A strain of Bacillus subtilis YJBS-26 and its application and microbial agent

Abstract

The present invention provides a strain of Bacillus subtilis YJBS-26 and its application and microbial agent, which relate to the field of microorganisms. The Bacillus subtilis YJBS-26 is deposited in the General Microbiological Center of the China Microorganism Culture Collection Administration Committee, and the deposit number is CGMCC No.27623. The strain has strong salt and alkali resistance, and can grow in an LB medium with a salt concentration of 16%, and can also grow in an LB medium with a pH value of 10.0. Under saline-alkali stress conditions, compared with the control group, the strain YJBS-26 has a significant growth-promoting function. Using the fermentation liquid of the strain to treat oat seeds or seedlings can increase the plant height, root length and fresh weight of the oat plants, and can increase the yield of the oat plants.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, in particular to a strain of Bacillus subtilis YJBS-26 and an application and a bacterial agent thereof. Background Art

[0002] Soil salinization is a global problem facing mankind. According to statistics, the area of ​​saline-alkali land in my country is nearly 100 million hm2. 2 , which seriously threatens my country's ecological environment and food security, and has become one of the main abiotic adversities limiting the improvement of crop yield and quality. People have adopted many methods to improve saline-alkali land, among which biological improvement of saline-alkali land is considered to be a green, environmentally friendly and effective measure. Studies in recent years have shown that the application of biological agents can promote plant growth under saline-alkali stress conditions and improve soil. Therefore, the isolation and acquisition of functional microorganisms with good salt-alkali tolerance and plant growth promotion is of great significance to the improvement of saline-alkali land and the development of new agents.

[0003] Plant growth promoting rhizobacteria (PGPR) are a type of bacteria that are widely present in the rhizosphere of plants and have multiple growth-promoting functions. They have important applications in plant growth and soil improvement. In terms of promoting plant growth in saline-alkali land and improving saline-alkali soil, salt-alkali tolerant PGPR has gradually become a research hotspot. Screening functional microorganisms with salt and alkali tolerance and using functional microorganisms to improve saline-alkali soil, promote crop growth, alleviate the damage of adverse stress to crops, and increase the diversity of soil microbial flora is one of the important ways to develop green agriculture. However, the current research on PGPR to improve saline-alkali soil and promote plant salt tolerance is still in its infancy, and there is a lack of strains that have strong tolerance to saline-alkali soil and can still promote plant growth under saline-alkali stress conditions. The present invention screens out a strain YJBS-26 with strong salt-alkali resistance and growth-promoting effect by separating plant rhizosphere microorganisms grown in saline-alkali land. The strain YJBS-26 is identified as Bacillus subtilis by molecular biology, and experiments prove that the strain YJBS-26 still has a significant growth-promoting effect on crops under saline-alkali stress. Therefore, the present invention can provide resources for the utilization and development and application of salt-alkali resistant growth-promoting bacteria. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a strain of Bacillus subtilis YJBS-26 and its application and microbial agent, which has strong salt-alkali resistance and can grow in an LB medium with a NaCl mass fraction of 16%, or in an LB medium with a pH value of 10.0. Under saline-alkali stress conditions, the strain YJBS-26 has a significant growth-promoting function compared with the control group. Using the fermentation liquid of the strain to treat oat seeds or seedlings can increase the plant height, root length and fresh weight of the oat plants, and can increase the yield of the oat plants.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The invention provides a strain of Bacillus subtilis YJBS-26. The Bacillus subtilis YJBS-26 is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, and the deposit number is CGMCC No.27623.

[0007] The present invention also provides the use of the Bacillus subtilis YJBS-26 described in the above technical solution in the preparation of indole-3-acetic acid.

[0008] The present invention also provides the use of the Bacillus subtilis YJBS-26 described in the above technical solution in promoting the growth of oats.

[0009] The present invention also provides the use of the Bacillus subtilis YJBS-26 described in the above technical solution in promoting the growth of oats under salt stress and / or alkali stress.

[0010] Preferably, the growth includes plant height, root length or fresh weight.

[0011] The present invention also provides the use of the Bacillus subtilis YJBS-26 described in the above technical solution in improving oat yield.

[0012] The present invention also provides the use of the Bacillus subtilis YJBS-26 described in the above technical solution in improving the yield of oats under salt stress and / or alkali stress.

[0013] The present invention also provides the use of the Bacillus subtilis YJBS-26 described in the above technical solution in improving the germination rate of oats under salt stress and / or alkali stress.

[0014] The present invention also provides the use of the Bacillus subtilis YJBS-26 described in the above technical solution in improving the thousand-grain weight of oats under salt stress and / or alkali stress.

[0015] The present invention also provides a bacterial agent, comprising the Bacillus subtilis YJBS-26 described in the above technical solution, wherein the effective viable count of Bacillus subtilis YJBS-26 in the bacterial agent is 1×10 7 cfu / mL.

[0016] The beneficial effects of the present invention are:

[0017] The Bacillus subtilis YJBS-26 provided by the present invention has strong salt-alkali resistance, can grow in an LB medium with a concentration of 16% NaCl, and can also grow in an LB medium with a pH value of 10.0, and has a certain ability to produce IAA. Under salt-alkali stress conditions, inoculation of the strain can improve the stress resistance of oats and significantly improve the emergence rate, biomass and field yield of the plants. Therefore, the present invention has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0019] Figure 1 This is the colony morphology of Bacillus subtilis YJBS-26;

[0020] Figure 2 The effect of strain YJBS-26 on oat growth under salt stress, the left is the control treatment, the right is the strain YJBS-26 inoculation treatment;

[0021] Figure 3 This is the effect of strain YJBS-26 on oat growth under alkaline stress. The left is the control treatment, and the right is the strain YJBS-26 inoculation treatment.

[0022] Biological Deposit Description

[0023] Bacillus subtilis YJBS-26, Latin name Bacillus subtilis, was deposited on June 14, 2023 at the General Microbiology Center of China Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCCNo.27623. DETAILED DESCRIPTION

[0024] The invention provides a strain of Bacillus subtilis YJBS-26. The Bacillus subtilis YJBS-26 is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, and the deposit number is CGMCC No.27623.

[0025] In the present invention, the colony and bacterial morphology of the Bacillus subtilis YJBS-26 are as follows: after being cultured on LB medium, the colony is observed to be nearly circular in shape, with wrinkles on the surface and milky white in color.

[0026] The present invention also provides the use of the Bacillus subtilis YJBS-26 described in the above technical solution in the preparation of indole-3-acetic acid. In the present invention, the bacterial solution containing Bacillus subtilis YJBS-26 turns red after adding Salkowski reagent, and has a strong ability to produce IAA, and the yield is measured to be 11.45 μg / mL.

[0027] The present invention also provides the use of the Bacillus subtilis YJBS-26 described in the above technical solution in promoting the growth of oats.

[0028] The present invention also provides the use of the Bacillus subtilis YJBS-26 described in the above technical solution in promoting the growth of oats under salt stress and / or alkali stress.

[0029] In the present invention, the growth preferably includes plant height, root length or fresh weight. In the present invention, the fresh weight refers to the weight of the whole plant. The application preferably includes: irrigating the roots of oat seedlings with a bacterial solution containing Bacillus subtilis YJBS-26, the amount of bacterial solution inoculated per plant is preferably 10 mL, and the effective viable bacteria count of Bacillus subtilis YJBS-26 in the bacterial solution is preferably 1×10 7 cfu / mL. The results showed that strain YJBS-26 had a growth-promoting effect on oat seeds in terms of germination rate, root length and stem length under different salt stresses.

[0030] The present invention also provides the use of the Bacillus subtilis YJBS-26 described in the above technical solution in improving oat yield.

[0031] The present invention also provides the use of the Bacillus subtilis YJBS-26 described in the above technical solution in improving the yield of oats under salt stress and / or alkali stress.

[0032] In the present invention, the application preferably comprises: dressing 500 g of oat seeds with 20 mL of a bacterial suspension containing Bacillus subtilis YJBS-26, wherein the effective viable count of Bacillus subtilis YJBS-26 in the bacterial suspension is preferably 1×10 7 cfu / mL, and the seeds were sown after drying. The results showed that the strain YJBS-26 treatment had a growth-promoting effect on the height of oat plants under field conditions, and could increase the thousand-grain weight and yield of oats.

[0033] The present invention also provides the use of the Bacillus subtilis YJBS-26 described in the above technical solution in improving the germination rate of oats under salt stress and / or alkali stress.

[0034] The present invention also provides the use of the Bacillus subtilis YJBS-26 described in the above technical solution in improving the thousand-grain weight of oats under salt stress and / or alkali stress.

[0035] The present invention also provides a bacterial agent, characterized in that it comprises the Bacillus subtilis YJBS-26 described in the above technical solution, and the effective viable count of Bacillus subtilis YJBS-26 in the bacterial agent is 1×10 7 The present invention has no special limitation on the preparation method of the bacterial agent, and those skilled in the art can prepare it according to conventional preparation methods.

[0036] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0037] Example 1

[0038] Isolation, purification and identification of Bacillus subtilis YJBS-26:

[0039] 1. Strain isolation and purification:

[0040] Collect the root soil of saline-alkali plants at multiple points, take 5g of the test soil and add it to a conical flask filled with 100mL of sterile water, shake it at 180r / min for 30min to fully disperse the soil in the sterile water to obtain a soil mixture. Dilute the soil mixture according to the gradient, and select a concentration gradient of 10 –2 , 10 –3 , 10 –4 , 10 –5 , 10 –6 The soil mixture was applied, and 100 μL of bacterial suspension was applied to LB solid medium containing 10% NaCl for microbial isolation. The plate was inverted and placed in a 28°C constant temperature incubator for 48 to 96 hours. A single microbial colony was picked and purified until a single strain was obtained. The purified strain was stored in a -80°C refrigerator.

[0041] 2. Identification of strain YJBS-26:

[0042] Pick a single colony of the strain to be identified in a 1.5mL centrifuge tube, centrifuge at 12000rpm for 1min, and collect the bacteria. Bacterial DNA was extracted by a bacterial genomic DNA extraction kit purchased from Tiangen Biochemical Technology Co., Ltd. For detailed steps, please refer to the product manual. 16S rDNA was amplified using universal primers 27F and 1492R. The PCR amplification reaction system was 25μL, including 3μL DNA template, 12.5μL 2×mix Taq enzyme, and 7.5μL ddH2O. PCR amplification reaction procedure: 94℃ pre-denaturation for 3min, 94℃ denaturation for 30s, 55℃ annealing for 1min, 72℃ extension for 1.5min, 30 cycles, and finally 72℃ extension for 10min, and stored at 4℃. The PCR product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing after 1% agarose gel electrophoresis. The sequencing result of sequence number OR708672 was compared and analyzed by the Blast program of NCBI database for similarity, and it was identified as Bacillus subtilis in combination with morphological characteristics.

[0043] Example 2

[0044] Determination of salt tolerance of Bacillus subtilis YJBS-26:

[0045] 1. Test method:

[0046] While keeping the pH value of the culture medium at 7.0, a salt concentration gradient was set, i.e., a culture medium with a mass percentage of NaCl of 10.0%, 11.0%, 12.0%, 13.0%, 14.0%, 15.0%, 16.0% and 17.0%. The test microorganisms were inoculated on the above salt concentration gradient culture medium plate and inverted at 28°C for 72 hours, and then the growth was observed.

[0047] 2. Test results:

[0048] As shown in Table 1, strain YJBS-26 can grow in LB solid medium with NaCl contents of 10.0%, 11.0%, 12.0%, 13.0%, 14.0%, 15.0% and 16.0%, respectively.

[0049] Table 1 Salt tolerance activity of strain YJBS-26

[0050]

[0051] Note: In the table, + means growth is possible; - means no growth.

[0052] Example 3

[0053] Alkali resistance determination of Bacillus subtilis YJBS-26:

[0054] 1. Test method:

[0055] While keeping the mass percentage of NaCl in the medium at 1%, the pH value of the medium was increased by 1 unit each time, that is, the pH values ​​were 7.0, 8.0, 9.0, 10.0 and 11.0 respectively. Bacillus subtilis YJBS-26 was inoculated on the above pH gradient medium plate and inverted at 28°C for 72 hours, and the growth was observed.

[0056] 2. Test results:

[0057] As shown in Table 2, strain YJBS-26 can grow in LB solid medium with pH values ​​of 7.0, 8.0, 9.0 and 10.0.

[0058] Table 2 Alkali resistance activity of strain YJBS-26

[0059]

[0060] Note: In the table, + means growth is possible; - means no growth.

[0061] Example 4

[0062] Quantitative detection of IAA production by Bacillus subtilis YJBS-26:

[0063] 1. Test method:

[0064] IAA quantitative determination was performed on strain YJBS-26. The strain was activated on an LB medium plate, punched into a cake using a 9mm puncher and placed in a 250mL triangular flask, each flask was filled with 100mL of liquid LB medium, and fermented on a shaker at 150-180r / min at 25℃-28℃ for 2-3 days. The fermentation liquid of the strain was centrifuged at 6000r / min for 5min, the supernatant was discarded after centrifugation, sterile water was added and shaken, and the suspension was suspended to a concentration of 10 7 cfu·mL -1 Add an equal volume of Salkowski colorimetric solution to the bacterial suspension, keep it in the dark for 30 minutes, and determine its OD600 value. Calculate the content of IAA in the fermentation broth per unit volume by comparing with the standard curve. The standard curve is prepared by gradient dilution of analytically pure IAA. IAA standard curve configuration: Accurately weigh 32.8 mg of IAA in a 100 mL volumetric flask and dilute it with methanol. The solution contains 328 mg / L of IAA. Then dilute it 1 times with methanol, and do it 4 times in a row to make 5 standard solutions with concentrations that differ by 1 times. When comparing colors, equal amounts of standard solution are added to equal amounts of colorimetric solution.

[0065] 2. Test results:

[0066] Table 3 IAA yield determination results

[0067] deal with IAA content (μg / mL) YJBS-26 11.45±0.02 CK —

[0068] Example 5

[0069] Indoor growth promotion experiment of Bacillus subtilis YJBS-26 under saline-alkali stress:

[0070] 1. Preparation of Bacillus subtilis YJBS-26 bacterial suspension:

[0071] The strain was activated on the LB medium plate, punched into a cake using a 9mm puncher and placed in a 250mL triangular flask, each flask was filled with 100mL of liquid LB medium, and fermented at 25℃~28℃, 150~180r / min on a shaking table for 2~3 days. The fermentation liquid of the strain was centrifuged at 6000r / min for 5min, the supernatant was discarded after centrifugation, and sterile water was added to shake and shake to prepare a concentration of 10 7 cfu·mL -1 of bacterial suspension.

[0072] 2. Salt stress test:

[0073] The soil for planting oats was sterilized at high temperature, and the soil, vermiculite, and nutrient soil were mixed in a volume ratio of 2:2:1, and potted for later use. The healthy oat seeds were rinsed, and then soaked in 5% sodium hypochlorite for 3 minutes, and rinsed with sterile water 4 times. Then 300 oat seeds were placed in 10 mL of the prepared strain YJBS-26 suspension (1×10 7 cfu / mL), shake in a shaking incubator at 28°C and 180rpm for 2h, and set oat seeds shaken in an equal amount of liquid LB for 2h as a control. Sprinkle the treated oat seeds evenly in pots, 30 seeds per pot, repeat 3 times, cover the surface with soil, and pour a solution with a mass percentage of 0.6% NaCl and a solution with pH 9.0 in the pots, culture in a greenhouse (25°C), and pour the same stress solution every other day. After growing for 3 weeks, measure the plant emergence rate, plant height and fresh weight, and analyze the effects of different endophytic bacteria on the growth of oat plants.

[0074] 3. Test results:

[0075] The results of the growth promotion test on indoor oat potted plants by inoculating YJBS-26 under 0.6% NaCl stress are shown in Table 4. Compared with the control group, the fresh weight of the YJBS-26-treated and control plants was 1.83 g and 0.93 g, respectively, which was 96.77% higher than that of the control plant; the root length was 14.53 cm and 7.00 cm, respectively, which was 107.57% higher than that of the control plant; the plant height was 27.93 cm and 15.33 cm, respectively, which was 82.19% higher than that of the control plant; the germination rate was 87% and 61%, respectively, which was 42.62% higher than that of the control plant; the germination potential was 61% and 38%, respectively, which was 60.53% higher than that of the control plant.

[0076] Table 4 Effects of inoculated strains on oats under 0.6% NaCl stress

[0077] deal with Plant height(cm) Root length (cm) Fresh weight(g) Germination rate (%) Germination potential YJBS-26 27.93±1.59a 14.53±0.55a 1.83±0.09a 0.87±0.02a 0.61±0.03a CK 15.33±0.63e 7.00±0.38c 0.93±0.09d 0.61±0.01d 0.38±0.02c

[0078] The results of the growth promotion test of indoor oat potted plants by inoculating YJBS-26 under pH 9.0 stress are shown in Table 5. Compared with the control group, the fresh weight of the YJBS-26 treatment and the control were 1.93g and 1.20g, respectively, which was 60.83% higher than that of the control; the root length was 16.47cm and 8.50cm, respectively, which was 93.76% higher than that of the control; the plant height was 31.93cm and 16.97cm, respectively, which was 88.16% higher than that of the control; the germination rate was 84.08% and 61.24%, respectively, which was 37.70% higher than that of the control; the germination potential was 52.19% and 37.37%, respectively, which was 40.54% higher than that of the control. The above results show that strain YJBS-26 has a good growth-promoting effect on indoor potted oats under moderate salt stress and moderate alkali stress ( Figure 2 , 3 ).

[0079] Table 5 Effects of inoculated strains on oats under pH 9 stress

[0080]

[0081] Example 6

[0082] Effect of Bacillus subtilis YJBS-26 on oat field yield:

[0083] 1. Test method:

[0084] The field test plot was set up in the Tumote Left Banner Forest Seedling Demonstration Base in Inner Mongolia Autonomous Region. The test was conducted from May to October 2022. The site was set up with Bacillus subtilis YJBS-26 treatment and 1 blank control. The Bacillus subtilis YJBS-26 treatment and blank control were repeated 3 times each, with a total of 6 plots. The variety of oat seeds was Baiyan No. 2. First, 20 mL of Bacillus subtilis YJBS-26 suspension (1×10 7 cfu / mL) were mixed with 500 g of oat seeds and sown after drying. Routine field management was performed. Plant height, yield and thousand-grain weight of oats were measured after harvest.

[0085] 2. Test results:

[0086] By analyzing the yield, 1000-grain weight and plant height of field oats, the plant height, yield and 1000-grain weight of oats treated with strain YJBS-26 were significantly higher than those of the control, as shown in Table 6. The yield of strain YJBS-26 treatment and control were 156.64kg and 110.92kg, respectively, and strain YJBS-26 treatment increased by 41.22% compared with the control; the 1000-grain weight of strain YJBS-26 treatment and control were 26.32g and 19.45g, respectively, and strain YJBS-26 treatment increased by 35.32% compared with the control; the plant height of strain YJBS-26 treatment and control were 121.45cm and 100.38cm, respectively, and strain YJBS-26 treatment increased by 20.99% compared with the control. The above results prove that strain YJBS-26 treatment has a good growth-promoting effect on oats under field conditions.

[0087] Table 6 Effect of Bacillus subtilis YJBS-26 on oat field yield

[0088] Strain number <![CDATA[Yield (kg / 667m 2 )]]> Thousand-grain weight (g) Plant height(cm) YJBS-26 156.64±6.93a 26.32±0.48a 121.45±2.13a CK 110.92±7.95b 19.45±0.46d 100.38±2.27c

[0089] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Bacillus subtilis YJBS-26, which is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration with a deposit number of CGMCC No. 27623.

2. Use of the Bacillus subtilis YJBS-26 according to claim 1 in the preparation of indole-3-acetic acid.

3. Use of the Bacillus subtilis YJBS-26 according to claim 1 in promoting the growth of oats.

4. Use of the Bacillus subtilis YJBS-26 according to claim 1 in promoting the growth of oats under salt stress and / or alkali stress.

5. The use according to claim 3 or 4, characterized in that: The growth includes plant height, root length or fresh weight.

6. Use of the Bacillus subtilis YJBS-26 according to claim 1 in increasing oat yield.

7. Use of the Bacillus subtilis YJBS-26 according to claim 1 in improving oat yield under salt stress and / or alkali stress.

8. Use of the Bacillus subtilis YJBS-26 according to claim 1 in improving the germination rate of oats under salt stress and / or alkali stress.

9. Use of the Bacillus subtilis YJBS-26 according to claim 1 in increasing the thousand-grain weight of oats under salt stress and / or alkali stress.

10. A bacterial agent, characterized in that: The invention comprises the Bacillus subtilis YJBS-26 of claim 1, wherein the effective viable count of the Bacillus subtilis YJBS-26 in the bacterial agent is 1×10 7 cfu / mL.

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