Saline-alkali tolerant growth-promoting bacterium ZIP1309 and application thereof

Abstract

The invention relates to a saline-alkaline tolerant growth-promoting bacterium ZIP1309 and application thereof, and belongs to the technical field of microorganisms, the strain ZIP1309 is preserved in the China General Microbiological Culture Collection Center on July 2, 2025, and the preservation number is CGMCC No.35073. The invention further discloses a preparation method of the saline-alkaline tolerant growth-promoting bacterium ZIP1309. The strain is high in salt and alkali resistance, can tolerate a salt environment of 10% and an alkali environment of which the pH value is 11 at most, has the effects of producing indoleacetic acid, siderophores, protease, amylase, lipase and cellulase, can promote corn growth in a salt and alkali environment of which the salt content is less than or equal to 0.5% and the pH value is less than or equal to 10, and is very wide in application prospect.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a salt- and alkali-tolerant growth-promoting bacterium ZIP1309 and an application thereof. Background Art

[0002] Soil salinization and excessive salt not only cause physiological drought in plants, disrupting ion balance and causing toxic effects, but also significantly inhibit plant photosynthesis and nutrient absorption, leading to a significant drop in crop yields or even complete crop failure. While traditional management methods, such as flood irrigation and the application of chemical amendments, can alleviate soil salinization to some extent, they carry drawbacks such as water waste, high costs, and potential environmental pollution. Therefore, there is an urgent need to explore new, environmentally friendly bioremediation approaches.

[0003] Plant growth-promoting rhizobacteria (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 and enhance plant tolerance to environmental stresses by secreting metabolites such as auxins, siderophores, and exopolysaccharides. Auxins, as key hormones that regulate plant root morphology and promote cell growth, can effectively enhance root vitality under saline-alkali stress. Siderophores, by specifically chelating iron ions in the environment, optimize plant iron supply and strengthen the plant's stress defense system.

[0004] Although numerous salt-tolerant growth-promoting strains have been isolated and identified, these strains generally experience a decrease in their growth-promoting activity in high-salt environments. Therefore, screening for novel strains with enhanced salt tolerance and increased biomass production has become a key to breaking through the bottleneck of salinized soil bioremediation technology. Summary of the Invention

[0005] In view of the deficiencies in the prior art, the present invention provides a salt- and alkali-tolerant growth-promoting bacterium ZIP1309 and its application.

[0006] The technical solutions of the present invention are as follows: A salt-alkali tolerant growth-promoting bacterium ( Exiguobacterium enclense ) ZIP1309, deposited on July 2, 2025, at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 35073.

[0007] Among them, the 16S rDNA sequence of the salt-alkali tolerant growth-promoting bacteria ZIP1309 is shown as SEQ ID NO.1.

[0008] The method for culturing the salt-alkali-tolerant growth-promoting bacteria ZIP1309 comprises the following steps: (1) The salt-alkali-tolerant growth-promoting bacteria ZIP1309 were inoculated onto NA solid medium and activated and cultured at 37±2℃ for 24-30h to obtain an activated strain; (2) The activated strain in step (1) was inoculated into NA liquid culture medium, and cultured under shaking conditions of 180-200 rpm and 37±2°C for 18-25 hours to obtain an activated bacterial solution; (3) The activated bacterial solution in step (2) was inoculated into NA liquid culture medium at an inoculum volume percentage of 5% to 10%, and cultured under shaking conditions of 180 to 200 rpm and 37 ± 2 ° C for 24 to 30 hours to obtain the salt-alkali tolerant growth-promoting bacteria ZIP1309 bacterial solution.

[0009] The salt-alkali tolerant growth-promoting bacteria ZIP1309 is used in producing indoleacetic acid, siderophore, protease, amylase, lipase and cellulase.

[0010] The application of the salt-alkali tolerant growth-promoting bacteria ZIP1309 in promoting corn growth.

[0011] The salt-alkali tolerant growth-promoting bacteria ZIP1309 is used to promote corn growth in a saline-alkali environment with a salt content of ≤5 g / kg and a pH of ≤10.

[0012] Preferably, the application method is to add the salt-alkali tolerant growth-promoting bacteria ZIP1309 bacterial solution at a rate of 2.5×10 6 ~ 3.5×10 6 The application amount of CFU / g was applied to the soil.

[0013] A live bacteria preparation with the salt-alkali tolerant growth-promoting bacteria ZIP1309 as an effective ingredient.

[0014] Beneficial effects of the present invention: The salt-alkali tolerant growth-promoting bacteria ZIP1309 provided by the present invention has strong salt-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, siderophore, protease, amylase, lipase, and cellulase. At the same time, the strain can also promote corn growth in a saline-alkali environment with a salt content of ≤0.5% and a pH of ≤10, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a colony picture of strain ZIP1309; Figure 2 This is a picture of the bacterial morphology of strain ZIP1309; Figure 3 is the phylogenetic tree of strain ZIP1309; Figure 4 This is a picture of the siderophore production effect of strain ZIP1309; Figure 5 This is a picture of the indoleacetic acid production effect of strain ZIP1309; Figure 6 This is a picture of the protease production effect of strain ZIP1309; Figure 7 This is a picture of the amylase production effect of strain ZIP1309; Figure 8 This is a picture of the lipase production effect of strain ZIP1309; Figure 9 This is a picture of the cellulase production effect of strain ZIP1309; Figure 10 The growth of strain ZIP1309 in high saline-alkaline medium; Figure 11 This is the growth-promoting effect of strain ZIP1309 on corn seedlings in saline-alkali environment. DETAILED DESCRIPTION

[0016] The following describes the process in conjunction with specific embodiments: Example 1: Isolation, screening and identification of salt-alkali-tolerant growth-promoting bacteria ZIP1309 A bacterial strain was screened from the rhizosphere soil of saline-alkali plants in Dongying City, Shandong Province, China, and named "ZIP1309". The morphology of the strain ZIP1309 obtained above is as follows Figures 1 and 2 As shown; Figure 1 As shown, the colony of ZIP1309 is yellow in color, round in shape, convex in the middle, and has a smooth and moist surface; Figure 2 As shown in the figure, after Gram staining, ZIP1309 bacteria were observed to be short rod-shaped, indicating that they were Gram-positive bacteria.

[0017] 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 the existing sequences in the NCBI database by BLAST analysis, and strains with similar homology were selected. The phylogenetic tree was constructed using MEGA X software using the neighbor-joining method. The results are shown in Figure 1. Figure 3 As shown. Figure 3 It can be seen that the taxonomic status of the strain ZIP1309 obtained above is the same as Exiguobacterium enclense In the same branch, combined with the physiological and biochemical characteristics of the strain, it was identified as Exiguobacterium enclense .

[0018] strain ( Exiguobacterium enclense) ZIP1309, deposited on July 2, 2025, at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 35073.

[0019] Example 2: Cultivation of salt-alkali tolerant growth-promoting bacteria ZIP1309 Follow these steps: (1) The salt-alkali-tolerant growth-promoting bacteria ZIP1309 were inoculated onto NA solid culture medium and activated at 37°C for 24 h to obtain an activated strain; (2) The activated strain in step (1) was inoculated into NA liquid culture medium, and cultured under shaking conditions of 180 rpm and 37°C for 18 h to obtain an activated bacterial solution; (3) The activated bacterial solution in step (2) was inoculated into NA liquid culture medium at a volume percentage of 5%, and cultured under shaking conditions of 180 rpm and 37 °C for 24 h to obtain a bacterial concentration of 1.7 × 10 9 CFU / mL of salt- and alkali-tolerant growth-promoting bacteria ZIP1309.

[0020] Example 3: Determination of the growth-promoting function of salt-alkali-tolerant growth-promoting bacteria ZIP1309 Activation of strains: Streak strain ZIP1309 onto NA solid medium and culture at 37°C for 24 h to obtain the activated strain.

[0021] (1) Determination of iron carrier production capacity: Use a sterile toothpick to pick up the activated strain and inoculate it into the CAS test medium, and culture it at 28℃ for 3 days. Figure 4 As shown in the figure, the color of the culture medium surrounding the strain turned transparent, indicating that ZIP1309 has the ability to produce siderophores. The diameter (D) of the transparent circle in the culture medium and the diameter (d) of the colony were measured with a vernier caliper, and the siderophore production ability of the strain was evaluated based on the D / d value.

[0022] The CAS assay medium components are as follows: chrome azurol 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; autoclave at 116°C for 30 min.

[0023] (2) Determination of indoleacetic acid production capacity: Use a sterile inoculation loop to pick up the activated strain and inoculate it into LB liquid culture medium containing 100 mg / L L-tryptophan. Cultivate at 30°C and 180 rpm for 48 hours to obtain fermentation liquid. Centrifuge the fermentation liquid at 4000 rpm for 10 minutes and collect the supernatant. Mix the supernatant with the same volume of Salkowski colorimetric reagent, and use a 20 mg / mL indoleacetic acid standard solution as a positive control (mix the indoleacetic acid standard solution with the Salkowski colorimetric reagent). After 30 minutes of reaction in the dark, observe the color change of the reaction liquid. The results are as follows. Figure 5 As shown, compared with the control group, the reaction solution turned red, indicating that indoleacetic acid was produced.

[0024] The indoleacetic acid standard solution was diluted to 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, and 50 mg / L, and indoleacetic acid was quantitatively determined by ultraviolet spectrometry to establish a standard curve of indoleacetic acid standard solution. 530 The absorbance was measured at 400 nm and the indoleacetic acid production of strain ZIP1309 was calculated based on the standard curve.

[0025] The results of the growth-promoting function assay of strain ZIP1309 are shown in Table 1 .

[0026] Table 1. Results of growth-promoting function assay of strain ZIP1309

[0027] As shown in Table 1, strain ZIP1309 has the ability to produce both siderophore and indoleacetic acid, and can stably synthesize 40.69 mg / L of indoleacetic acid.

[0028] Example 4: Evaluation of enzyme production capacity of salt-alkali tolerant growth-promoting bacteria ZIP1309 Activation of strains: Streak strain ZIP1309 onto NA solid medium and culture at 37°C for 24 h to obtain the activated strain.

[0029] (1) Determination of protease production capacity: Use a sterilized toothpick to pick up the activated strain and inoculate it into skim milk powder culture medium. Incubate at 28℃ for 5 days. After the incubation, observe the color change of the culture medium around the strain. Figure 6 As shown, the color of the culture medium surrounding the strain changed from yellow-white to transparent, indicating that ZIP1309 has the ability to produce protease. The diameter (D) of the transparent circle in the culture medium and the diameter (d) of the colony were measured with a vernier caliper, and the protease production ability of the strain was evaluated based on the D / d value.

[0030] The components of the skim milk powder medium are as follows: 0.5 g beef extract, 1 g peptone, 0.5 g NaCl, 3 g skim milk powder, 2 g agar, and 100 mL distilled water; pH 7.0.

[0031] (2) Determination of amylase production capacity: Use a sterilized toothpick to pick up the activated strain and inoculate it into the starch culture medium. Incubate at 28℃ for 5 days. After the incubation, add Lugol's iodine solution and observe the color change of the culture medium around the strain. Figure 7 As shown, the color of the culture medium surrounding the strain changed from blue-purple to transparent, indicating that ZIP1309 has the ability to produce amylase. The diameter (D) of the transparent circle in the culture medium and the diameter (d) of the colony were measured with a vernier caliper, and the amylase production ability of the strain was evaluated based on the D / d value.

[0032] The starch culture medium comprises the following components: 2 g soluble starch, 10 g peptone, 5 g beef extract, 5 g sodium chloride, and 20 g agar; the pH is 7.2; the volume is adjusted to 1 L with distilled water; and the culture medium is sterilized at 121° C. for 30 min.

[0033] Among them, the components of Lugol's iodine solution are as follows: 1g iodine tablets, 2g potassium iodide, and 300mL distilled water.

[0034] (3) Determination of lipase production capacity: Use a sterilized toothpick to pick up the activated strain and inoculate it into the fat culture medium. Incubate it at 28℃ for 5 days. After the incubation, observe the color change of the culture medium around the strain. Figure 8 As shown, the color of the culture medium surrounding the strain changed from white to transparent, indicating that ZIP1309 has the ability to produce lipase. The diameter (D) of the transparent circle in the culture medium and the diameter (d) of the colony were measured with a vernier caliper, and the lipase production ability of the strain was evaluated based on the D / d value.

[0035] The components of the fat culture medium are as follows: peptone 10 g, yeast powder 5 g, NaCl 10 g, tributyrin 2 mL, agar 20 g, distilled water 1 L; pH 7.5.

[0036] (4) Determination of cellulase production capacity: Use a sterilized toothpick to pick up the activated strain and inoculate it into the cellulose culture medium. Incubate at 28℃ for 5 days. After the incubation, observe the color change of the culture medium around the strain. Figure 9 As shown, the color of the culture medium surrounding the strain changed from red (stained with Congo red) to transparent, indicating that ZIP1309 has the ability to produce cellulase. The diameter (D) of the transparent circle in the culture medium and the diameter (d) of the colony were measured with a vernier caliper, and the cellulase production ability of the strain was evaluated based on the D / d value.

[0037] The components of the cellulose culture medium are as follows: 10 g of peptone, 5 g of yeast powder, 10 g of sodium chloride, 10 g of sodium carboxymethyl cellulose, 20 g of agar, and 1 L of distilled water.

[0038] The results of the enzyme production ability evaluation of strain ZIP1309 are shown in Table 2.

[0039] Table 2. Evaluation results of enzyme production capacity of strain ZIP1309

[0040] As shown in Table 2, strain ZIP1309 has the ability to produce protease, amylase, lipase and cellulase.

[0041] Example 5: Determination of salt-alkali tolerance of salt-alkali tolerant growth-promoting bacteria ZIP1309 Activation of strains: Streak strain ZIP1309 onto NA solid medium and culture at 37°C for 24 h to obtain the activated strain.

[0042] Preparation of high-salt-alkali culture medium: Heat and melt the 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 a high-salt-alkali culture medium; pour the medium onto a plate and cool to solidify.

[0043] After the strain ZIP1309 was streaked on a high-salt-alkali medium, it was cultured at 37°C for 24 hours and the growth of ZIP1309 was observed. Figure 10 As shown. Figure 10 It was found that ZIP1309 could grow vigorously in a high saline-alkali environment with a salinity of 10% and a pH of 11.

[0044] Example 6: Growth-promoting effect of salt-alkali tolerant growth-promoting bacteria ZIP1309 on corn in saline-alkali soil environment The corn seed variety is Zhengdan 958. The corn seeds were soaked in water at 28°C for 6 hours and then grown in a light incubator at a temperature of 24°C.

[0045] Prepare 30 cultivation pots, each containing 1.5 ± 0.1 kg of soil, including cultivated soil and vermiculite, with a volume ratio of 1:1. Sodium chloride and Na2CO3 were added to make the soil salinity in the pots 0.5% (5 g / kg) and the pH 10.

[0046] The experiment was carried out when the corn seedlings were in the true leaf stage. The above-mentioned cultivation pots were divided into two groups, namely the ZIP1309 group and the control group, with 15 pots in each group. Among them, the ZIP1309 group used ZIP1309 bacterial solution to irrigate the corn seedlings every day, with an application rate of 3.0×10 6 CFU / g soil; the control group used the same volume of PB buffer to irrigate the maize seedlings daily. The experimental period was 10 days. After the experiment, the growth of the maize seedlings was observed and measured.

[0047] The growth of corn seedlings Figure 11 As shown in the figure, the three basins on the left are the control group, and the three basins on the right are 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.

[0048] The growth-promoting effect of strain ZIP1309 on corn seedlings is shown in Table 3.

[0049] Table 3. Growth-promoting effect of ZIP1309 on maize seedlings

[0050] As shown in Table 3, strain ZIP1309 significantly promoted plant height, stem diameter, root length, fresh weight, and dry weight of maize seedlings compared to the control group. Specifically, compared to the control group, the ZIP1309 group showed an increase in wheat plant height by 27.42%, stem diameter by 132.11%, root length by 108.72%, fresh weight by 132.11%, and dry weight by 111.76%.

Claims

1. A salt- and alkali-tolerant growth-promoting bacterium ( Exiguobacterium enclense ) ZIP1309, characterized in that, It was deposited on July 2, 2025 in the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 35073.

2. The method for cultivating the salt-alkali-tolerant growth-promoting bacteria ZIP1309 according to claim 1, characterized in that: The steps include: (1) The salt-alkali-tolerant growth-promoting bacteria ZIP1309 were inoculated onto NA solid medium and activated and cultured at 37±2℃ for 24-30h to obtain an activated strain; (2) The activated strain in step (1) was inoculated into NA liquid culture medium, and cultured under shaking conditions of 180-200 rpm and 37±2°C for 18-25 hours to obtain an activated bacterial solution; (3) The activated bacterial solution in step (2) was inoculated into NA liquid culture medium at an inoculum volume percentage of 5% to 10%, and cultured under shaking conditions of 180 to 200 rpm and 37 ± 2 ° C for 24 to 30 hours to obtain the salt-alkali tolerant growth-promoting bacteria ZIP1309 bacterial solution.

3. The use of the salt-alkali tolerant growth-promoting bacteria ZIP1309 according to claim 1, characterized in that: It is used to produce indoleacetic acid, siderophore, protease, amylase, lipase and cellulase.

4. The use of the salt-alkali tolerant growth-promoting bacteria ZIP1309 according to claim 1, characterized in that: Used to promote corn growth.

5. The use according to claim 4, characterized in that The salt-alkali tolerant growth-promoting bacteria ZIP1309 promotes corn growth in a saline-alkali environment with a salt content of ≤5 g / kg and a pH of ≤10.

6. The use according to claim 4, characterized in that The application method is to add the salt-alkali tolerant growth-promoting bacteria ZIP1309 bacterial solution at a rate of 2.5×10 6 ~ 3.5×10 6 The application amount of CFU / g was applied to the soil.

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

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