Saline-alkaline tolerant phosphate solubilizing bacteria and growth promoting effect thereof in adversity stress

Abstract

The invention provides a salt and alkali tolerant phosphate solubilizing bacterium and a growth promoting effect in adversity stress, and belongs to the technical field of biocontrol bacteria. The invention provides a salt and alkali tolerant phosphate solubilizing bacterium, the phosphate solubilizing bacterium comprises enterobacter CCZ3-8, and the phosphate solubilizing bacterium can activate insoluble phosphorus in soil, including inorganic phosphorus and organic phosphorus. The strain CCZ3-8 disclosed by the invention has strong stress resistance, can quickly grow when the NaCl concentration is below 7%, and can obviously slow down the growth rate when the NaCl concentration is greater than 9%; the strain can normally proliferate in a pH range of 4-10, and has relatively strong tolerance to a certain degree of acid and alkali environments at the same time. The phosphate solubilizing bacteria can promote plant growth under a single condition, can promote plant growth in a complex soil environment, can improve the adversity resistance of plants, and has a huge application value.

Description

Technical Field

[0001] This invention belongs to the field of biocontrol bacteria technology, specifically relating to a salt-tolerant phosphate-solubilizing bacterium and its growth-promoting effect under adverse abiotic stress. Background Technology

[0002] Phosphorus is an essential macronutrient for plant metabolism, growth, and development. However, recent statistics indicate that the yield-increasing efficiency of chemical fertilizers is gradually decreasing, and excessive use of phosphate fertilizers has led to severe eutrophication of water bodies. Currently, many arable lands suffer from high total phosphorus content and low available phosphorus content, with a low proportion of absorbable phosphorus in the total phosphorus. This is mainly due to the soil's high phosphorus fixation capacity, which reduces soil phosphorus availability. Most phosphate fertilizers are rapidly fixed, primarily in the form of calcium-fixed insoluble inorganic phosphates such as calcium phosphate, as well as phytates, nucleic acids, and other organophosphates. These forms are difficult for plants to directly utilize in the soil. Low levels of available nutrients in soils under saline-alkali stress are a major factor limiting plant growth and productivity. Therefore, activating in-situ insoluble phosphorus in the soil is key to effectively increasing the available phosphorus content in saline-alkali soils.

[0003] Phosphate-solubilizing bacteria are a class of microorganisms that can dissolve inorganic phosphates or mineralize organic phosphates in soil, promoting plant growth. They can promote the conversion of insoluble phosphorus into available phosphorus for plant use by secreting organic acids, phosphatases, protons, extracellular polysaccharides, and siderophores. Currently reported phosphate-solubilizing bacteria are mainly concentrated in several genera, including Bacillus, Pseudomonas, Enterobacter, Rhizobium, Bacillus, and Arthrobacter. While reports indicate varying phosphate-solubilizing effects between different genera and even different species within the same genera, their actual performance in soil is poor, making it difficult to replicate laboratory results. Summary of the Invention

[0004] This invention provides a salt-tolerant phosphate-solubilizing bacterium and its growth-promoting effect under abiotic stress. The strain has a strong phosphate-solubilizing ability and is suitable for activating phosphorus in arable soil. It can significantly increase the content of available phosphorus in arable soil, reduce the application of phosphate fertilizer, and promote plant growth under abiotic stress.

[0005] This invention provides a salt-tolerant phosphate-solubilizing bacterium, said phosphate-solubilizing bacterium including Enterobacteriaceae (… Enterobacter sp. The Enterobacter CCZ3-8 is described in the form of CGMCC No. 35746.

[0006] In a preferred embodiment of the present invention, the nucleotide sequence of the 16S rDNA of the phosphate-solubilizing bacteria is shown in SEQ ID No. 1.

[0007] This invention also provides the application of the above-mentioned phosphate-solubilizing bacteria in increasing the available phosphorus content in soil.

[0008] In a preferred embodiment of the present invention, the source of available phosphorus in the soil includes insoluble organic phosphorus and / or inorganic phosphorus in the soil.

[0009] The present invention also provides the application of the above-mentioned phosphate-solubilizing bacteria in promoting plant growth in abiotic stress environments.

[0010] In a preferred embodiment of the present invention, the abiotic stress environment includes at least one of the following: salt, alkali and acid.

[0011] The present invention also provides the application of the above-mentioned phosphate-solubilizing bacteria in reducing the amount of phosphate fertilizer used in agriculture.

[0012] The present invention also provides a biocontrol agent prepared using the above-mentioned phosphate-solubilizing bacteria.

[0013] The present invention also provides the application of the above-mentioned biocontrol agent in at least one of the following: degrading insoluble phosphorus in soil, increasing the available phosphorus content in soil, antagonizing abiotic stress, and promoting plant growth.

[0014] The present invention also provides a method for promoting plant growth, comprising applying the above-mentioned biocontrol agent to the soil.

[0015] Beneficial effects: This invention screened a strain of Ludwig's Enterobacter from soil (… Enterobacter ludwigii ) and Colby Enterobacter ( Enterobacter kobei The sequence similarity was 100% and it was identified as Enterobacter ( Enterobacter sp. The phosphate-solubilizing bacteria were identified and named CCZ3-8. These bacteria can activate insoluble phosphorus in the soil, such as calcium phosphate (inorganic phosphorus) and calcium phytate (organic phosphorus).

[0016] The strain CCZ3-8 described in this invention exhibits strong stress resistance, growing rapidly at NaCl concentrations below 7%, while its growth rate slows significantly at concentrations above 9%. This strain can proliferate normally within a pH range of 4-10 and also demonstrates strong tolerance to certain levels of acidity and alkalinity. The phosphate-solubilizing bacteria described in this invention can not only promote plant growth under single conditions but also enhance plant growth and improve their resilience in complex soil environments, thus possessing significant application value.

[0017] Biological Preservation Information Enterobacteriaceae ( Enterobacter sp. CCZ3-8 was deposited on August 26, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 35746, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. Attached Figure Description

[0018] Figure 1 The growth curves of strain CCZ3-8 at different NaCl concentrations are shown. Figure 2 The growth curves of strain CCZ3-8 at different pH values ​​are shown. Figure 3 Figure 1 shows the results of the CCZ3-8 strain promoting rice growth and improving salt tolerance. In the figure, a: phenotype; b: plant height; c: root length; d: aboveground fresh weight; e: root fresh weight. Detailed Implementation

[0019] This invention provides a salt-tolerant phosphate-solubilizing bacterium, said phosphate-solubilizing bacterium including Enterobacteriaceae (… Enterobacter sp. The Enterobacter CCZ3-8 is described in the form of CGMCC No. 35746.

[0020] The strain CCZ3-8 described in this invention was isolated from soil and has white, round colonies. It has functions such as producing H2O2 enzyme, hydrolyzing citrate, solubilizing potassium, fixing nitrogen, producing ammonia, and producing IAA.

[0021] The nucleotide sequence of the 16S rDNA of the phosphate-solubilizing bacteria described in this invention is shown in SEQ ID No. 1:

[0022] This invention also provides the application of the above-mentioned phosphate-solubilizing bacteria in increasing the available phosphorus content in soil.

[0023] The sources of available phosphorus in soil described in this invention include insoluble organic phosphorus and / or inorganic phosphorus in the soil. In one embodiment of this invention, the phosphorus solubilization capacity in a culture medium containing calcium phosphate (inorganic phosphorus) and calcium phytate (organic phosphorus) was verified. After inoculating the CCZ3-8 strain in a culture medium containing 5 g / L calcium phosphate for 4 days, the soluble phosphorus concentration reached 241 mg / L; after inoculating the CCZ3-8 strain in a culture medium containing 5 g / L calcium phytate for 4 days, the soluble phosphorus concentration reached 370 mg / L.

[0024] The present invention also provides the application of the above-mentioned phosphate-solubilizing bacteria in promoting plant growth in abiotic stress environments.

[0025] The abiotic stress environment described in this invention includes at least one of the following: saline-alkali and acidic environments. The strain CCZ3-8 described in this invention exhibits strong stress resistance, growing rapidly at NaCl concentrations below 7%, with a significantly slower growth rate at concentrations above 9%. This strain can proliferate normally within a pH range of 4-10 and also demonstrates strong tolerance to certain levels of acidity and alkalinity.

[0026] The present invention also provides the application of the above-mentioned phosphate-solubilizing bacteria in reducing the amount of phosphate fertilizer used in agriculture.

[0027] The phosphorus-solubilizing bacteria described in this invention can directly activate insoluble phosphorus in the soil, converting it into available phosphorus that can be absorbed and utilized by plants, thereby reducing the amount of phosphate fertilizer used.

[0028] The present invention also provides a biocontrol agent prepared using the above-mentioned phosphate-solubilizing bacteria.

[0029] The biocontrol agent of the present invention can be a resuspension of the phosphate-solubilizing bacteria, and in one embodiment, it is resuspended in sterile water, and the OD of the bacterial resuspension is... 600 It ranges from 0.8 to 1.0.

[0030] The phosphate-solubilizing bacteria of the present invention are cultured on nutrient broth NB medium. In one embodiment, the nutrient broth NB medium comprises the following components at concentrations of: 10 g / L peptone, 3 g / L beef extract and 5 g / L sodium chloride.

[0031] In this invention, the phosphate-solubilizing bacteria are inoculated onto the nutrient broth NB medium and cultured at a temperature of 30°C, with shaking occurring during the culture process at a frequency of 200 rpm, until the culture reaches OD. 600Centrifugation was performed at a pH of 0.8–1.0, and the bacterial cells were collected. In one embodiment, centrifugation was performed at 5000 rpm for 5 minutes. After discarding the supernatant, the bacterial cells were collected and washed with sterile water. The collected bacterial cells were then resuspended in bacterial water at an OD value of 0.8–1.0. 600 The value is 0.8~1.2, such as OD. 600 When the value is 1, the biological agent is obtained.

[0032] The present invention also provides the application of the above-mentioned biocontrol agent in at least one of the following: degrading insoluble phosphorus in soil, increasing the available phosphorus content in soil, antagonizing abiotic stress, and promoting plant growth.

[0033] The present invention, when applied to normal soil, can promote plant growth, especially increasing the fresh weight of the aboveground parts, as well as the root length and fresh weight; under salt-stressed soil, it can promote plant growth, especially increasing the plant height and fresh weight of the aboveground parts, as well as the fresh weight of the roots.

[0034] The present invention also provides a method for promoting plant growth, comprising applying the above-mentioned biocontrol agent to the soil.

[0035] When using the biocontrol agent described in this invention, it needs to be diluted before being applied directly to the soil. In one embodiment of this invention, the biocontrol agent is diluted 500 to 5000 times, and the application includes applying it once every 10 to 14 days, with each application amount being 10 to 100 mL per kilogram of soil, or approximately 30 to 100 L per acre in the field.

[0036] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a salt-tolerant phosphate-solubilizing bacterium provided by the present invention and its growth-promoting effect under adverse stress, should not be construed as limiting the scope of protection of the present invention.

[0037] The composition of the culture medium used in the embodiments of the present invention is as follows: Nutrient broth NB medium: peptone 10 g / L, beef extract 3 g / L and sodium chloride 5 g / L; Inorganic phosphorus medium: sucrose 10 g / L, calcium phosphate 5 g / L, magnesium chloride 5 g / L, magnesium sulfate 0.25 g / L, potassium chloride 0.2 g / L and ammonium sulfate 0.1 g / L; Organic phosphorus culture medium: sucrose 10 g / L, calcium phytate 5 g / L, magnesium chloride 5 g / L, magnesium sulfate 0.25 g / L, potassium chloride 0.2 g / L and ammonium sulfate 0.1 g / L; The above are all liquid culture medium formulations. For the corresponding solid culture medium, add 10g / L of agar.

[0038] Example 1 (1) Source of strain The bacterial samples were collected from paddy fields at the Changxing Experimental Station in Zhejiang Province (30°59′24″N, 119°54′36″E). The screening steps are as follows: Take 25g of soil, add 1L of pure water to prepare a suspension, and then dilute it serially by 10 times, 100 times, and 10 times. 3 times, 10 4 times, 10 5 times, 10 6 times, 10 7 Diluted solutions of various concentrations were transferred to 96-well plates containing inorganic and organic phosphorus liquid media, respectively, with 15 replicates per plate. The plates were incubated statically for 4 days. For plates where approximately one-third of the bacterial culture had grown, a small amount of the culture was streaked onto NB solid medium. Single colonies were picked, shaken, and streaked again on NB liquid medium for purification. This purification process was repeated three times. The culture was then sequenced and identified. Fresh culture was mixed with 50% glycerol at a 1:1 ratio and stored at -80°C.

[0039] (2) Strain identification A small amount of the preserved bacterial culture was activated by shaking in NB liquid medium and sent to Ruiboxing Biotechnology Co., Ltd. for 16S rDNA sequencing identification. The obtained gene sequence was compared with the nucleotide sequence BLAST on the website of the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov), and it was found to be similar to Ludwig C. Enterobacter ludwigii ) and Colby Enterobacter ( Enterobacter kobei The sequence similarity was 100%, and the isolated strain was identified as Enterobacter (Enterobacter). Enterobacter sp. The sequencing sequence is shown in SEQ ID No. 1.

[0040] 16S rDNA sequencing primers: 27F (SEQ ID No. 2): 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R (SEQ ID No. 3): 5'-GGTTACCTTGTTACGACTT-3'.

[0041] Example 2 Phosphate solubilization capacity determination of strains Solid and liquid calcium phosphate inorganic phosphorus medium and calcium phytate organic phosphorus medium were prepared respectively. The preserved bacterial culture was activated in NB liquid medium, centrifuged at 5000 rpm for 5 min, the supernatant was discarded, and the culture was washed three times with sterile water and resuspended in sterile water to adjust the OD. 600Approximately 1 μL of bacterial suspension was inoculated into inorganic phosphorus / organic phosphorus solid medium and incubated at 28°C for 4 days. The phosphorus solubility index was calculated as the ratio of the phosphorus solubility zone diameter to the colony diameter. A 1% inoculum was then inoculated into inorganic phosphorus / organic phosphorus liquid medium and incubated at 28°C with shaking at 200 rpm for 4 days. Simultaneously, an equal volume of sterile water was used as a negative control. Each treatment was repeated in triplicate. After centrifugation at 6000 rpm for 10 minutes, a suitable amount of supernatant was collected, and the available phosphorus content was determined using the molybdenum antimony colorimetric method.

[0042] Provided high-efficiency phosphate-solubilizing bacteria Enterobacter sp. After culturing CCZ3-8 in calcium phosphate (inorganic phosphorus) medium for 4 days, the soluble phosphorus concentration reached 241 mg / L; after culturing in calcium phytate (organic phosphorus) medium for 4 days, the phosphorus solubility was 370 mg / L.

[0043] Example 3 Determination of stress resistance of strain CCZ3-8 Using nutrient broth NB medium as the base medium, different salt concentrations (0%, 3%, 5%, 7%, 9%) and pH values ​​(4, 6, 8, 9) were prepared (3 replicates per group).

[0044] After activating the preserved bacterial strain, the seed culture was centrifuged, washed three times with sterile water, and resuspended at OD. 600 Approximately 1, at an inoculum rate of 1%, was transferred to NB medium and cultured at 28℃ and 180 rpm on a shaker. OD was measured every 1 hour. 600 Value. Result as follows Figure 1 , Figure 2 As shown.

[0045] Depend on Figure 1 It can be seen that this strain has strong salt tolerance and can grow rapidly at NaCl concentrations below 7%, while the growth rate slows down significantly at concentrations above 9%.

[0046] Depend on Figure 2 It can be seen that this strain can proliferate normally in the pH range of 4 to 10, and it also has strong tolerance to certain acidic and alkaline environments.

[0047] Example 4 Effects of bacterial strains on plant salt tolerance Ishikari Hakuge, a representative japonica rice variety from Hokkaido, Japan, was selected. Rice seeds were germinated in pure water at 37℃ for 2 days, then inoculated into a modified Hogland rice nutrient solution for seedling cultivation. After seven days of growth, the seedlings were transplanted into paddy soil and watered once daily. They were then grown in a greenhouse for 7 days. Subsequently, control groups, salt treatment groups, bacterial treatment groups, and bacterial-salt treatment groups were established. The salt treatment involved adding NaCl solution at a mass fraction of 0.5% of the soil mass. The bacterial treatment followed the method described in Example 2, where the bacterial solution was washed and then applied to the paddy soil at a ratio of 1:1000 to water. Results were observed after 4 days.

[0048] The stock solution formula is as follows: ammonium chloride 39.58 g / L, sodium dihydrogen phosphate 31.2 g / L, potassium sulfate 15.68 g / L, calcium nitrate tetrahydrate 87.38 g / L, magnesium sulfate heptahydrate 134.8 g / L, potassium nitrate 18.20 g / L, ferric sodium ethylenediaminetetraacetate 36.7 g / L, manganese chloride tetrahydrate 0.99 g / L, boric acid 1.86 g / L, ammonium molybdate tetrahydrate 1.236 g / L, zinc sulfate heptahydrate 1.15 g / L, and copper sulfate pentahydrate 0.518 g / L. Before use, dilute 1000 times and adjust the pH to 5.5-6.5 with 4 mol / L hydrochloric acid and sodium hydroxide.

[0049] like Figure 3 It can be seen that when this strain is applied to the soil, it can promote plant growth. The height of the aboveground parts does not change significantly, but the fresh weight increases by 20.2%, while the fresh weight of the roots increases by 17.9% and 33.9%, respectively. Under salt stress, the height and fresh weight of the aboveground parts increase by 13% and 26.5%, respectively, while the root length does not change significantly, but the fresh weight increases by 80.2%.

[0050] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A salt-tolerant phosphate-solubilizing bacterium, characterized in that, The phosphate-solubilizing bacteria include Enterobacteriaceae ( Enterobacter sp. The Enterobacter CCZ3-8 is described in the form of CGMCC No. 35746.

2. The phosphate-solubilizing bacteria according to claim 1, characterized in that, The nucleotide sequence of the 16S rDNA of the phosphate-solubilizing bacteria is shown in SEQ ID No.

1.

3. The application of the phosphate-solubilizing bacteria described in claim 1 or 2 in increasing the available phosphorus content in soil.

4. The application according to claim 3, characterized in that, The sources of available phosphorus in the soil include insoluble organic phosphorus and / or inorganic phosphorus in the soil.

5. The application of the phosphate-solubilizing bacteria according to claim 1 or 2 in promoting plant growth in abiotic stress environments.

6. The application according to claim 5, characterized in that, The abiotic stress environment includes at least one of the following: salt, alkali and acid.

7. The application of the phosphate-solubilizing bacteria as described in claim 1 or 2 in reducing the amount of phosphate fertilizer used in agriculture.

8. A biocontrol agent prepared using the phosphate-solubilizing bacteria according to claim 1 or 2.

9. The use of the biocontrol agent according to claim 8 in at least one of the following, characterized in that, include: It degrades insoluble phosphorus in soil, increases available phosphorus content in soil, antagonizes abiotic stress, and promotes plant growth.

10. A method for promoting plant growth, characterized in that, This includes applying the biocontrol agent of claim 8 to the soil.

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