application of the fermentation liquid of salt-tolerant strain C5 alone can promote the growth of pepper compared with the blank control group.

[0121] Table 7 Effects of C5 on pepper seedling growth under salt stress

[0122]

[0123]

[0124] Example 12: Effect of strain C5 fermentation broth on antioxidant enzymes in tobacco seedlings

[0125] 1. Experimental methods:

[0126] Plant 2 tobacco seeds per hole in a hole tray filled with peat soil, cover with 0.5 cm of peat soil, place the hole tray in a tray, add 1 L of water, wait for the seeds to germinate, water twice a week, each time with 1 L of water, wait for the tobacco seedlings to grow to the four-leaf and one-heart stage, transplant them into pots (10 cm high, 10 cm in diameter at the top), and transplant them into large pots (20 cm high, 20 cm in diameter at the top) again after one week for a potted experiment.

[0127] The following four treatment groups were set up for tobacco:

[0128] (1) Blank control group (no treatment);

[0129] (2) salt stress treatment group (peaty soil treated with 150 mmol / L NaCl saline, soil salt content of 4‰ as measured by a salinometer);

[0130] (3) Blank control + strain C5 fermentation liquid treatment (28°C, 160 r / min culture for 20 h, then diluted 10 times for root irrigation)

[0131] (4) Salt stress treatment group + strain C5 fermentation liquid treatment (28°C, 160r / min culture for 20h, then diluted 10 times and irrigated with roots)

[0132] The tobacco seedlings treated with the above treatments were irrigated with a fixed amount of 50 ml, and the tobacco was treated twice. Antioxidant enzymes were measured after the tobacco had grown for 4 weeks. The SOD enzyme activity was determined by referring to the nitroblue tetrazolium (NBT) method described by Li et al. (2021); the POD enzyme activity was determined by referring to the guaiacol method described by Yu et al. (2020); the CAT enzyme activity was determined by referring to Shen et al. (2021). 2. Experimental results and analysis:

[0133] Antioxidant enzymes include SOD, POD and CAT. Antioxidant enzymes are one of the important indicators of crop resistance to abiotic stress. The enzyme activities of POD, SOD and CAT are measured. Fig.11 It can be seen that under the condition of 4‰ salt content stress, the enzyme activity of POD increased by 63.39% compared with the control after the fermentation liquid of strain C5 was treated, the enzyme activity of SOD increased by 12.85%, and the enzyme activity of CAT increased by 28.85%. This shows that the antioxidant enzyme activity in tobacco leaves was significantly increased after the treatment of strain C5 liquid under salt stress. This shows that the treatment of strain C5 liquid can improve the salt tolerance of tobacco seedlings by increasing the antioxidant enzyme activity of tobacco seedlings.

[0134] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. A strain of Bacillus velez Bacillus velezensis ), characterized in that, It is Bacillus Velez C5, and its deposit number is CGMCC No. 27764.

2. A bacterial fermentation liquid prepared by using the Bacillus Velez C5 described in claim 1.

3. A method for culturing Bacillus Velezii according to claim 1, characterized in that: The strain C5 seed solution was inoculated into 20 mL LB medium at a 5% inoculum size and cultured at 28°C and 160 r / min for 20 h.

4. A microbial agent, characterized in that: The microbial agent includes the Bacillus Velez C5 according to claim 1 and / or the bacterial fermentation broth according to claim 2.

5. A microbial agent according to claim 4, characterized in that: The microbial agent also includes other bacterial species that play a complementary or synergistic role with Bacillus Velez C5.

6. A microbial fertilizer, characterized in that: The microbial fertilizer comprises the Bacillus Velez C5 according to claim 1 and / or the bacterial fermentation broth according to claim 2.

7. A microbial fertilizer according to claim 6, characterized in that: The microbial fertilizer also includes other bacteria species that play a complementary or synergistic role with Bacillus Velez C5.

8. Use of the Bacillus Velez C5 according to claim 1, the bacterial fermentation liquid according to claim 2, the microbial agent according to claim 4, and the microbial fertilizer according to claim 6 in salt stress tolerance and / or growth promotion of corn, tobacco, and pepper.

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