Growth-promoting streptomyces strain with saline-alkaline tolerance and application of growth-promoting streptomyces strain

Abstract

The invention discloses a growth-promoting streptomyces strain with saline-alkaline tolerance and application thereof, and belongs to the technical field of microorganisms. In order to excavate streptomyces capable of improving the saline-alkali stress resistance of alfalfa, a strain of Streptomyces Beauvianus Jrh8-9 is screened from rhizosphere soil of ranunculus japonicus growing in saline-alkali soil, and a saline-alkali tolerance experiment, a germination accelerating experiment and a potting experiment prove that the Streptomyces Beauvianus can survive and grow in a high-salt and high-pH environment, and the Streptomyces Beauvianus Jrh8-9 has the advantages that the Streptomyces Beauvianus Jrh8-9 can be used for improving the saline-alkali stress resistance of alfalfa; and germination of alfalfa seeds and growth of alfalfa seedlings under saline-alkali stress can be promoted. In addition, under the non-stress condition, the strain Jrh8-9 also has a certain promotion effect on the growth of alfalfa seedlings. The invention provides a valuable strain resource for promoting the growth of alfalfa under saline-alkali stress.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a growth-promoting streptomyces strain with salt-alkali resistance and application thereof. Background Art

[0002] Agricultural soils in arid and semi-arid regions around the world are reduced by about 1-2% each year due to salinization. In addition, global climate change and unreasonable farming and irrigation have exacerbated the salinization problem, causing the area of ​​salinized soil to continue to expand. At present, a large amount of saline-alkali wasteland can be developed into arable land. However, the salt and alkalinity in saline-alkali wasteland severely restrict the growth of saline-alkali sensitive plants and even affect some saline-alkali tolerant plants, resulting in a significant reduction in crop yields. In addition, there are many salinized arable lands in the existing arable land that need to be transformed. To meet this challenge, the application of soil saline-alkali tolerant microorganisms has been found to effectively promote the growth of a variety of plants under saline-alkali stress. Microbial inoculation has the advantages of low cost, high efficiency, quick effect and environmental friendliness. Therefore, using beneficial microorganisms to improve plant salt tolerance is an important strategy to improve the utilization of saline-alkali land and develop saline-alkali agriculture.

[0003] Recent studies have shown that some Streptomyces have significant functions in enhancing plant tolerance to abiotic stress. For example, Streptomyces such as Streptomyces padanus AOK-30, Streptomyces coelicolor DE07, Streptomyces olivaceus DE10 and Streptomyces geysiriensis DE27 have been found to significantly improve the adaptability of plants to drought and saline-alkali environments. However, there are few studies on Streptomyces that can improve the tolerance of alfalfa to salt-alkali stress. Therefore, exploring Streptomyces that can improve the tolerance of alfalfa to salt-alkali stress will be of great significance for promoting the growth of alfalfa in saline-alkali land. Summary of the invention

[0004] In order to explore Streptomyces that can improve the salt-alkali stress tolerance of alfalfa, the present invention screened a beautiful Streptomycete (Streptomycete bellus) Jrh8-9 from the rhizosphere soil of alkali buttercup grown in saline-alkali land, and proved through salt-alkali tolerance experiments, germination experiments and pot experiments that the Streptomyces can survive and grow in an environment with high salt and high pH, ​​and can promote the germination of alfalfa seeds and the growth of alfalfa seedlings under salt-alkali stress; in addition, it is also found that under non-stress conditions, the strain Jrh8-9 also has a certain promoting effect on the growth of alfalfa seedlings.

[0005] In order to solve the above technical problems and achieve corresponding technical effects, the present invention specifically provides the following technical solutions:

[0006] The first object of the present invention is to provide a strain of Streptomyces bellus Jrh8-9, which is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No. 29440, a deposit date of December 28, 2023, and an address of the depository unit at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0007] The second object of the present invention is to provide the use of the above-mentioned Streptomyces beautifulus Jrh8-9 in promoting the germination of alfalfa seeds under saline-alkali stress.

[0008] In one embodiment of the present invention, the saline-alkali stress includes neutral salt and alkaline salt stress.

[0009] The third object of the present invention is to provide the use of the above-mentioned Streptomyces beautifulus Jrh8-9 in promoting the growth of alfalfa seedlings under saline-alkali stress.

[0010] In one embodiment of the present invention, the saline-alkali stress includes neutral salt and alkaline salt stress.

[0011] The fourth object of the present invention is to provide the use of the above-mentioned Streptomyces beautifulus Jrh8-9 in promoting the growth of alfalfa seedlings under non-stress conditions.

[0012] The fifth object of the present invention is to provide the use of the above-mentioned Streptomyces beautifulus Jrh8-9 in the preparation of alfalfa growth-promoting agent or alfalfa salt-alkali tolerant growth-promoting agent.

[0013] The sixth object of the present invention is to provide a bacterial agent containing the above-mentioned Streptomyces beautifulus Jrh8-9.

[0014] The seventh object of the present invention is to provide the use of the above-mentioned bacterial agent in promoting the germination of alfalfa seeds under saline-alkali stress.

[0015] The eighth object of the present invention is to provide the use of the above-mentioned bacterial agent in promoting the growth of alfalfa seedlings under saline-alkali stress or non-stress.

[0016] In one embodiment of the present invention, the above-mentioned bacterial agent is to inoculate strain Jrh8-9 into ISP2 liquid culture medium for cultivation (30°C, 150rpm, cultivation for 120h). After cultivation, the bacteria are collected by centrifugation, and then the bacteria are resuspended in sterile water and diluted to OD 600 A value of 1.0 is used to obtain the above mentioned inoculum.

[0017] The ninth object of the present invention is to provide a method for improving the germination rate of alfalfa seeds under saline-alkali stress, wherein the method comprises soaking the alfalfa seeds in the bacterial solution of the above-mentioned Streptomyces beautifulis Jrh8-9.

[0018] The tenth object of the present invention is to provide a method for increasing alfalfa yield under saline-alkali stress, wherein the method comprises applying the bacterial solution of the above-mentioned Streptomyces beautifulis Jrh8-9 to the root system during the seedling stage.

[0019] Beneficial effects of the present invention:

[0020] The present invention screens an actinomycete from the rhizosphere soil of alkali buttercup grown in saline-alkali land, and the actinomycete is identified as Streptomycete bellus Jrh8-9 through molecular biological identification. The salt-alkali tolerance experiment proves that the Streptomycete can survive and grow in an environment with high salt and high pH. The germination experiment proves that soaking alfalfa seeds with the bacterial solution of the Streptomycete can improve the germination rate under neutral salt and alkaline salt stress during the seed period. The potted experiment proves that applying the bacterial solution of Streptomycete Jrh8-9 to the root system during the alfalfa seedling period can significantly improve the plant height, leaf area, aboveground fresh weight and root fresh weight of alfalfa under neutral salt and alkaline salt stress; and under non-stress, Streptomycete Jrh8-9 also has a certain promoting effect on the leaf area, aboveground fresh weight and root fresh weight of alfalfa. The present invention provides valuable bacterial species resources for promoting the growth of alfalfa under salt-alkali stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the phylogenetic tree analysis result of the 16S rRNA gene sequence of strain Jrh8-9;

[0022] Figure 2 is a growth curve of strain Jrh8-9 in liquid culture medium containing different concentrations of NaCl;

[0023] Figure 3 It is the growth curve of strain Jrh8-9 in liquid culture medium with different pH;

[0024] Figure 4 This is the result diagram of the effect of strain Jrh8-9 on alfalfa seed germination rate under different saline-alkali environments;

[0025] Figure 5 The results of the effect of strain Jrh8-9 on the growth status of alfalfa seedlings under different saline-alkali environments; Figure 5 A in the figure shows the effect of strain Jrh8-9 on the height of alfalfa seedlings under different saline-alkali environments. Figure 5 B in the figure is the effect of strain Jrh8-9 on the leaf area of ​​alfalfa seedlings under different saline-alkali environments. Figure 5 C in the figure shows the effect of strain Jrh8-9 on the aboveground fresh weight of alfalfa seedlings under different saline-alkali environments. Figure 5 D in the figure is the effect of strain Jrh8-9 on the fresh weight of alfalfa seedling roots under different saline-alkali environments. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific implementation methods and the accompanying drawings of the specification. The embodiments described in the present invention are not all embodiments of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The experimental methods used in the following embodiments are conventional methods unless otherwise specified, and the materials, reagents and instruments used are conventional materials, reagents and instruments in the field unless otherwise specified, and those skilled in the art can obtain them through commercial channels.

[0027] The beautiful Streptomyces (Streptomyces bellus) Jrh8-9 screened by the present invention is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.29440 and a deposit date of December 28, 2023. The address of the depository unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0028] The culture medium involved in the present invention is as follows:

[0029] NA solid culture medium: weigh 3 g beef extract powder and 10 g peptone, add to distilled water and stir until completely dissolved, and dilute to 1 L; adjust the pH value of the culture medium to about 7.2, add 1.5% agar by mass fraction, and then sterilize at high temperature and high pressure.

[0030] ISP2 liquid culture medium: 1% malt extract powder, 0.4% yeast extract powder, 0.4% glucose, and the balance is distilled water; the pH value of the culture medium is adjusted to about 7.2, and then sterilized by high temperature and high pressure.

[0031] Example 1: Isolation and identification of Streptomyces beautifulis Jrh8-9

[0032] 1. Isolation of strain Jrh8-9

[0033] 5 g of soil sample (collected from the rhizosphere soil of Ranunculus alkali growing in saline-alkali land) was added to a triangular flask containing 45 mL of sterile water and shaken on a shaker at 150 rpm for 30 min. Then, 1 mL of the suspension was added to 9 mL of sterile water and mixed to obtain 10 -1 Follow the same method to make a gradient dilution until 10 -5 Take 10 -3 , 10 -4 and 10 -5100 μL of each dilution was spread on NA solid medium and cultured in an incubator at 28° C. for 7 days. Then, a single colony on the NA solid medium was picked for purification, and a strain of bacteria was named Jrh8-9.

[0034] 2. Identification of strain Jrh8-9

[0035] Jrh8-9 strain was inoculated into ISP2 liquid medium and cultured at 150rpm and 30℃ for 72h, then centrifuged to collect the bacteria. Genomic DNA was extracted using a bacterial genome extraction kit, and the 16S rRNA gene sequence was amplified using universal primers 27F and 1492R using genomic DNA as a template. The purified PCR products were connected and transformed, and the positive clones were sent to Sangon Biotech Co., Ltd. for sequencing. The obtained 16S rRNA gene sequence was submitted to the EzBioCloud website for comparison. Typical strains with close kinship were selected for phylogenetic analysis based on the 16SrRNA gene sequence.

[0036] The sequencing results showed that the 16S rRNA gene sequence of strain Jrh8-9 was as shown in SEQ ID NO.1 (1469nt). The 16S rRNA gene sequence of strain Jrh8-9 was analyzed by EzBioCloud website, and the results showed that the strain with the highest similarity was Streptomyces bellus ISP 5185 T , with a similarity of 99.5%. Then, the Streptomyces with the top 21 similarities were selected for phylogenetic analysis based on the 16S rRNA gene sequence together with strain Jrh8-9. The results are as follows Figure 1 As shown, strain Jrh8-9 and Streptomyces bellus ISP 5185 T In an independent branch. Based on the results of 16SrRNA gene sequence analysis, strain Jrh8-9 was finally identified as Streptomyces bellus and deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number CGMCC No. 29440 and the deposit date December 28, 2023. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0037] SEQ ID NO.1:

[0038]

[0039] Example 2: Detection of salt and alkali tolerance of Streptomyces beautifulis Jrh8-9

[0040] The strain Jrh8-9 was inoculated into ISP2 liquid medium and cultured at 150 rpm and 30°C for 72 h to obtain seed solution. The seed solution was transferred to ISP2 liquid medium with NaCl concentrations of 0, 0.3, 0.6, 0.9, 1.2, and 1.5 mol / L, and ISP2 liquid medium with pH values ​​of 5, 6, 7.2, 9, 10, and 11, respectively, at a ratio of 1% to evaluate the strain's tolerance range to NaCl and pH.

[0041] The results of salt tolerance test are as follows Figure 2 As shown in Figure 2, with the increase of NaCl concentration in the liquid culture medium, the growth of strain Jrh8-9 gradually weakened. However, after 3 days of cultivation, the OD of strain Jrh8-9 in the liquid culture medium with a NaCl concentration of 1.2 mol / L was 600 The value can still reach 0.77, but when the NaCl concentration in the liquid culture medium is increased to 1.5 mol / L, the OD 600 The value was reduced to 0.15. It can be seen that strain Jrh8-9 can grow well in an environment with a salt concentration not exceeding 1.2 mol / L. Figure 3 As shown in Figure 2, when the pH of the liquid culture medium was 7.2, the strain Jrh8-9 grew most vigorously. As the pH of the liquid culture medium increased or decreased, the growth state of the strain Jrh8-9 gradually weakened. When the pH of the liquid culture medium was 9, the OD of the strain Jrh8-9 after 3 days of cultivation was 600 The value reached 3.08, but when the pH of the liquid medium was increased to 10, the OD of strain Jrh8-9 600 The value decreased to 0.47. It can be seen that strain Jrh8-9 can grow well in an environment where the pH does not exceed 9. These results show that strain Jrh8-9 can survive and grow in saline-alkali soil with high salt and high pH.

[0042] Example 3: Application of Streptomyces beautifulis Jrh8-9 in promoting alfalfa seed germination under saline-alkali stress

[0043] Soak the seeds of alfalfa in 75% ethanol by volume for 10 minutes, shaking them intermittently during the process. Then discard the ethanol solution, wash with 75% ethanol by volume for 5 minutes and discard the solution. Wash the seeds twice with anhydrous ethanol and finally blow dry in a sterile operating table. Inoculate strain Jrh8-9 into ISP2 liquid culture medium, culture at 150rpm and 30℃ for 120h, and then collect the cells by centrifugation. Resuspend the cells with sterile water and dilute to OD600 The value is 1.0, which is used as the bacterial suspension. The sterilized alfalfa seeds were soaked in the bacterial suspension for 24 h, and then placed on sterile filter paper moistened with sterile water, 60 mM NaCl solution, and 40 mM NaHCO3 solution, respectively, and germinated for 5 days under room temperature and dark conditions, and then the seed germination rate of each treatment group was counted.

[0044] The results are as follows Figure 4 As shown in the figure, both 60mM NaCl and 40mM NaHCO3 treatments significantly inhibited the germination of alfalfa seeds. Under 60mM NaCl treatment, the germination rate of seeds soaked with Jrh8-9 suspension increased by 25.7% compared with seeds not treated with Jrh8-9 suspension; while under 40mM NaHCO3 treatment, Jrh8-9 suspension treatment increased the germination rate of seeds by 23.8%.

[0045] Example 4: Application of Streptomyces beautifulis Jrh8-9 in promoting the growth of alfalfa seedlings under saline-alkali stress

[0046] The surface of alfalfa seeds was sterilized according to the method in Example 3. Then the seeds were placed in sterile water and soaked for 24 hours, then transferred to moist gauze and germinated at room temperature for 2 days. Seeds with consistent germination were selected and sown in pots filled with sterilized soil (peat soil: vermiculite = 2:1). When the seedlings grow to three leaves and one heart, the bacterial suspension was inoculated into the roots of the seedlings at a ratio of 10mL of bacterial suspension per seedling (preparation method is the same as in Example 3). One week after inoculation, the soil was irrigated with 125mM NaHCO3 solution and 150mM NaCl solution respectively until the solution seeped out from the bottom of the flowerpot. The stress solution was irrigated once every 3 days, and the growth indicators of the seedlings were counted after 3 weeks of treatment. The entire cultivation process was carried out in a light culture room, with day and night temperatures of 25 and 20°C respectively, a photoperiod of 16 hours of light and 8 hours of darkness, and a light intensity of 150μmol / m 2 / s, relative humidity is 70%.

[0047] The results are as follows Figure 5As shown in the figure, under the treatment of 150mM NaCl, the plant height, leaf area, aboveground fresh weight and root fresh weight of alfalfa were significantly reduced; compared with the plants not inoculated with bacterial solution, the plant height, leaf area, aboveground fresh weight and root fresh weight of alfalfa plants added with Jrh8-9 bacterial solution increased by 35.8%, 31.4%, 58.3% and 69.0%, respectively. Under the treatment of 125mM NaHCO3, the plant height, leaf area, aboveground fresh weight and root fresh weight of alfalfa were also significantly reduced; but the addition of Jrh8-9 bacterial solution increased the plant height, leaf area, aboveground fresh weight and root fresh weight of alfalfa plants by 42.7%, 76.6%, 111.3% and 316.8%, respectively. In addition, under normal conditions, the addition of Jrh8-9 bacterial solution also significantly increased the leaf area, aboveground fresh weight and root fresh weight of alfalfa. It can be seen that strain Jrh8-9 can not only promote the growth of alfalfa seedlings under saline-alkali stress, but also promote the growth of alfalfa seedlings under non-salt-alkali stress.

[0048] Although the present invention has been disclosed as above in the form of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Streptomyces bellus Jrh8-9, characterized in that: The deposit number is CGMCC No.29440.

2. Use of the beautiful Streptomyces Jrh8-9 described in claim 1 in promoting alfalfa seed germination under saline-alkali stress.

3. Use of the beautiful Streptomyces Jrh8-9 described in claim 1 in promoting the growth of alfalfa seedlings under saline-alkali stress.

4. Use of the beautiful Streptomyces Jrh8-9 described in claim 1 in promoting the growth of alfalfa seedlings under non-stress conditions.

5. Use of the beautiful Streptomyces Jrh8-9 described in claim 1 in the preparation of an alfalfa growth-promoting agent or an alfalfa salt-alkali tolerant growth-promoting agent.

6. A bacterial agent containing the beautiful Streptomyces Jrh8-9 according to claim 1.

7. Use of the bacterial agent according to claim 6 in promoting germination of alfalfa seeds under saline-alkali stress.

8. Use of the bacterial agent according to claim 6 in promoting the growth of alfalfa seedlings under saline-alkali stress or non-stress.

9. A method for improving the germination rate of alfalfa seeds under saline-alkali stress, characterized in that: The alfalfa seeds are soaked in the bacterial liquid of the beautiful Streptomyces Jrh8-9 described in claim 1.

10. A method for increasing alfalfa yield under saline-alkali stress, characterized in that: During the seedling stage, the bacterial solution of the beautiful Streptomyces Jrh8-9 of claim 1 is applied to the root system.

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