Salt-tolerant bacterial strain and application thereof

Abstract

The application discloses a salt and alkali tolerant bacterial strain and application thereof. The salt and 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, and Planococcus sp. with a preservation number of CGMCC No. 32648, which are all preserved in the China General Microbiological Culture Collection Center (CGMCC) on November 15, 2024, and the preservation address is No. 1, Xibanchenli, Chaoyang District, Beijing, China. All of them have the abilities of nitrogen fixation and indole acetic acid production; can significantly promote the growth of plants in salt and alkali, improve the content of chlorophyll and proline in rice, improve the antioxidant enzyme activity, and reduce the content of malondialdehyde; in addition, the Providencia sp. has the ability of nitrogen fixation, the Pseudoclavibacter sp. has the ability of iron production, and the Planococcus sp. has the abilities of iron production and phosphorus dissolution.

Description

Technical Field

[0001] This invention relates to salt-tolerant bacterial strains and their applications, belonging to the field of agricultural biological product manufacturing technology. Background Technology

[0002] Salt stress is a major abiotic stress faced by plants. It causes damage 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 restoring ion and osmotic balance, reducing cell damage caused by salt stress, and promoting crop growth under salt stress. Therefore, utilizing rhizosphere growth-promoting bacteria to enhance crop salt tolerance provides an environmentally friendly and efficient new strategy for improving the productivity of saline-alkali agriculture. Currently, there are few strains that are salt-tolerant and promote plant growth, and their effectiveness is limited. Summary of the Invention

[0003] Purpose of the invention: The purpose of this invention is to provide salt-tolerant bacteria strains and their applications.

[0004] Technical Solution: The present invention provides a salt-tolerant bacterial strain, wherein the salt-tolerant bacterial strain is selected from any one or more of the following: *Pseudoclavibacter sp.*, accession number CGMCC No. 32646; *Providencia sp.*, accession number CGMCC No. 32647; and *Planococcus sp.*, accession number CGMCC No. 32648, all of which were deposited on November 15, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China.

[0005] Furthermore, the 16S rRNA nucleotide sequence of *Pseudomonas pseudophlebsiella* is shown in SEQ ID NO.1, the 16S rRNA nucleotide sequence of *Providencia* is shown in SEQ ID NO.2, and the 16S rRNA nucleotide sequence of *Aggregatibacter* is shown in SEQ ID NO.3.

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

[0007] This invention also provides the application of the above-mentioned salt-tolerant bacteria strains and growth-promoting bacteria agents in promoting the salt-tolerant growth of plants.

[0008] Furthermore, the application includes promoting plant seed germination and promoting plant seedling height and root growth.

[0009] Furthermore, the method for promoting the plant height and root growth of crop seedlings is to drench the roots of the plants with a bacterial suspension containing the aforementioned salt-tolerant bacterial strains.

[0010] The present invention also provides the application of the above-mentioned salt-tolerant bacteria strains in the preparation of products that promote chlorophyll synthesis in rice leaves.

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

[0012] The present invention also provides the application of the above-mentioned salt-tolerant bacteria strains in the preparation of products that produce indoleacetic acid or plant resistance inducers.

[0013] The present invention also provides the application of the above-mentioned salt-tolerant bacterial strains in the preparation of iron-producing products, wherein the strains are Pseudoclavibacter sp. and / or Planococcus sp.

[0014] The present invention also provides the application of the above-mentioned salt-tolerant bacterial strain in the preparation of products for phosphate solubilization, wherein the strain is Plantococcus sp.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following outstanding advantages: The pseudo-pseudo-pheloides, *Providencia*, and *Aggregatibacterium* isolated by the present invention all have the ability to produce indoleacetic acid; they can significantly promote the salt-alkali tolerance of plants, increase the chlorophyll and proline content in rice, increase the activity of antioxidant enzymes, and reduce the malondialdehyde content; in addition, *Providencia* has nitrogen-fixing ability, *Pseudo-pheloides* has iron-producing ability, and *Aggregatibacterium* has iron-producing and phosphorus-solubilizing ability. Attached Figure Description

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

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

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

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

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

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

[0022] Figure 7 Gram staining for RC1.

[0023] Figure 8 Gram staining for RC2.

[0024] Figure 9 Gram staining for RC4.

[0025] Figure 10 The growth of RC2 on Assumption solid medium.

[0026] Figure 11 This is the standard curve for indoleacetic acid.

[0027] Figure 12 The growth of RC1 on solid culture medium was detected using CAS.

[0028] Figure 13 The growth of RC4 on solid culture medium was detected using CAS.

[0029] Figure 14 The growth of RC4 on Monkina inorganic phosphorus solid medium.

[0030] Figure 15 The growth status of rice under the RC1 inoculation and non-inoculation treatments.

[0031] Figure 16 The growth status of rice under the RC2 inoculation and non-inoculation treatments.

[0032] Figure 17 The growth status of rice under the RC4-free and RC4-inoculated treatments.

[0033] Figure 18 This is the standard curve for proline. Detailed Implementation

[0034] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0035] Example 1: Isolation and Identification of Strains

[0036] (I) Isolation and purification of strains

[0037] 10g of rhizosphere soil from rice planted in coastal saline-alkali soil was weighed and placed in an Erlenmeyer flask containing 90mL of sterile water to prepare a suspension. The suspension was then serially diluted, and 100μL of each dilution was evenly spread onto Luria-Bertani solid medium and incubated upside down at 28℃ for 48h. After incubation, single colonies were selected based on their morphological characteristics and further purified by streak plating to obtain pure strains RC1, RC2, and RC4. RC1, RC2, and RC4 grew 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'). The amplification system consisted of 1 μL of 27F (20 μmol), 1 μL of 1492R (20 μmol), 4 μL of dNTPs, 5 μL of 10×Buffer, 0.25 μL of Taq DNA polymerase, 2 μL of DNA, and ddH2O to a final volume of 50 μL. The amplification program was as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 40 s, for 35 cycles, followed by a final extension at 72℃ for 10 min. The purified PCR products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequences were then assembled using DNAMAN software, and the assembled sequences are shown below.

[0041]

[0042]

[0043]

[0044] The assembled sequences were compared with sequences in the GenBank database of NCBI, and a phylogenetic tree was constructed using the neighbor-joining method with MAGE 5.2 software to connect the strain sequences with highly similar type strain sequences. Figure 1 As shown, RC1 belongs to the same clade as Pseudoclavibacter sp., therefore RC1 was identified as Pseudoclavibacter. Figure 2 As shown, RC2 belongs to the same smallest clade as Providencia sp. PROV170, therefore RC2 was identified as Providencia. Figure 3 As shown, strain RC4 belongs to the same clade as Planococcus sp., therefore, RC4 was identified as a zoococcus. The strains RC1, RC2, and RC4 are all *Pseudoclavibacter* sp., *Providencia* sp., and *Planococcus* sp., respectively. The preservation information is as follows: *Pseudoclavibacter* sp., preservation number CGMCC No. 32646; *Providencia* sp., preservation number CGMCC No. 32647; and *Planococcus* sp., preservation number CGMCC No. 32648, were all deposited on November 15, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China.

[0045] 2. Morphological observation

[0046] RC1, RC2, and RC4 were inoculated onto nutrient broth solid medium, and the morphological characteristics of the RC1, RC2, and RC4 colonies were observed. Figures 4-6 As shown, RC1 colonies on nutrient broth solid medium are round, yellow, opaque, with smooth edges, and a moist, raised surface; RC2 colonies on nutrient broth solid medium are round, off-white, opaque, with smooth edges, and a moist, raised surface; RC4 colonies on nutrient broth solid medium are round, orange, opaque, with smooth edges, and a moist, raised surface. Gram staining was performed on RC1, RC2, and RC4, and the results are as follows. Figures 7-9 As shown, RC1 and RC4 stain purple with Gram staining, while RC2 stains red with Gram staining, indicating that RC1 and RC4 are Gram-positive bacilli, and RC2 is a Gram-negative bacillus.

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

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

[0049] RC1, RC2, and RC4 were streaked onto Assumption solid medium and incubated upside down at 28°C for 72 hours. After incubation, the growth of the strains was observed; good growth indicated nitrogen-fixing ability. RC2 was able to grow normally on nitrogen-free Assumption solid medium. Figure 10 This indicates that RC2 has nitrogen-fixing capabilities.

[0050] 2. Identification of the strain's ability to produce indoleacetic acid

[0051] Prepare a series of indoleacetic acid (IAA) standard solutions (concentrations of 0, 5, 10, 20, 30, 40, 50, and 60 mg / L). Add an equal volume of Salkowski colorimetric solution to each standard solution, mix well, and react in the dark for 30 min. Measure the absorbance (OD530) at 530 nm using a UV spectrophotometer. Plot an IAA standard curve. The standard curve is shown below. Figure 11 As shown.

[0052] 200 μL of RC1, RC2, or RC4 bacterial cultures were inoculated into nutrient broth containing 100 mg / L L-tryptophan, respectively. After incubation at 30°C and 150 rpm for 48 h, the supernatant was collected by centrifugation at 4000 rpm for 10 min, and an equal volume of Salkowski colorimetric solution was added and mixed thoroughly. A 50 mg / L indoleacetic acid (IAA) standard was used as a positive control, and sterile culture medium was used as a negative control. The reaction was carried out in the dark for 30 min, and the color change was observed. A red reaction indicated that the strain could produce IAA. Furthermore, the absorbance of the reaction solution was measured at 530 nm using a UV spectrophotometer, and the indoleacetic acid production capacity of the strain was quantified according to a standard curve. The results are shown in Table 1. Each mL of RC1 culture medium contained 9.99 μg of IAA, each mL of RC2 culture medium contained 25.48 μg of IAA, and each mL of RC4 culture medium contained 9.28 μg of IAA.

[0053] Table 1. Indoleacetic acid production capacity of strain RC2

[0054]

[0055]

[0056] 3. Identification of the strain's ability to produce siderophores

[0057] RC1, RC2, and RC4 were spot-inoculated onto CAS detection solid medium and incubated upside down at 28°C for 48 hours. After incubation, the presence of orange-yellow halos around the colonies was observed. The presence of orange-yellow halos indicated the colonies' ability to produce siderophores. The results showed that RC1 and RC4 exhibited orange-yellow halos around their colonies on the CAS detection solid medium. Figure 12 and Figure 13 This indicates that RC1 and RC4 have the ability to produce iron carriers.

[0058] 4. Identification of the strain's phosphate-solubilizing ability

[0059] RC1, RC2, and RC4 were inoculated onto Monkina inorganic phosphorus solid medium and incubated upside down at 28°C for 48 hours. After incubation, the formation of a clear zone around the colonies was observed. The presence of a clear zone indicated that the strain possessed phosphate-solubilizing function. The results showed that a clear zone existed around strain RC4 on Monkina organic phosphorus medium. Figure 14 This indicates that strain RC4 has the ability to solubilize phosphorus.

[0060] Example 2: Effects of strain on rice under salt stress

[0061] 1. Effects of bacterial strains on rice germination period

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

[0063] Surface-sterilized rice seeds were soaked in bacterial suspensions of RC1, RC2, and RC4 for 24 hours, respectively, with a sterile water soaking treatment as a control (CK). After soaking, the rice seeds were cultured in sterile moistened filter paper containing 0 mM NaCl and 100 mM NaCl, respectively, with 40 seeds per dish. Germination rate, root length, and shoot length were measured on day 8 of culture. The relevant indicators are calculated using the following formulas: Germination rate = (Number of seeds germinated in the first 8 days / Total number of seeds tested) × 100%; Relative germination rate = (Germination rate of 100mM NaCl treatment / Germination rate of 0mM NaCl treatment) × 100%; Relative salt damage rate = [(Germination rate of 0mM NaCl treatment - Germination rate of 100mM NaCl treatment) / Germination rate of 0mM NaCl treatment] × 100%; Relative root length = (Root length of 100mM NaCl treatment / Root length of 0mM NaCl treatment) × 100%; Relative shoot length = (Shoot length of 100mM NaCl treatment / Shoot length of 0mM NaCl treatment) × 100%; Root-shoot ratio = (Root length / Shoot length) × 100%; Relative root-shoot ratio = (Root-shoot ratio of 100mM NaCl treatment / Root-shoot ratio of 0mM NaCl treatment) × 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-to-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 soaking rice seeds with RC1, RC2 and RC4 can alleviate the negative impact of salt stress on rice seed germination.

[0064] Table 2. Relevant Indicators for Rice Germination Stage

[0065] 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 bacterial strains on rice seedling stage

[0067] Rice seedlings with uniform growth were selected and transplanted into coastal saline-alkali soil with a salt content of 1.5 g / kg. After 7 days of cultivation, RC1, RC2, and RC4 bacterial suspensions were evenly poured around the rice roots, with an inoculation concentration of 10. 8 CFU / g soil. A control group was also included, irrigated with an equal volume of sterile water. Rice seedlings were cultivated under natural conditions. Watering was monitored daily, maintaining a water layer of 2-3 cm. Various physiological and biochemical indicators of the rice were measured after 30 days of growth.

[0068] (1) Biomass determination

[0069] Select representative cultures for photography. Figures 15-17 It was found that rice inoculated with RC1(T4), RC2(T2), and RC4(T3) grew better than the control rice.

[0070] Intact rice plants were harvested, and the soil around the roots was rinsed off with clean water. Residual moisture was then absorbed with filter paper. The seedling height and taproot length were measured and recorded using a ruler. The rice roots, stems, and leaves were separated and placed in a constant-temperature drying oven at 115℃ for 30 minutes to kill the greening agent, followed by drying at 80℃ to constant weight. The dry weight of the roots and stems was weighed using an electronic balance and recorded. The results are shown in Table 3. Rice plants inoculated with RC1, RC2, and RC4 showed higher root length, plant height, root dry weight, and aboveground dry matter weight than the control group, indicating that inoculation with RC1, RC2, and RC4 promoted rice growth under salt stress.

[0071] Table 3. Rice root length, plant height, root dry weight, and aboveground dry weight

[0072] 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) Chlorophyll content determination

[0074] The contents of chlorophyll a, chlorophyll b, and chlorophyll in rice leaves were determined by ethanol extraction colorimetric method. Table 4 shows the contents of chlorophyll a, chlorophyll b, and chlorophyll in rice leaves. 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 photosynthesis in rice under salt stress.

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

[0076] 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 using a sulfosalicylic acid extraction-acid ninhydrin colorimetric method. The standard curve is shown below. Figure 18 As shown in Table 5, the proline content in rice leaves and roots was higher than that in the control. This indicates that inoculation with RC1, RC2 and RC4 enhanced the rice's tolerance to salt stress, with RC4 showing the most significant effect.

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

[0080] 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) Antioxidant enzyme activity assay

[0082] The activities of superoxide dismutase (SOD), peroxidase (PCP), and catalase in rice tissues (roots and leaves) were detected using the nitroblue tetrazolium method, the guaiacol method, and the ultraviolet absorption method, respectively. The antioxidant enzyme activities are shown in Table 6. The activities of SOD, PCP, and PCP in rice leaves and roots inoculated with RC1, RC2, and RC4 were higher than those in the control treatment, indicating that inoculation with RC1, RC2, and RC4 can protect rice leaf and root 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 (MDA) content in rice roots and leaves was determined using the thiobarbituric acid colorimetric method. The MDA content results are shown in Table 7. Compared with the control, the MDA content in the leaves and roots of rice inoculated with RC1, RC2, and RC4 was lower, indicating that inoculation with RC1, RC2, and RC4 can alleviate the damage to the membrane system under salt stress.

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

[0089] 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-tolerant bacterial strain, characterized in that, The salt-tolerant bacteria strain is *Pseudoflavinus* (…). Pseudoclavibacter The specimen (spe.) with accession number CGMCC No.32646 was deposited on November 15, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China.

2. The salt-tolerant bacterial strain according to claim 1, characterized in that, The 16S rRNA nucleotide sequence of the pseudophlebacterium is shown in SEQ ID NO.

1.

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

4. The application of the salt-tolerant bacteria strains according to any one of claims 1 to 2 and the growth-promoting bacteria agent according to claim 3 in promoting the salt-tolerant growth of plants.

5. The application according to claim 4, characterized in that, The applications include promoting plant seed germination and promoting plant seedling height and root growth.

6. The use of the salt-tolerant bacteria strain according to any one of claims 1 to 2 in the preparation of products that promote chlorophyll synthesis in rice leaves.

7. The use of the salt-tolerant bacterial strain according to any one of claims 1 to 2 in the preparation of products producing indoleacetic acid.

8. The use of the salt-tolerant bacteria strain according to any one of claims 1 to 2 in the preparation of products that produce siderophores.

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