Saline-alkali tolerant bacteria strain and application thereof

Abstract

The invention discloses a saline-alkaline tolerant bacterium strain and application thereof. The saline-alkaline tolerant bacteria strain is selected from any one or more of the following bacteria: Pseudoclavibacter sp., the preservation number of which is CGMCC No.32646, the preservation number of which is CGMCC No.32646, the preservation number of which is CGMCC No.32646, and the preservation number of which is CGMCC No.32646. The preservation number of the providencia sp. Is CGMCC (China General Microbiological Culture Collection Center) No.32647, and the preservation number of the providencia sp. Is CGMCC No.32647. The preservation number of the planococcus sp. Is CGMCC No.32648, the planococcus sp. Is preserved in the China General Microbiological Culture Collection Center (CGMCC) on November 15, 2024, and the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China. The bacillus amyloliquefaciens and the bacillus amyloliquefaciens have the capabilities of fixing nitrogen The saline-alkaline tolerant growth of plants can be obviously promoted, the content of chlorophyll and proline in rice is increased, the activity of antioxidant enzyme is improved, and the content of malondialdehyde is reduced; in addition, the providencia has nitrogen fixation capacity, the pseudoochrobactrum pseudococcum has iron production capacity, and the motility coccus has iron production and phosphorus solubilizing capacity.

Description

Technical Field

[0001] The invention relates to a salt-alkali resistant bacterial strain and application thereof, belonging to the technical field of agricultural biological product production. Background Art

[0002] Salt stress is a major abiotic stress faced by plants. Salt stress can cause damage to plants such as osmotic stress, oxidative stress, and ion toxicity, inhibiting normal plant growth and leading to reduced crop yields. Studies have shown that rhizosphere growth-promoting bacteria can enhance crop salt tolerance by assisting plants in rebuilding ion and osmotic balance, reducing cell damage to crop plants caused by salt stress, and promoting the growth of crops under salt stress. Therefore, using rhizosphere growth-promoting bacteria to enhance crop salt tolerance provides an environmentally friendly and efficient new strategy for improving saline-alkali agricultural productivity. At present, there are few strains that can tolerate salt and alkali and promote plant growth, and the effect is not good. Summary of the invention

[0003] Purpose of the invention: The purpose of the present invention is to provide salt-alkali tolerant bacterial strains and applications thereof.

[0004] Technical solution: The present invention provides a salt-alkali tolerant bacterial strain, which is selected from any one or more of the following: Pseudoclavibacter sp., with a preservation number of CGMCC No.32646; Providencia sp., with a preservation number of CGMCC No.32647; Planococcus sp., with a preservation number of CGMCC No.32648, all of which were deposited in the General Microbiology Center of China National Committee for the Preservation of Microorganisms (CGMCC) on November 15, 2024, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China.

[0005] Furthermore, the 16S rRNA nucleotide sequence of Pseudomonas aeruginosa is shown as SEQ ID NO.1, the 16S rRNA nucleotide sequence of Providencia is shown as SEQ ID NO.2, and the 16S rRNA nucleotide sequence of Moinecoccus is shown as SEQ ID NO.3.

[0006] The present invention also provides a growth-promoting bacterial agent, the active ingredient of the growth-promoting bacterial agent is the above-mentioned salt-alkali tolerant bacterial strain.

[0007] The present invention also provides the use of the salt-alkali tolerant bacterial strain and growth-promoting bacterial agent in promoting the salt-alkali tolerant growth of plants.

[0008] Furthermore, the application includes promoting the germination of plant seeds and promoting the height and root growth of plant seedlings.

[0009] Furthermore, the method for promoting plant height and root growth of crop seedlings is to irrigate the roots of plants with a bacterial suspension containing the above-mentioned salt-alkali tolerant bacterial strain.

[0010] The present invention also provides the use of the salt-alkali tolerant bacteria strain in the preparation of a product for promoting the synthesis of chlorophyll in rice leaves.

[0011] The present invention also provides the use of the salt-alkali tolerant bacterial strain in the preparation of nitrogen-fixing products, wherein the strain is Providencia sp.

[0012] The present invention also provides the use of the salt-alkali tolerant bacterial strain in the preparation of products producing indoleacetic acid or plant resistance inducers.

[0013] The present invention also provides the use of the salt-alkali tolerant bacterial strain in the preparation of iron-producing products, wherein the bacterial strain is Pseudoclavibacter sp. and / or Planococcus sp.

[0014] The present invention also provides the use of the salt-alkali tolerant bacterial strain in the preparation of phosphorus-dissolving products, wherein the strain is Planococcus sp.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following outstanding and significant advantages: the Pseudo-pseudo-flavonoids, Providencia and Motile cocci separated by the present invention all have the ability to produce indoleacetic acid; can significantly promote the salt-alkali tolerance growth of plants, increase the content of chlorophyll and proline in rice, increase the activity of antioxidant enzymes, and reduce the content of malondialdehyde; in addition, Providencia and have nitrogen fixation ability, Pseudo-pseudo-flavonoids has iron production ability, and Motile cocci has iron production and phosphorus dissolving ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the phylogenetic tree of RC1.

[0017] Figure 2 This is the phylogenetic tree of RC2.

[0018] Figure 3 This is the phylogenetic tree of RC4.

[0019] Figure 4 The colonies of RC1 grown on nutrient broth solid medium.

[0020] Figure 5 The colonies of RC2 grown on nutrient broth solid medium.

[0021] Figure 6 The colonies of RC4 on nutrient broth solid medium.

[0022] Figure 7 Gram stain for RC1.

[0023] Figure 8 Gram stain for RC2.

[0024] Fig. 9 Gram stain for RC4.

[0025] Fig.10 The growth of RC2 on Axubei solid medium.

[0026] Fig.11 The standard curve of indoleacetic acid.

[0027] Fig.12 The growth of RC1 on CAS solid medium.

[0028] Fig.13 The growth of RC4 on CAS solid medium.

[0029] Fig.14 The growth of RC4 on Montana inorganic phosphate solid medium.

[0030] Fig.15 This is the growth status of rice under the treatments of no inoculation and inoculation with RC1.

[0031] Fig.16 This is the growth status of rice under the treatments of no inoculation and inoculation with RC2.

[0032] Fig.17 This is the growth status of rice under the treatments of no inoculation and inoculation with RC4.

[0033] Fig.18 The standard curve of proline. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0035] Example 1 Isolation and identification of strains

[0036] (I) Isolation and purification of strains

[0037] Weigh 10g of rice rhizosphere soil planted in coastal saline-alkali soil and place it in a triangular flask containing 90mL of sterile water to make a suspension and perform gradient dilution. Take 100μL of different gradient dilutions and evenly spread them on Luria-Bertani solid medium, and invert and culture at 28℃ for 48h. After the culture, single colonies were selected according to the morphological characteristics of the colonies, and further streaked and purified to obtain pure strains RC1, RC2 and RC4. RC1, RC2 and RC4 grow well on Luria-Bertani solid medium containing 5% sodium chloride.

[0038] (II) Identification of strains

[0039] 1. Molecular identification

[0040] The 16S rRNA sequences of RC1, RC2 and RC4 were amplified using primers 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3'. Amplification system: 27F (20 μmol) 1 μL, 1492R (20 μmol) 1 μL, dNTP 4 μL, 10×Buffer 5 μL, Taq DNA polymerase 0.25 μL, DNA 2 μL, ddH2O to 50 μL. Amplification program: 95℃ pre-denaturation for 3 min, 95℃ deformation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 40 s, 35 cycles, 72℃ extension for 10 min. After purification, the PCR product was sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing. The sequences were spliced ​​using DNAMAN software, and the spliced ​​sequences are shown below.

[0041]

[0042]

[0043]

[0044] The spliced ​​sequences were compared with the sequences in the GenBank database of NCBI, and the strain sequences and their highly similar model strain sequences were used to construct a phylogenetic tree using the neighbor-joining method using MAGE5.2 software. Figure 1 As shown in the figure, RC1 belongs to the same branch as Pseudoclavibacter sp., so RC1 was identified as Pseudoclavibacter sp. Figure 2 As shown in the figure, RC2 and Providencia sp. PROV170 belong to the same smallest branch, so RC2 was identified as Providencia sp. Figure 3 As shown, strain RC4 belongs to the same branch as Planococcus sp., so RC4 is identified as Planococcus sp. The strain RC1 is Pseudoclavibacter sp., RC2 is Providencia sp., and RC4 is Planococcus sp. The preservation information is: Pseudoclavibacter sp., the preservation number is CGMCC No.32646; Providencia sp., the preservation number is CGMCC No.32647; Planococcus sp., the preservation number is CGMCC No.32648, all of which were deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration on November 15, 2024, and the preservation address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China.

[0045] 2. Morphological observation

[0046] RC1, RC2 and RC4 were inoculated on nutrient broth solid medium, and the morphology of RC1, RC2 and RC4 colonies was observed. Figures 4 to 6 As shown, the colonies of RC1 on the nutrient broth solid medium are round, yellow, opaque, with smooth edges, and the surface of the colonies is moist and convex; the colonies of RC2 on the nutrient broth solid medium are round, off-white, opaque, with smooth edges, and the surface of the colonies is moist and convex; the colonies of RC4 on the nutrient broth solid medium are round, orange, opaque, with smooth edges, and the surface of the colonies is moist and convex. Gram staining of the RC1, RC2 and RC4 is as follows Figure 7 to Figure 9 As shown, RC1 and RC4 were stained purple by Gram staining, and RC2 was stained red by Gram staining, indicating that RC1 and RC4 were Gram-positive rods, and RC2 was a Gram-negative bacterium.

[0047] (III) Identification of growth-promoting properties of strains

[0048] 1. Identification of nitrogen fixation ability of strains

[0049] RC1, RC2 and RC4 were streaked onto Axubei solid medium and incubated at 28°C for 72 hours. After the culture, the growth of the strain was observed. If the growth was good, it was identified as having nitrogen fixation ability. Among them, RC2 could grow normally on Axubei nitrogen-free solid medium ( Fig.10 ), indicating that RC2 has the ability to fix nitrogen.

[0050] 2. Identification of the ability of strains to produce indoleacetic acid

[0051] Prepare a series of indoleacetic acid standard solutions (concentrations are 0, 5, 10, 20, 30, 40, 50, 60 mg / L), take different standard solutions and add equal volumes of Salkowski colorimetric solution, mix well and react in the dark for 30 minutes, use an ultraviolet spectrophotometer to measure the absorbance (OD530) at a wavelength of 530 nm, and draw an indoleacetic acid standard curve. The standard curve is as follows Fig.11 shown.

[0052] 200 μL of RC1, RC2 or RC4 bacterial liquid was inoculated into a nutrient broth liquid medium containing 100 mg / L L-tryptophan, and after shaking at 30°C and 150 r / min for 48 h, the supernatant was collected by centrifugation at 4000 r / min for 10 min, and an equal volume of Salkowski colorimetric solution was added and mixed. At the same time, a 50 mg / L indoleacetic acid standard was used as a positive control, and a sterile culture medium was used as a negative control. After 30 min of light-proof reaction, the color change was observed. If a red reaction was produced, it means that the strain can produce IAA. Further, the absorbance of the reaction solution was measured at a wavelength of 530 nm using an ultraviolet spectrophotometer, and the ability of the strain to produce indoleacetic acid was quantified according to the standard curve. The results are shown in Table 1. Each milliliter of RC1 culture medium contained 9.99 μg of indoleacetic acid, each milliliter of RC2 culture medium contained 25.48 μg of indoleacetic acid, and each milliliter of RC4 culture medium contained 9.28 μg of indoleacetic acid.

[0053] Table 1 Indoleacetic acid production ability of strain RC2

[0054]

[0055]

[0056] 3. Identification of the ability of strains to produce siderophores

[0057] RC1, RC2 and RC4 were spotted onto CAS solid culture medium and inverted at 28°C for 48 hours. After the culture was completed, the colonies were observed to see if an orange-yellow halo was formed around them. If an orange-yellow halo was formed, it was identified as having the ability to produce iron carriers. The results showed that there was an orange-yellow halo around RC1 and RC4 on the CAS solid culture medium ( Fig.12 and Fig.13 ), indicating that RC1 and RC4 have the ability to produce siderophores.

[0058] 4. Identification of the Phosphate Solubilizing Ability of the Strain

[0059] RC1, RC2 and RC4 were inoculated onto the Montkina inorganic phosphorus solid medium and inverted for 48 hours at 28°C. After the incubation, observe whether a transparent circle can be formed around the colony. If there is a transparent circle, it is identified that the strain has the ability to dissolve phosphorus. The results showed that there was a transparent circle around the strain RC4 on the Montkina organic phosphorus medium ( Fig.14 ), indicating that strain RC4 has the ability to solubilize phosphate.

[0060] Example 2 Effect of strains on rice under salt stress

[0061] 1. Effects of strains on rice germination

[0062] RC1, RC2 and RC4 were inoculated into Luria-Bertani medium respectively, and cultured at 28°C and 180 r / min for 24 h, and then the cells were collected, washed 2-3 times with sterile water, and resuspended to an absorbance of OD600 = 1.0.

[0063] The surface-sterilized rice seeds were placed in RC1, RC2 and RC4 bacterial suspensions for 24 h, and a sterile water seed soaking treatment was set as a control (CK). After soaking, the rice seeds were placed in culture dishes containing sterile wet filter paper containing 0 mM NaCl and 100 mM NaCl, with 40 seeds per dish. The seed germination rate, root length and shoot length were measured on the 8th day of culture. The calculation formulas of relevant indicators are as follows: germination rate = (number of seeds germinated in the first 8 days / total number of tested seeds) × 100%, relative germination rate = (germination rate treated with 100mM NaCl / germination rate treated with 0mM NaCl) × 100%, relative salt damage rate = [(germination rate treated with 0mM NaCl-germination rate treated with 100mM NaCl) / germination rate treated with 0mM NaCl] × 100%, relative root length = (root length treated with 100mM NaCl / root length treated with 0mM NaCl) × 100%, relative shoot length = (shoot length treated with 100mM NaCl / shoot length treated with 0mM NaCl) × 100%; root-to-crown ratio = (root length / shoot length) × 100%, relative root-to-crown ratio = (root-to-crown ratio treated with 100mM NaCl / root-to-crown ratio treated with 0mM NaCl) × 100%. The results are shown in Table 2. Compared with the control, the relative germination rate, relative root length, relative shoot length and relative root-shoot ratio of rice inoculated with RC1, RC2 and RC4 were significantly increased, while the relative salt damage rate was significantly decreased; this indicates that RC1, RC2 and RC4 seed soaking can alleviate the negative impact of salt stress on rice seed germination.

[0064] Table 2 Related indicators of rice germination period

[0065] deal with Relative germination rate Relative salt damage rate Relative root length Relative bud length Relative root-shoot ratio CK 86.67 15.38 39.20 79.41 49.36 RC1 97.56 2.50 82.66 82.63 100.04 RC2 97.73 2.33 154.44 87.62 176.27 RC4 96.63 3.49 128.05 97.98 130.69

[0066] 2. Effects of strains on rice seedlings

[0067] Select rice seedlings with consistent growth and transplant them into coastal saline-alkali soil with a salt content of 1.5 g / kg. After 7 days of cultivation, take RC1, RC2 and RC4 bacterial suspensions and evenly irrigate around the rice roots. The inoculation concentration is 10 8 CFU / g soil. At the same time, the same amount of sterile water was used as a control. The rice seedlings were cultured under natural conditions. Watering was observed every day to maintain a 2-3 cm water layer. After 30 days of growth, various physiological and biochemical indicators of rice were measured.

[0068] (1) Biomass determination

[0069] Select representative cultures for photography ( Figure 15 to Figure 17 ), and found that the growth of rice inoculated with RC1(T4), RC2(T2), and RC4(T3) was better than that of rice inoculated with the control treatment.

[0070] Take the complete rice plant, rinse the soil around the root with clean water, then use filter paper to absorb the residual water, and then use a ruler to measure the seedling height and taproot length and record them. The rice roots, stems and leaves were separated and placed in a constant temperature drying oven at 115°C for 30 minutes, and then dried at 80°C to constant weight. The dry weight of the roots and stems was weighed and recorded using an electronic balance. The results are shown in Table 3. The root length, plant height, root dry weight and aboveground dry matter weight of rice inoculated with RC1, RC2 and RC4 were all higher than those of the control treatment, indicating that inoculation with RC1, RC2 and RC4 promoted the growth of rice under salt stress.

[0071] Table 3 Root length, plant height, root dry weight and aboveground dry matter weight of rice

[0072] deal with Root length (cm) Plant height(cm) Root dry weight (g / hole) Aboveground dry matter weight (g / hole) CK 14.1±0.2 31.3±1.8 0.27±0.04 1.00±0.07 RC1 20.6±1.8 38.7±0.6 0.57±0.11 1.90±0.26 RC2 19.4±1.5 34.7±0.8 0.45±0.10 1.37±0.23 RC4 21.3±1.8 36.8±1.6 0.63±0.06 1.85±0.13

[0073] (2) Determination of chlorophyll content

[0074] The ethanol extraction colorimetric method was used to determine the contents of chlorophyll a, chlorophyll b and chlorophyll in rice leaves. The contents of chlorophyll a, chlorophyll b and chlorophyll in rice leaves are shown in Table 4. Compared with the control, the contents of chlorophyll a and chlorophyll b in rice leaves inoculated with RC1, RC2 and RC4 were higher than those in the control treatment, indicating that inoculation with RC1, RC2 and RC4 improved the photosynthesis of rice under salt stress.

[0075] Table 4 Chlorophyll content of rice leaves (mg / g FW)

[0076] deal with Chlorophyll a Chlorophyll b Chlorophyll CK 0.89±0.07 0.82±0.04 1.71±0.08 RC1 1.06±0.07 1.08±0.26 2.15±0.22 RC2 0.93±0.08 1.19±0.23 2.12±0.16 RC4 0.91±0.13 1.36±0.16 2.27±0.03

[0077] (3) Free proline content

[0078] The free proline content in rice leaves and roots was determined by sampling sulfosalicylic acid extraction-acid ninhydrin colorimetry. The standard curve is shown in Fig.18 The proline content in rice leaves and roots is shown in Table 5. Compared with the control, the proline content in rice leaves and roots inoculated with RC1, RC2 and RC4 was higher, indicating that inoculation with RC1, RC2 and RC4 enhanced the tolerance of rice to salt stress, among which the effect of RC4 was the most prominent.

[0079] Table 5 Proline content in rice leaves and roots (mg / g FW)

[0080] deal with Proline content in leaves Proline content in roots CK 15.21±1.78 4.73±0.46 RC1 39.45±3.44 18.03±1.62 RC2 34.78±2.86 8.73±0.83 RC4 61.16±2.98 26.30±2.38

[0081] (4) Determination of antioxidant enzyme activity

[0082] The superoxide dismutase activity, peroxidase activity and catalase activity in rice tissues (roots and leaves) were detected by nitroblue tetrazolium method, guaiacol method and ultraviolet absorption method respectively. Antioxidant enzyme activity As shown in Table 6, the superoxide dismutase, catalase and peroxidase activities of rice leaves and roots inoculated with RC1, RC2 and RC4 were higher than those of the control treatment, indicating that inoculation with RC1, RC2 and RC4 can protect rice leaf and root plant cells from oxidative damage.

[0083] Table 6 Antioxidant enzyme activities in rice leaves and roots (U / g FW)

[0084]

[0085]

[0086] (5) Determination of malondialdehyde content

[0087] The malondialdehyde content in rice roots and leaves was determined by thiobarbituric acid colorimetry. The malondialdehyde content results are shown in Table 7. Compared with the control, the malondialdehyde content in rice leaves and roots inoculated with RC1, RC2 and RC4 was lower, indicating that inoculation with RC1, RC2 and RC4 can reduce the degree of damage to the membrane system under salt stress.

[0088] Table 7 Malondialdehyde content in rice leaves and roots (mg / g FW)

[0089] deal with Malondialdehyde content in leaves Malondialdehyde content in roots CK 41.10±0.27 42.04±2.65 RC1 33.62±0.27 25.63±0.35 RC2 34.55±1.62 29.32±0.26 RC4 31.22±0.41 24.95±0.43 .

Claims

1. A salt-alkali tolerant bacterial strain, characterized in that: The salt-alkali tolerant bacterial strain is selected from any one or more of the following: Pseudoclavibacter sp., with a preservation number of CGMCC No. 32646; Providencia sp., with a preservation number of CGMCC No. 32647; Planococcus sp., with a preservation number of CGMCC No. 32648, all of which were deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration (CGMCC) on November 15, 2024, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China.

2. The salt-alkali tolerant bacteria according to claim 1, characterized in that The 16S rRNA nucleotide sequence of Pseudomonas aeruginosa is shown as SEQ ID NO.1, the 16S rRNA nucleotide sequence of Providencia is shown as SEQ ID NO.2, and the 16S rRNA nucleotide sequence of Moinecoccus is shown as SEQ ID NO.

3.

3. A growth-promoting bacterial agent, characterized in that: The active ingredient of the growth-promoting bacterial agent is the salt-alkali-tolerant bacterial strain described in any one of claims 1 to 2.

4. Use of the salt-alkali tolerant bacterial strain according to any one of claims 1 to 2 and the growth-promoting bacterial agent according to claim 3 in promoting the salt-alkali tolerant growth of plants.

5. The use according to claim 4, characterized in that: The application includes promoting the germination of plant seeds and promoting the height and root growth of plant seedlings.

6. Use of the salt-alkali tolerant bacterial strain according to any one of claims 1 to 2 in the preparation of a product for promoting the synthesis of chlorophyll in rice leaves.

7. Use of the salt-tolerant alkali bacteria strain according to any one of claims 1 to 2 in the preparation of nitrogen-fixing products, characterized in that: The strain is Providencia sp.

8. Use of the salt-alkali tolerant bacterial strain according to any one of claims 1 to 2 in the preparation of products producing indoleacetic acid or plant resistance inducers.

9. Use of the salt-alkali tolerant bacterial strain according to any one of claims 1 to 2 in the preparation of iron-producing products, characterized in that: The strains are Pseudoclavibacter sp. and / or Planococcus sp.

10. Use of the salt-alkali tolerant bacterial strain according to any one of claims 1 to 2 in the preparation of a phosphate-dissolving product, characterized in that: The strain is Planococcus sp.

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