Bacillus with growth promoting function on plant salt and alkali and heavy metal stress and application thereof

Abstract

The invention discloses bacillus with a growth promoting function on plant salt and alkali and heavy metal stress and application, and belongs to the technical field of microorganisms. The strain is preserved in China Center for Type Culture Collection, the preservation address is Wuhan University, Wuchang District, Wuhan City, Hubei Province, the preservation date is January 14, 2026, the preservation number is CCTCC NO: M2026103, and the strain is classified and named as Bacillus thuringiensis BL01. The strain provided by the invention has a probiotic effect of promoting germination and growth of plant seeds under stress of salt, alkali and heavy metals, can be used for phytoremediation of soil with high salt, alkali and heavy metal concentration, and has the advantages of low cost and high efficiency; the fertilizer can be used as a bio-fertilizer, achieves the purpose of improving the yield and quality, and has the advantages of low cost, no pollution to the environment, safety to human and livestock and the like.

Description

Technical Field

[0001] This invention belongs to the field of microbial application technology, specifically relating to a Bacillus species that promotes plant growth under salt and heavy metal stress and its application. Background Technology

[0002] Plant growth-promoting rhizobacteria (PGPR) can stimulate root development and enhance root growth, mitigating the adverse effects of soil salinization and heavy metals. They can also help plants increase yield and stress resistance, reducing the damage caused by biotic and abiotic stresses. However, in Northeast my country, due to abundant mineral resources, mining and subsequent processing easily lead to excessive accumulation of heavy metals in the soil. In the remediation of saline-alkali soils, heavy metal stress restricts the practical application of microbial remediation technologies, thus affecting the remediation effect. Furthermore, heavy metal stress reduces seed germination rates, inhibits the function of protective enzyme systems within plant cells, disrupts intracellular redox balance, resulting in low seed germination rates, oxidative damage to cells, and even cell death. Summary of the Invention

[0003] Based on the above problems, the purpose of this invention is to provide a Bacillus that promotes plant growth under salt and alkali and heavy metal stress, thereby solving the problem of low seed germination rate caused by heavy metal stress. Its colony characteristics are: round, milky white, opaque, rough and raised.

[0004] The technology used in this invention is as follows: A Bacillus species with growth-promoting function against plant salinity and heavy metal stress is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. The deposit date is January 14, 2026, and the accession number is CCTCC NO: M2026103. The classification name is Bacillus thuringiensis BL01 (Bacillus sp. BL01).

[0005] Another object of the present invention is to provide a method for culturing Bacillus that promotes plant growth under salt and heavy metal stress, as described above, as follows: Take OD 600 =1.0 seed culture was added to the culture medium at a volume ratio of 0.1%, and cultured on a shaker for 24 hours at a temperature of 28℃, 180 r / min, and pH=7.0.

[0006] Furthermore, the culture medium is formulated as follows: sodium chloride 10 g / L, yeast extract 5 g / L, tryptone 10 g / L, agar 15 g / L, pH=7.0.

[0007] Another object of the present application is to provide an application of the bacillus having the plant salt and heavy metal stress promoting function as a microbial product for promoting seed germination of plants under salt and heavy metal stress.

[0008] Further, the plant seed is red clover.

[0009] Another object of the present application is to provide a promoter for promoting seed germination of plants under salt and heavy metal stress, containing the bacillus having the plant salt and heavy metal stress promoting function as described above.

[0010] Further, the bacillus liquid is adjusted to OD 600 =0.50±0.02, and the liquid is treated for seed soaking at a ratio of 10ml / 100 red clover seeds, for 1h, at a temperature of 28℃, and pH=7.0.

[0011] The bacillus strain of the present application has the probiotic effect of promoting seed germination and growth of plants under salt and heavy metal stress, can be used for plant remediation of heavy metal and high salt and alkali concentration soil, has the advantages of low cost and high efficiency, can be used as a biological fertilizer to improve yield and quality, has the advantages of low cost, no pollution to the environment, safety to humans and animals, etc. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 It is a bacterial colony morphological feature diagram;

[0013] Figure 2 It is a bacterial cell gram-positive microscopic feature diagram;

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

[0015] Figure 4 It is a red clover seed germination phenotype diagram under compound salt and heavy metal stress;

[0016] Figure 5 It is a columnar diagram of the influence of the strain on the red clover seed germination potential under compound salt and heavy metal stress;

[0017] Figure 6 It is a columnar diagram of the influence of the strain on the red clover seed germination rate under compound salt and heavy metal stress;

[0018] Figure 7 It is a columnar diagram of the influence of the strain on the red clover seed radicle length under compound salt and heavy metal stress;

[0019] Figure 8 It is a columnar diagram of the influence of the strain on the red clover seed plumule length under compound salt and heavy metal stress;

[0020] Figure 9 Figure 4 is a column chart showing the effect of the strain on the seed germination index of red clover under the combined stress of salinity and heavy metals;

[0021] Figure 10 Figure 5 is a column chart showing the effect of the strain on the seed vigor index of red clover under the combined stress of salinity and heavy metals. DETAILED DESCRIPTION

[0022] The following examples further illustrate the present application, but it should be understood that the specific embodiments described are merely illustrative of the present application and are not meant to be limiting in any way.

[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 soil contained 4.5 g / Kg of lead. 5 g of the rhizosphere soil of red clover was placed in a sterile water container with 45 mL of sterile water and continuously shaken for 30 min. At this time, the concentration of the bacterial suspension was 10 -1 , and then diluted by 10 times in gradient for 5 times to a concentration of 10 -5 . From the dilutions marked 10 -3 , 10 -4 , and 10 -5 , 100 μL of bacterial suspension was taken out, and the bacterial liquid was evenly spread on LB medium (sodium chloride 10 g / L, yeast extract 5 g / L, tryptone 10 g / L, agar 15 g / L, pH=7.0) plates using a sterilized spreader. The culture dishes were sealed and inverted in a constant temperature incubator at 28°C for 48 h. The growth state of the colonies was observed, and after the colonies grew, preliminary screening was performed according to the morphology, size, and color of the colonies. Single colonies of different types were picked, and after several plate streaking purifications, single purified strains were obtained. The purified strains were stored in a -80°C refrigerator with glycerol. The strains obtained by the above isolation and screening were subjected to determination of growth-promoting ability.

[0025] Example 2: Identification of bacteria

[0026] The strain was subjected to morphological, physiological and biochemical characteristics, and 16S rDNA sequence analysis. The steps of 16S rDNA molecular identification were as follows: 1 ml of bacterial liquid culture with OD 600 =1-1.5 was collected in a 2 ml centrifuge tube, centrifuged at 12000 x g for 30 s, and the supernatant was discarded. The strain DNA was extracted using a kit.

[0027] The extracted DNA was used as a template, bacterial genome PCR amplification was performed using 27F ("5-AGAGTTTGATCCTGGCTCAG-3") and 1492R ("5-CTACGGCTACCTTGTTACGA-3") universal primers, and the sample was sent to Shanghai Shenguo Biotechnology Co., Ltd. for bacterial 16S rDNA sequencing. According to the sequence results obtained by sequencing, sequence alignment analysis was performed in the BLAST program in the NCBI website, and MEGA7.0 was used to construct a phylogenetic tree (Fig. 1) Figure 2 ), and the results showed that the bacteria were Bacillus thuringiensis.

[0028] Table 1 Physiological and biochemical test characteristics of the test strains

[0029]

[0030] Example 3 Effect of the strains on the germination of red clover under the stress of compound saline-alkali and heavy metals

[0031] The purified target strain was inoculated into LB liquid medium, and cultured at 28°C, 180 r / min shaking bed for 24 h. The bacterial cells were collected by centrifugation, and the bacterial solution was adjusted to OD 600=0.50±0.02, the bacteria liquid is treated according to the proportion of 10 ml / 100 red clover seeds, the time is 1h, the temperature is 28℃, and pH=7.0. The simulation northeast Songnen plain soil is configured with a composite saline-alkali solution (NaCl:Na2SO4:NaHCO3:Na2CO3=1:9:9:1 molar ratio), and one liter of 25 mmol / L, 50 mmol / L, 75 mmol / L and 100 mmol / L saline-alkali solution is prepared, and 200 mg of potassium pyroantimonate is added as a heavy metal pollution source. The sterilized “Qinggang” red clover seeds are divided into two parts, one part is soaked in the bacteria suspension for 1h (recorded as BL01); the other part is placed in sterile water solution for the same time as the control (recorded as X). Appropriate size of culture dishes are selected, and two layers of filter paper are placed as the germination bed, and the filter paper is soaked with the composite solution. The red clover seeds with appropriate size and full grains are selected, and 25 seeds are placed in each culture dish. The treatment is as follows: 25℃ without stress culture (CK), 25 mmol / L composite saline-alkali + 200 mg / L potassium pyroantimonate (S), 50 mmol / L composite saline-alkali + 200 mg / L potassium pyroantimonate (L), 75 mmol / L composite saline-alkali + 200 mg / L potassium pyroantimonate (M), and 100 mmol / L composite saline-alkali + 200 mg / L potassium pyroantimonate (N), and three repetitions are set for each treatment. According to the evaporation rate of water, sterile water or composite solution is added to the culture dish in time to ensure the germination of the seeds, and the temperature of the incubator is adjusted for seed germination culture experiment. From the beginning of seed inoculation, the germination potential is counted at 4d, and the germination rate is counted at 7d, then the scale is used to measure the radicle length and plumule length of the seeds, the germination index (GI)=∑Gt / Dt (Gt refers to the number of germinations within t time, and Dt refers to the germination days); the vigor index=germination index×radicle length.

[0032] Table 2 Influence of strains on germination potential of red clover seeds under different saline-alkali and heavy metal stress

[0033]

[0034] Table 3 Influence of strains on germination rate of red clover seeds under different saline-alkali and heavy metal stress

[0035]

[0036] Table 4 Influence of strains on radicle length of red clover seeds under different saline-alkali and heavy metal stress

[0037]

[0038] Table 5 Influence of strains on plumule length of red clover seeds under different saline-alkali and heavy metal stress

[0039]

[0040] Table 6 Influence of strains on seed germination index of red clover under different saline-alkali and heavy metal stress

[0041]

[0042] Table 7 Influence of strains on seed vigor index of red clover under different saline-alkali and heavy metal stress

[0043]

[0044] Through the above experiments, the results show that under low concentration of saline-alkali stress (25 mmol / L compound salt-alkali + 200 mg / L potassium pyroantimonate), the germination rate, germination potential, radicle length and plumule length, germination index and vigor index can be significantly improved (P<0.05), which indicates that low concentration of saline-alkali can promote plant growth. Using Bacillus thuringiensis BL01 liquid to treat red clover seeds can significantly improve the germination rate, germination potential, radicle length and plumule length, germination index and vigor index under high concentration of compound salt-alkali and heavy metal stress (P<0.05). It shows that Bacillus thuringiensis BL01 can be used as a seed germination promoter to alleviate the growth inhibition of plants under saline-alkali and heavy metal stress and promote the growth of plants.

Claims

1. A Bacillus species with growth-promoting function against plant salinity and heavy metal stress is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, on January 14, 2026, with accession number CCTCC NO: M 2026103, and classified as Bacillus thuringiensis BL01.

2. The method for culturing Bacillus species with growth-promoting function against plant salinity and heavy metal stress according to claim 1, characterized in that, The method is as follows: Take OD 600 =1.0 seed culture was added to the culture medium at a volume ratio of 0.1%, and cultured on a shaker for 24 hours at a temperature of 28℃, 180 r / min, and pH=7.

0.

3. A method for culturing Bacillus species with growth-promoting functions against plant salinity and heavy metal stress according to claim 2, characterized in that, The culture medium has the following formula: sodium chloride 10 g / L, yeast extract 5 g / L, tryptone 10 g / L, agar 15 g / L, pH=7.

0.

4. The application of the Bacillus species with growth-promoting function against salt-alkali and heavy metal stress as described in claim 1 as a microbial product for promoting seed germination in plants under salt-alkali and heavy metal stress.

5. The application according to claim 4, characterized in that: The plant seeds mentioned are red clover.

6. A seed germination promoter for plants under saline-alkali and heavy metal stress, characterized in that: It contains a Bacillus species as described in claim 1 that has a growth-promoting function against plant salinity and heavy metal stress.

7. A seed germination promoter for plants under heavy metal stress according to claim 6, characterized in that: The bacterial culture of the aforementioned Bacillus was adjusted to OD. 600 =0.50±0.02, the bacterial solution was used to soak 100 red clover seeds at a ratio of 10 ml: 100 seeds for 1 hour, at a temperature of 28℃ and a pH of 7.0.

Images