A salt-tolerant and alkali-tolerant growth-promoting bacterium, ZIP1309, and its application.

Abstract

This invention relates to a salt-tolerant and alkali-resistant growth-promoting bacterium, ZIP1309, and its applications, belonging to the field of microbial technology. The strain ZIP1309 was deposited on July 2, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 35073. This strain exhibits strong salt and alkali resistance, tolerating environments with a maximum salt concentration of 10% and an alkaline environment at pH 11. It produces indoleacetic acid, siderophores, proteases, amylases, lipases, and cellulases. Furthermore, this strain can promote maize growth in saline-alkali environments with a salt content ≤0.5% and a pH ≤10, demonstrating broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a salt-tolerant and alkali-resistant growth-promoting bacterium, ZIP1309, and its applications. Background Technology

[0002] Soil salinization, with its excessive salt content, not only causes physiological drought in plants and disrupts ion balance leading to toxic effects, but also significantly inhibits photosynthesis and nutrient absorption, resulting in a sharp decline in crop yields or even crop failure. Traditional remediation methods, such as flood irrigation and the application of chemical amendments, can alleviate soil salinization to some extent, but they also have drawbacks such as water waste, high costs, and potential environmental pollution. Therefore, there is an urgent need to explore new environmentally friendly biological remediation pathways.

[0003] Plant rhizosphere growth-promoting bacteria (PGPRs) have become a research hotspot in the field of bioremediation due to their unique growth-promoting mechanisms and environmental adaptability. These microorganisms can regulate plant growth and development processes and enhance plant tolerance to abiotic stress by secreting metabolites such as auxins, siderophores, and extracellular polysaccharides. Among them, auxins, as key hormones that regulate root morphogenesis and promote cell growth, can effectively enhance root vitality under saline-alkali stress; siderophores optimize iron nutrient supply to plants by specifically chelating iron ions in the environment, thereby strengthening the plant's stress resistance and defense system.

[0004] Although numerous salt-tolerant and growth-promoting bacterial strains have been isolated and identified, these strains generally exhibit reduced growth-promoting activity under high-salt environments. Therefore, screening for novel strains with stronger salt tolerance and higher production capacity of growth-promoting substances has become crucial to overcoming the bottlenecks in bioremediation technology for saline-alkali soils. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a salt-tolerant and alkali-resistant growth-promoting bacterium, ZIP1309, and its applications.

[0006] The technical solution of this invention is as follows:

[0007] A salt-tolerant growth-promoting bacterium ( Exiguobacterium enclense ZIP1309 was deposited on July 2, 2025 at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35073.

[0008] The 16S rDNA sequence of the salt-tolerant growth-promoting bacterium ZIP1309 is shown in SEQ ID NO.1.

[0009] The method for culturing the salt-tolerant and alkali-tolerant growth-promoting bacterium ZIP1309 includes the following steps:

[0010] (1) The salt-tolerant growth-promoting bacteria ZIP1309 was inoculated onto NA solid medium and activated at 37±2℃ for 24~30h to obtain the activated strain;

[0011] (2) Inoculate the activated strain from step (1) into NA liquid culture medium and culture it with shaking at 180~200 rpm and 37±2℃ for 18~25 h to obtain activated bacterial solution;

[0012] (3) Inoculate the activated bacterial solution from step (2) into NA liquid culture medium at an inoculation amount of 5%~10% by volume, and culture at 180~200 rpm and 37±2℃ for 24~30 h with shaking to obtain salt-tolerant growth-promoting bacterial solution ZIP1309.

[0013] Applications of the salt-tolerant and alkali-tolerant growth-promoting bacterium ZIP1309 in the production of indoleacetic acid, siderophores, proteases, amylases, lipases, and cellulases.

[0014] Application of the salt-tolerant growth-promoting bacterium ZIP1309 in promoting maize growth.

[0015] Application of the salt-tolerant growth-promoting bacterium ZIP1309 in promoting maize growth in saline-alkali environments with a salt content ≤5g / kg and pH ≤10.

[0016] Preferably, the application method is to mix the salt-tolerant growth-promoting bacteria ZIP1309 solution at a concentration of 2.5 × 10⁻⁶. 6 ~ 3.5×10 6 The CFU / g dosage is applied to the soil.

[0017] A live bacterial preparation with the salt-tolerant growth-promoting bacterium ZIP1309 as the active ingredient.

[0018] The beneficial effects of this invention are:

[0019] The salt-tolerant growth-promoting bacterium ZIP1309 provided by this invention has strong salt and alkali resistance, and can tolerate a salt environment of up to 10% and an alkaline environment of pH 11. It has the functions of producing indoleacetic acid, siderophores, proteases, amylases, lipases and cellulases. At the same time, this strain can also promote the growth of corn in saline-alkali environments with a salt content of ≤0.5% and a pH of ≤10, and has a very broad application prospect. Attached Figure Description

[0020] Figure 1 Image of bacterial colonies of strain ZIP1309;

[0021] Figure 2 Images showing the cell morphology of strain ZIP1309;

[0022] Figure 3 Phylogenetic tree of strain ZIP1309;

[0023] Figure 4 Images showing the effect of siderophore production by strain ZIP1309;

[0024] Figure 5 Images showing the effect of strain ZIP1309 in producing indoleacetic acid;

[0025] Figure 6 Images showing the protease production effect of strain ZIP1309;

[0026] Figure 7 Images showing the amylase production effect of strain ZIP1309;

[0027] Figure 8 Images showing the lipase production effect of strain ZIP1309;

[0028] Figure 9 Images showing the cellulase production effect of strain ZIP1309;

[0029] Figure 10 The growth of strain ZIP1309 in a high-salt-alkaline medium;

[0030] Figure 11 The effect of strain ZIP1309 on the growth promotion of maize seedlings under saline-alkali conditions. Detailed Implementation

[0031] The following description is based on specific embodiments:

[0032] Example 1: Isolation, screening and identification of salt-tolerant growth-promoting bacteria ZIP1309

[0033] A bacterial strain was screened from the rhizosphere soil of plants in saline-alkali land in Dongying City, Shandong Province, China, and named "ZIP1309". The morphology of the strain ZIP1309 obtained above is as follows: Figures 1-2 As shown; Figure 1 As shown, ZIP1309 colonies are yellow in color, round in shape, with a raised center, and a smooth, moist surface; Figure 2 As shown, after Gram staining, ZIP1309 cells were observed to be short rod-shaped, indicating that they are Gram-positive bacteria.

[0034] The 16S rDNA gene sequence of strain ZIP1309 was sequenced, and the sequencing results are shown in SEQ ID NO.1. The obtained 16S rDNA sequence was compared with existing sequences in the NCBI database using BLAST analysis. Strains with similar homology were selected, and a phylogenetic tree was constructed using MEGA X software with a Neighbor-joining method. The results are as follows: Figure 3 As shown. By Figure 3 Therefore, the taxonomic position of the strain ZIP1309 obtained from the above screening is consistent with... Exiguobacterium enclense Based on the fact that they belong to the same branch and the physiological and biochemical characteristics of the strains, they were identified as... Exiguobacterium enclense .

[0035] strain ( Exiguobacterium enclense ZIP1309 was deposited on July 2, 2025 at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35073.

[0036] Example 2: Cultivation of salt-tolerant and alkali-tolerant growth-promoting bacteria ZIP1309

[0037] Follow these steps:

[0038] (1) The salt-tolerant growth-promoting bacteria ZIP1309 was inoculated onto NA solid medium and activated at 37°C for 24 h to obtain the activated strain;

[0039] (2) Inoculate the activated strain from step (1) into NA liquid culture medium and culture it at 180 rpm and 37°C for 18 h with shaking to obtain activated bacterial solution;

[0040] (3) The activated bacterial solution from step (2) was inoculated into NA liquid medium at an inoculation rate of 5% by volume, and cultured with shaking at 180 rpm and 37°C for 24 h to obtain a bacterial concentration of 1.7 × 10⁻⁶. 9 Salt-tolerant and alkali-resistant growth-promoting bacteria ZIP1309 bacterial suspension at CFU / mL.

[0041] Example 3: Determination of the growth-promoting function of salt-tolerant growth-promoting bacteria ZIP1309

[0042] Strain activation: The strain ZIP1309 was streaked onto NA solid medium and incubated at 37°C for 24 hours to obtain the activated strain.

[0043] (1) Siderophore production capacity determination: Activated strains were inoculated onto CAS test medium using a sterile toothpick and cultured at 28℃ for 3 days. Photos of the cultured strains after incubation are shown below. Figure 4 As shown, the culture medium around the strain turns transparent, indicating that ZIP1309 has the ability to produce siderophores; the diameter (D) of the transparent zone and the diameter (d) of the colony in the culture medium are measured with vernier calipers, and the siderophore production ability of the strain is evaluated based on the D / d value.

[0044] The CAS detection culture medium components are as follows: Chromium Azurite S 60.5 mg, cetyltrimethylammonium bromide 72.9 mg, ferric chloride hexahydrate 2.645 mg, sodium dihydrogen phosphate dihydrate 295.25 mg, disodium hydrogen phosphate dodecahydrate 1213.5 mg, ammonium chloride 62.5 mg, potassium dihydrogen phosphate 37.5 mg, sodium chloride 62.5 mg, agar 9 g, water 1000 mL; pH 6.8; autoclaved at 116℃ for 30 min.

[0045] (2) Determination of indoleacetic acid production capacity: Activated strains were inoculated into LB liquid medium containing 100 mg / L L-tryptophan using a sterile inoculation loop and cultured at 30℃ and 180 rpm for 48 h to obtain fermentation broth. The fermentation broth was centrifuged at 4000 rpm for 10 min, and the supernatant was collected. The supernatant was mixed with an equal volume of Salkowski colorimetric reagent, and a 20 mg / mL indoleacetic acid standard solution was used as a positive control (the indoleacetic acid standard solution was mixed with the Salkowski colorimetric reagent). The reaction was carried out in the dark for 30 min, and the color change of the reaction solution was observed. The results are as follows: Figure 5 As shown, compared to the control group, the reaction solution turned red, indicating that indoleacetic acid was produced.

[0046] Indoleacetic acid (IAA) standard solutions were diluted to concentrations of 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, and 50 mg / L, respectively. IAA was then quantitatively determined using ultraviolet (UV) spectrophotometry. A standard curve for IAA was established. The above reaction solution was then subjected to OD240 microscopy. 530 The absorbance was measured, and the indoleacetic acid yield of strain ZIP1309 was calculated by combining the absorbance with the standard curve.

[0047] The results of the growth-promoting function assay of strain ZIP1309 are shown in Table 1.

[0048] Table 1. Results of growth-promoting function assay of strain ZIP1309

[0049]

[0050] As shown in Table 1, strain ZIP1309 has the ability to produce siderophores and indoleacetic acid, and can stably synthesize 40.69 mg / L of indoleacetic acid.

[0051] Example 4: Evaluation of the enzyme production capacity of salt-tolerant and alkali-tolerant growth-promoting bacteria ZIP1309

[0052] Strain activation: The strain ZIP1309 was streaked onto NA solid medium and incubated at 37°C for 24 hours to obtain the activated strain.

[0053] (1) Protease production capacity determination: Activated strains were inoculated into skim milk powder culture medium using a sterile toothpick and cultured at 28℃ for 5 days. After the culture was completed, the color change of the culture medium surrounding the strain was observed. Photos after the culture were taken as follows: Figure 6 As shown, the color of the culture medium around the strain changed from yellowish-white to transparent, indicating that ZIP1309 has the ability to produce protease. The diameter (D) of the transparent zone and the diameter (d) of the colony in the culture medium were measured with vernier calipers, and the protease production ability of the strain was evaluated based on the D / d value.

[0054] The components of the skim milk powder culture medium are as follows: 0.5g beef extract, 1g peptone, 0.5g NaCl, 3g skim milk powder, 2g agar, and 100mL distilled water; pH 7.0.

[0055] (2) Amylase production capacity assay: Activated strains were inoculated into starch medium using a sterile toothpick and cultured at 28℃ for 5 days. After the culture was completed, Lugol's iodine solution was added, and the color change of the medium surrounding the strain was observed. The results are as follows: Figure 7 As shown, the color of the culture medium around the strain changed from blue-purple to transparent, indicating that ZIP1309 has the ability to produce amylase. The diameter (D) of the transparent zone and the diameter (d) of the colony in the culture medium were measured with vernier calipers, and the amylase production capacity of the strain was evaluated based on the D / d value.

[0056] The starch culture medium consists of the following components: 2g soluble starch, 10g peptone, 5g beef extract, 5g sodium chloride, and 20g agar; pH 7.2; diluted to 1L with distilled water; and sterilized at 121℃ for 30min.

[0057] The components of Lugos' iodine solution are as follows: 1g iodine tablets, 2g potassium iodide, and 300mL distilled water.

[0058] (3) Lipase production capacity assay: Activated strains were inoculated into fat culture medium using a sterile toothpick and cultured at 28℃ for 5 days. After the culture period, the color change of the culture medium surrounding the strain was observed. The results are as follows: Figure 8 As shown, the color of the culture medium around the strain changed from white to transparent, indicating that ZIP1309 has the ability to produce lipase. The diameter (D) of the transparent zone and the diameter (d) of the colony in the culture medium were measured with vernier calipers, and the lipase production ability of the strain was evaluated based on the D / d value.

[0059] The fat culture medium consists of the following components: 10g peptone, 5g yeast extract, 10g NaCl, 2mL glyceryl tartrate, 20g agar, and 1L distilled water; pH 7.5.

[0060] (4) Cellulase production capacity assay: Activated strains were inoculated into cellulose medium using a sterile toothpick and cultured at 28℃ for 5 days. After the culture period, the color change of the medium surrounding the strain was observed. The results are as follows: Figure 9 As shown, the color of the culture medium around the strain changed from red (staining with Congo red) to transparent, indicating that ZIP1309 has the ability to produce cellulase; the diameter (D) of the transparent zone and the diameter of the colony (d) in the culture medium were measured with vernier calipers, and the cellulase production capacity of the strain was evaluated based on the D / d value.

[0061] The cellulose culture medium consists of the following components: 10g peptone, 5g yeast extract, 10g sodium chloride, 10g sodium carboxymethyl cellulose, 20g agar, and 1L distilled water.

[0062] The enzyme production capacity evaluation results of strain ZIP1309 are shown in Table 2.

[0063] Table 2. Evaluation results of enzyme production capacity of strain ZIP1309

[0064]

[0065] As shown in Table 2, strain ZIP1309 has the ability to produce protease, amylase, lipase and cellulase.

[0066] Example 5: Determination of the salt and alkali tolerance of salt- and alkali-tolerant growth-promoting bacteria ZIP1309

[0067] Strain activation: The strain ZIP1309 was streaked onto NA solid medium and incubated at 37°C for 24 hours to obtain the activated strain.

[0068] Preparation of high-salt-alkaline culture medium: Heat and melt NA solid culture medium, then adjust the pH to 11 with Na2CO3, and add sodium chloride to a final concentration of 10% (0.1 g / mL) to obtain high-salt-alkaline culture medium; pour into plates and cool to solidify.

[0069] After streaking ZIP1309 onto a high-salt-alkali medium, it was incubated at 37℃ for 24 hours. The growth of ZIP1309 was then observed, and the results are as follows: Figure 10 As shown. By Figure 10 It can be seen that ZIP1309 can grow vigorously in a high salinity environment with a salinity of 10% and a pH of 11.

[0070] Example 6: Growth-promoting effect of salt-tolerant and alkali-tolerant growth-promoting bacteria ZIP1309 on maize in saline-alkali soil environment

[0071] The corn seed variety was Zhengdan 958. The corn seeds were soaked in water at 28℃ for 6 hours and then raised in a light incubator at a temperature of 24℃.

[0072] Prepare 30 cultivation pots, each containing a total soil weight of 1.5±0.1kg, including topsoil and vermiculite in a 1:1 volume ratio; add sodium chloride and Na2CO3 to make the soil salinity in the pots 0.5% (5g / kg) and pH 10.

[0073] The experiment was conducted when the maize seedlings were in the true leaf stage. The cultivation pots were divided into two groups: the ZIP1309 group and the control group, with 15 pots in each group. The ZIP1309 group received daily root irrigation with ZIP1309 bacterial solution at a rate of 3.0 × 10⁻⁶ pots. 6 The soil concentration was CFU / g; the control group received daily root irrigation with the same volume of PB buffer for maize seedlings. The experiment lasted 10 days, and the growth of the maize seedlings was observed and measured after the experiment.

[0074] The growth status of corn seedlings is as follows: Figure 11 As shown in the image, the three basins on the left represent the control group, and the three basins on the right represent the ZIP1309 group. Figure 11 It can be seen that, compared with the control group, the corn seedlings in the ZIP1309 group grew significantly better, with higher plant height and more vigorous leaf growth.

[0075] The growth-promoting effect of strain ZIP1309 on maize seedlings is shown in Table 3.

[0076] Table 3. Growth-promoting effect of ZIP1309 on maize seedlings

[0077]

[0078] Table 3 shows that, compared to the control group, strain ZIP1309 significantly promoted the growth of maize seedlings in terms of plant height, stem diameter, root length, fresh weight, and dry weight. Specifically, compared to the control group, the ZIP1309 group showed an increase of 27.42% in wheat plant height, 132.11% in stem diameter, 108.72% in root length, 132.11% in fresh weight, and 111.76% in dry weight.

Claims

1. A salt-tolerant and alkali-tolerant growth-promoting bacterium ( Exiguobacterium enclense ZIP1309, characterized in that, It was deposited on July 2, 2025 at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35073.

2. The method for cultivating the salt-tolerant and alkali-resistant growth-promoting bacterium ZIP1309 according to claim 1, characterized in that, Includes the following steps: (1) The salt-tolerant growth-promoting bacteria ZIP1309 was inoculated onto NA solid medium and activated at 37±2℃ for 24~30h to obtain the activated strain; (2) Inoculate the activated strain from step (1) into NA liquid culture medium and culture it with shaking at 180~200 rpm and 37±2℃ for 18~25 h to obtain activated bacterial solution; (3) Inoculate the activated bacterial solution from step (2) into NA liquid culture medium at an inoculation amount of 5%~10% by volume, and culture at 180~200 rpm and 37±2℃ for 24~30 h with shaking to obtain salt-tolerant growth-promoting bacterial solution ZIP1309.

3. The application of the salt-tolerant and alkali-resistant growth-promoting bacterium ZIP1309 according to claim 1, characterized in that, It is used for the production of indoleacetic acid, siderophores, proteases, amylases, lipases, and cellulases.

4. The application of the salt-tolerant and alkali-resistant growth-promoting bacterium ZIP1309 according to claim 1, characterized in that, It is used to promote corn growth.

5. The application as described in claim 4, characterized in that, The salt-tolerant growth-promoting bacterium ZIP1309 promotes maize growth in a saline-alkali environment with a salt content ≤5g / kg and pH ≤10.

6. The application as described in claim 4, characterized in that, The application method involves mixing the salt-tolerant and alkali-tolerant growth-promoting bacteria ZIP1309 solution at a concentration of 2.5 × 10⁻⁶. 6 ~ 3.5×10 6 The CFU / g dosage is applied to the soil.

7. A live bacteria preparation, characterized in that, The active ingredient is the salt-tolerant and alkali-resistant growth-promoting bacterium ZIP1309 as described in claim 1.

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