Bacillus with growth-promoting function for plants under low temperature and salt stress and application thereof

Abstract

The present invention discloses a bacillus having a growth-promoting function for plant low temperature and salt stress and its application, belonging to the field of microbial technology. The strain is deposited in the China Center for Type Culture Collection, with a deposit address of Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, on May 27, 2024. The deposit number is CCTCC NO: M 20241065, and the classification name is Bacillus cereus X16. The strain has significantly higher tolerance to low temperature and salt than other existing Pseudomonas, and also has the function of secreting protease, cellulase and iron carrier, and can be used for plant repair of high salt concentration soil, with the advantages of low cost and high efficiency.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial application, and particularly relates to a bacillus having a growth-promoting function for plants under low temperature and salt stress and an application thereof. Background Art

[0002] Plant growth promoting rhizobacteria (PGPR) can stimulate plant root development and enhance root growth, alleviate the adverse effects of soil salinization and low temperature, and help plants increase yield and stress resistance, and reduce the harm of plants to biological and abiotic stresses. In Northeast my country, due to the low annual average temperature and long and cold winters, the low temperature environment has restricted the practical application of many remediation methods, especially microbial remediation technology, in the process of saline-alkali soil treatment, thus affecting the remediation effect. In addition, low temperature stress can reduce the germination rate of plant seeds and inhibit the function of the protective enzyme system inside plant cells, thereby causing cell oxidative damage or even death. Summary of the invention

[0003] Based on the above problems, the purpose of the present invention is to provide a Bacillus that has a growth-promoting function on plants under low temperature and salt stress, and its colony characteristics are: round, milky white, opaque, smooth surface, and convex; the strain can produce iron carriers and secrete proteases and cellulases, and has beneficial effects such as promoting plant seed germination and growth under salt stress and low temperature.

[0004] The technology adopted in the present invention is as follows: a Bacillus with growth-promoting function for plant low temperature and salt stress, deposited in the China Type Culture Collection, the deposit address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code: 430072, the deposit date is May 27, 2024, the deposit number is CCTCC NO: M 20241065, and the classification name is Bacillus cereus X16.

[0005] Another object of the present invention is to provide a method for culturing the above-mentioned Bacillus having the function of promoting plant growth under low temperature and salt stress, as follows: taking OD 600 =1.0 seed liquid was inoculated into the culture medium at a volume ratio of 0.1%, and cultured on a shaking table for 24 hours under the conditions of 28°C, 180r / min, and pH=7.0.

[0006] Furthermore, the culture medium adopts LB liquid culture medium with the formula of: 10 g / L sodium chloride, 5 g / L yeast extract, 10 g / L tryptone, 15 g / L agar, pH=7.0.

[0007] Another object of the present invention is to provide a Bacillus having a growth-promoting function against plant low temperature and salt stress as a microbial product for promoting plant seed germination under low temperature and salt stress.

[0008] Furthermore, the plant seeds are alfalfa.

[0009] Another object of the present invention is to provide a promoter for promoting plant seed germination under low temperature and salt stress, comprising the above-mentioned Bacillus having the function of promoting plant growth under low temperature and salt stress.

[0010] Further, the bacterial solution of the Bacillus was adjusted to OD 600 =0.50±0.02, the bacterial solution was used for soaking alfalfa seeds at a ratio of 10 ml / 100 alfalfa seeds for 1 hour at a temperature of 28°C and a pH of 7.0.

[0011] Advantages and beneficial effects of the present invention: The strain of the present invention has the beneficial effect of promoting the germination and growth of plant seeds under low temperature and salt stress, and also has the function of secreting protease, cellulase and iron carrier, and can be used for plant repair of high salt concentration soil, with the advantages of low cost and high efficiency. It can be used as a biological fertilizer to achieve the purpose of improving its yield and quality, with the advantages of low cost, no pollution to the environment, and safety for humans and animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a characteristic diagram of bacterial colony morphology;

[0013] Figure 2 This is a microscopic feature diagram of bacterial cells;

[0014] Figure 3 This is a bacterial phylogenetic tree diagram;

[0015] Figure 4 This is a diagram of the germination phenotype of alfalfa seeds under low temperature and salt stress;

[0016] Figure 5 This is a bar graph showing the effect of the strain on the germination potential of alfalfa seeds under single and combined stresses of salt and low temperature;

[0017] Figure 6 This is a bar graph showing the effect of the strain on the germination rate of alfalfa seeds under single stress of salt and low temperature and combined stress;

[0018] Figure 7 This is a bar graph showing the effect of the strain on the radicle length of alfalfa seeds under single stress of salt and low temperature and combined stress;

[0019] Figure 8This is a bar graph showing the effect of the strain on the embryo length of alfalfa seeds under single stress of salt and low temperature and combined stress;

[0020] Fig. 9 This is a bar graph showing the effect of the strain on the germination index of alfalfa seeds under single stress of salt and low temperature and combined stress;

[0021] Fig.10 This is a bar graph showing the effect of this strain on the vigor index of alfalfa seeds under single stress and combined stress of salt and low temperature. DETAILED DESCRIPTION

[0022] The following embodiments further illustrate the present invention in detail, but it should be noted that the specific embodiments described herein are only used to illustrate and explain the purpose, technical solutions and advantages of the present invention, and do not constitute any limitation to the present invention.

[0023] Example 1 Isolation and screening of bacteria

[0024] The Bacillus provided in this example was isolated from the rhizosphere soil of red clover plants in the campus of Northeast Agricultural University in Harbin, Heilongjiang Province. The lead content of the soil was 4.5 g / kg. 5 g of red clover rhizosphere soil was placed in 45 mL of sterile water and shaken continuously for 30 minutes. At this time, the concentration of the bacterial suspension was 10 -1 Then, 10-fold serial dilution was performed 5 times to a concentration of 10 -5 . From Mark 10 -3 , 10 -4 , 10 -5 100 μL of bacterial suspension was extracted from the dilution solution, and the bacterial suspension was evenly spread on LB solid culture medium (10 g / L sodium chloride, 5 g / L yeast extract, 10 g / L tryptone, 15 g / L agar, pH = 7.0) plates with a sterilized applicator. The culture dish was sealed and inverted in a 28°C constant temperature incubator for 48 hours. The growth state of the colony was observed. After the colony grew out, it was preliminarily screened according to the morphology, size, color, etc. of the colony, and single colonies of different types were picked. After several plate streaking purifications, a single purified strain was obtained, and the purified strain was stored in a -80°C refrigerator with glycerol. The growth-promoting ability of the isolated and purified strains was determined, including but not limited to protease, cellulase production and iron carrier. Through the above separation and screening, the strain of the present invention was obtained for the next step of research.

[0025] Example 2 Identification of bacteria

[0026] The strain was subjected to morphological, physiological and biochemical characteristics and 16S rDNA sequencing analysis. The 16S rDNA molecular identification steps are as follows: 1 ml OD 600=1-1.5 bacterial culture, centrifuge at 12,000×g for 30s, discard the supernatant and use a kit to extract strain DNA. Using the extracted DNA as a template, 27F ("5-AGAGTTTGATCCTGGCTCAG-3") and 1492R ("5-CTACGGCTACCTTGTTACGA-3") bacterial universal primers were used for bacterial genome PCR amplification, and the samples were sent to Shanghai Paisono Biotechnology Co., Ltd. for bacterial 16Sr DNA sequencing. According to the sequence results obtained by sequencing, the sequence alignment analysis was performed in the BLAST program on the NCBI website, and the phylogenetic evolution tree was constructed using MEGA7.0 ( Figure 2 ), the results showed that the bacteria was Bacillus cereus.

[0027] Table 1 Physiological and biochemical characteristics of the tested strains

[0028]

[0029] Example 3 Protease production ability of strains

[0030] The strains obtained by screening were inoculated into LB liquid medium (10 g / L sodium chloride, 5 g / L yeast extract, 10 g / L tryptone, 15 g / L agar, pH = 7) with an inoculation loop, and cultured on a shaking table for 24 hours at 28°C, 180 r / min, and pH = 7.0 to obtain seed solution. The seed solution was diluted to OD 0. 600 =0.5 and then set aside. The activated strains were inoculated onto the protease screening medium with sterilized inoculation loops in the clean bench, the culture dishes were sealed, and inverted in a 28°C constant temperature incubator for 48 hours. The outer circle of the colony was observed to see if a transparent light ring was produced. If a transparent light ring was produced, it means that the strain has the ability to produce protease. The maximum diameter of the transparent circle (D) and the colony diameter (d) were measured, and the (D / d) value was calculated. The results are shown in Table 2.

[0031] Table 2 D / d values ​​of strains on protease medium

[0032]

[0033] Example 4 Iron carrier capacity of strains

[0034] Prepare bacterial solution in the same way as above. Dilute the seed solution to OD 600=0.5 and then set aside. The activated target strain was inoculated onto the CAS detection medium with a sterilized inoculation loop in the clean bench, the culture dish was sealed, and it was inverted in a 28°C constant temperature incubator. After one week of culture, the color of the outer circle of the colony medium changed from blue to orange-yellow. The appearance of the iron carrier secretion circle indicated that the strain had the ability to produce iron carriers. The maximum diameter of the transparent circle (D) and the colony diameter (d) were measured, and the (D / d) value was calculated. The results are shown in Table 3.

[0035] Table 3 D / d values ​​of strains on CAS test medium

[0036]

[0037] Example 5 Cellulase production ability of strains

[0038] Prepare bacterial solution in the same way as above. Dilute the seed solution to OD 600 =0.5 and then set aside. The activated target strain was inoculated onto Congo red medium with a sterilized inoculation loop in a clean bench, the culture dish was sealed, and placed upside down in a 28°C constant temperature incubator. After one week of culture, observe whether a transparent light ring is produced in the outer circle of the colony. The production of a transparent light ring indicates that the strain has the ability to produce cellulase. Measure the maximum diameter of the transparent ring (D) and the colony diameter (d), and calculate the (D / d) value. The results are shown in Table 4.

[0039] Table 4 D / d values ​​of strains on Congo red medium

[0040]

[0041] Example 6 Effect of strains on germination of alfalfa under low temperature stress

[0042] The purified target strain was inoculated into LB liquid culture medium and cultured at 28°C and 180 r / min for 24 h. The bacteria were collected by centrifugation and the bacterial solution was adjusted to OD 600=0.50±0.02, soak the alfalfa seeds at a ratio of 10ml / 100 alfalfa seeds for 1h, 28℃, and pH=7.0. Divide the disinfected "Weishen" alfalfa seeds into two parts, one part is placed in the X16 bacterial suspension and soaked for 1h (recorded as X16); the other part is placed in a sterile 0.03mol / L MgSO4 solution and soaked for the same time as a control (recorded as N). Select a culture dish of appropriate size, put two layers of filter paper as a germination bed, and soak the filter paper with sterile water or saline solution. Select alfalfa seeds of appropriate size and full grains, put them in a culture dish, and place 50 seeds in each culture dish. The treatments are as follows: 25℃ culture (CK), 25℃+0.6% NaCl (S), 4℃ culture (L), 4℃+0.6% NaCl (S+L), and each treatment is repeated three times. According to the evaporation rate of water, sterile water or salt is added to the culture dish in time to ensure the germination of seeds, and the temperature of the incubator is adjusted to carry out the seed germination culture experiment. Starting from the completion of seed inoculation, the germination potential is counted at 4 days after the seeds germinate, and the germination rate is counted at 7 days. Then the length of the radicle and the length of the embryo are measured with a ruler. The germination index (GI) = ∑Gt / Dt (Gt refers to the number of germinations within t time, and Dt refers to the number of germination days); Vitality index = germination index × radicle length.

[0043] Table 5 Effects of strains on germination potential of alfalfa seeds under different low temperature and salt stresses

[0044]

[0045] Table 6 Effects of strains on germination rate of alfalfa seeds under different low temperature and salt stress

[0046]

[0047] Table 7 Effects of strains on radicle length of alfalfa seeds under different low temperature and salt stresses

[0048]

[0049] Table 8 Effects of strains on the embryo length of alfalfa seeds under different low temperature and salt stresses

[0050]

[0051]

[0052] Table 9 Effects of strains on germination index of alfalfa seeds under different low temperature and salt stress

[0053]

[0054] Table 10 Effects of strains on alfalfa seed vigor index under different low temperature and salt stresses

[0055]

[0056] Through the above experiments, the results show that: under low temperature stress (4℃), soaking alfalfa seeds with Bacillus cereus X16 can significantly improve its germination rate, germination potential, radicle length and plumule length, germination index and vitality index (P<0.05). This shows that Bacillus cereus X16 can be used as a seed germination promoter to alleviate the growth inhibition of plants under low temperature stress and promote plant growth.

[0057] Example 7 Effect of strains on germination of alfalfa under salt stress

[0058] Prepare bacterial solution in the same way as above. Divide the sterilized alfalfa seeds into two parts at room temperature, one part is placed in X16 bacterial suspension and soaked for 1 hour (recorded as X16); the other part is placed in sterile 0.03mol / L MgSO4 solution and soaked for the same time as control (recorded as N). Choose a culture dish of appropriate size, put two layers of filter paper in the bottom layer as the germination bed, and soak the filter paper with sterile water. Then put the soaked "Weishen" alfalfa seeds into the culture dish, with 50 seeds in each culture dish. The treatments are as follows: 25℃ culture (CK), 25℃+0.6% NaCl (S), 4℃ culture (L), 4℃+0.6% NaCl (S+L), and each treatment is repeated three times. According to the evaporation rate of water, add sterile water or salt to the culture dish in time to ensure the germination of seeds. Starting from the completion of seed inoculation, the germination potential is counted at 4 days after seed germination, and the germination rate is counted at 7 days. Then the seed radicle length and plumule length are measured with a ruler. Germination index (GI) = ∑Gt / Dt (Gt refers to the number of germinations within t time, Dt refers to the number of germination days); Vitality index = Germination index × radicle length.

[0059] Through the above experiment, the results show that: under salt stress (0.6% NaCl), the use of Bacillus cereus X16 bacterial solution to soak alfalfa seeds can significantly improve its germination rate, germination potential, radicle length and plumule length, germination index and vitality index (P<0.05). This shows that Bacillus cereus X16 can be used as a seed germination promoter to alleviate the growth inhibition of plants under salt stress and promote plant growth.

Claims

1. A Bacillus that has the function of promoting plant growth under low temperature and salt stress, deposited in the China Type Culture Collection, the deposit address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, the deposit date is May 27, 2024, the deposit number is CCTCC NO: M 20241065, and the classification name is Bacillus cereus X16.

2. The method for culturing a bacillus having a growth-promoting function against plant low temperature and salt stress according to claim 1, characterized in that: The method is as follows: Take OD 600 =1.0 seed liquid was inoculated into the culture medium at a volume ratio of 0.1%, and cultured on a shaking table for 24 hours under the conditions of 28°C, 180r / min, and pH=7.

0.

3. Use of the Bacillus according to claim 1 that has the function of promoting plant growth under low temperature and salt stress as a microbial product for promoting seed germination of alfalfa seeds under low temperature and salt stress.

4. An accelerator for promoting seed germination of alfalfa seeds under low temperature and salt stress, characterized in that: The invention contains the Bacillus spores as claimed in claim 1 which has the function of promoting plant growth under low temperature and salt stress.

5. The accelerator for promoting seed germination of alfalfa seeds under low temperature stress according to claim 4, characterized in that: The Bacillus culture solution was adjusted to OD 600 =0.50±0.02, the bacterial solution was used for soaking alfalfa seeds at a ratio of 10 ml / 100 alfalfa seeds for 1 hour at a temperature of 28°C and a pH of 7.0.

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