Elaeagnus angustifolia rhizosphere growth-promoting bacterium and salt-tolerant induction method thereof

Abstract

The invention relates to the technical field of microbial technology and agricultural application, and discloses an elaeagnus angustifolia rhizosphere growth-promoting bacterium and a salt-tolerant induction method thereof. The elaeagnus angustifolia rhizosphere growth-promoting bacterium SZ1 is preserved in the China General Microbiological Culture Collection Center on October 21, 2024, the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO.32296. The elaeagnus angustifolia rhizosphere growth-promoting bacterium SZ1 disclosed by the invention has the advantages that the preservation number is CGMCC NO.32296; the elaeagnus angustifolia rhizosphere growth-promoting bacterium SZ1 simultaneously has six growth-promoting functions of decomposing organic phosphorus and inorganic phosphorus, producing iron carriers, producing IAA, fixing nitrogen and producing ammonia, can remarkably promote the growth and development of plants, has relatively strong NaCl tolerance after induction, can normally grow under the condition of 10.0% of NaCl, and has a good application prospect. A new strain resource is provided for development of a novel plant growth promoting technology, a new thought is provided for improvement of plant growth and salt tolerance in a saline-alkali environment, and the strain has a wide application prospect in saline-alkali soil plant growth and ecological restoration.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology and agricultural application technology, and in particular to a rhizosphere growth-promoting bacterium of Elaeagnus angustifolia and a method for inducing salt tolerance thereof. Background Art

[0002] Elaeagnus angustifolia has strong resistance to drought, cold and salt, and can maintain strong growth vitality in harsh environments. Therefore, Elaeagnus angustifolia plays an important role in windbreak and sand fixation, soil improvement, and ecological restoration. Especially in Xinjiang, Elaeagnus angustifolia is a key plant to improve soil salinization and restore the ecological environment in Xinjiang. However, with global climate change and long-term unreasonable agricultural farming methods, the problem of soil salinization in Xinjiang has become increasingly serious, resulting in the restriction of the root growth of Elaeagnus angustifolia, which seriously affects the absorption of water and nutrients by Elaeagnus angustifolia, thus significantly weakening the growth of Elaeagnus angustifolia and greatly reducing the effect of ecological restoration.

[0003] Plant growth-promoting rhizobacteria (PGPR) can not only provide essential ions to the soil, regulate the imbalance of ions in the soil, improve the rhizosphere environment of plants, and enhance the salt tolerance of plants, but also provide plants with rich nutrients, which are absorbed and utilized by plants, thus playing an active role in the restoration of vegetation and the improvement of saline-alkali land. In this context, the research and development of plant growth-promoting rhizobacteria for Elaeagnus angustifolia is of great significance.

[0004] However, due to the special climate and soil environment in Xinjiang, most of the rhizosphere growth-promoting bacteria of Elaeagnus angustifolia outside Xinjiang have poor adaptability in Xinjiang and their application effect is not significant. Therefore, screening rhizosphere growth-promoting bacteria of Elaeagnus angustifolia suitable for the climate and soil environment in Xinjiang is beneficial to the growth of saline-alkali land plants and ecological restoration in Xinjiang. Summary of the invention

[0005] The purpose of the present invention is to provide a rhizosphere growth-promoting bacterium of Elaeagnus angustifolia and a method for inducing salt tolerance thereof. The rhizosphere growth-promoting bacterium of Elaeagnus angustifolia can be suitable for the special climate and soil environment in Xinjiang, and is beneficial to the growth of saline-alkali land plants and ecological restoration in Xinjiang.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] One of the technical solutions of the present invention:

[0008] A strain of rhizospheric growth-promoting bacteria SZ1 (Pantoea rodasii) of Elaeagnus angustifolia was deposited in the General Microbiology Center of China Microorganism Culture Collection on October 21, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.32296.

[0009] The second technical solution of the present invention:

[0010] The above-mentioned rhizosphere growth-promoting bacteria SZ1 of Elaeagnus angustifolia is used in promoting plant growth and ecological restoration in saline-alkali land.

[0011] The third technical solution of the present invention:

[0012] The above-mentioned method for inducing salt tolerance of the rhizosphere growth-promoting bacteria SZ1 of Elaeagnus angustifolia comprises the following steps:

[0013] 1) Prepare NB broth shake flasks with different NaCl concentration gradients;

[0014] 2) inoculating the fermentation liquid of the rhizosphere growth-promoting bacteria SZ1 of Elaeagnus angustifolia into the shaking bottle of the low-concentration NaCl broth prepared in step 1), and culturing by shaking to obtain a bacterial liquid;

[0015] 3) Determine the concentration of the bacterial solution obtained in step 2) and select OD 600 When the bacterial solution reaches 1.0, confirm the NaCl concentration and inoculate it into the NB broth shake flask with the next concentration gradient prepared in step 1), shake and culture to obtain the induced bacterial solution;

[0016] 4) Repeat the salt tolerance induction of step 3) for the induced bacterial solution obtained in step 3) until it is cultured in a NB broth shake flask containing 10% NaCl to obtain a salt-tolerant strain of Elaeagnus angustifolia rhizosphere growth-promoting bacteria that tolerates a NaCl concentration of 10.0%.

[0017] Furthermore, in step 1), the different NaCl concentration gradients are specifically: 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 6.0%, 7.0%, 8.0%, 9% and 10%.

[0018] Furthermore, in step 2), the preparation of the fermentation liquid of the Elaeagnus angustifolia rhizosphere growth-promoting bacteria SZ1 comprises the following steps:

[0019] The rhizosphere growth-promoting bacteria SZ1 of Elaeagnus angustifolia was inoculated into 100 mL of NB culture medium, and cultured in a constant temperature shaker at 37° C. and 180 r / min for 18 hours to obtain the fermentation liquid of the rhizosphere growth-promoting bacteria SZ1 of Elaeagnus angustifolia.

[0020] Furthermore, in step 2), the inoculation amount of the fermentation liquid of the Elaeagnus angustifolia rhizospheric growth-promoting bacteria SZ1 is 1%.

[0021] Furthermore, in step 2), the concentration of NaCl in the NaCl low-concentration NB broth shake flask is 0.5%, 1.0% or 1.5%.

[0022] Furthermore, in step 3), the inoculation amount of the bacterial solution is 1%.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The SZ1 rhizosphere growth-promoting bacteria of Elaeagnus angustifolia provided by the present invention simultaneously possesses six growth-promoting functions of decomposing organic and inorganic phosphorus, producing siderophores, producing IAA, fixing nitrogen and producing ammonia, and can significantly promote the growth and development of plants. Moreover, the SZ1 rhizosphere growth-promoting bacteria of Elaeagnus angustifolia provided by the present invention has a strong NaCl tolerance after induction, and can grow normally under 10.0% NaCl conditions, which provides a new bacterial resource for the development of new plant growth-promoting technologies, provides a new idea for improving plant growth and salt tolerance in saline-alkali environments, and has broad application prospects in plant growth and ecological restoration in saline-alkali lands. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. Moreover, the same reference symbols are used throughout the accompanying drawings to represent the same components. In the accompanying drawings:

[0026] Figure 1 : are the plate images of the rhizosphere growth-promoting bacteria SZ1 of Elaeagnus angustifolia and the salt-tolerant strain SZ1NY of the rhizosphere growth-promoting bacteria of Elaeagnus angustifolia, wherein A is the plate image of the rhizosphere growth-promoting bacteria SZ1 of Elaeagnus angustifolia in the NA nutrient agar plate, and B is the plate image of the salt-tolerant strain SZ1NY of the rhizosphere growth-promoting bacteria of Elaeagnus angustifolia in the NA nutrient agar plate;

[0027] Figure 2 The results of the nitrogen fixation ability test of the rhizosphere growth-promoting bacteria SZ1 and the salt-tolerant strain SZ1NY of the rhizosphere growth-promoting bacteria of Elaeagnus angustifolia.

[0028] Figure 3 The results of the siderophore production test of the rhizosphere growth-promoting bacteria SZ1 and the salt-tolerant strain SZ1NY of the rhizosphere growth-promoting bacteria of the angustifolia.

[0029] Figure 4 The results of the test of the ability of SZ1, a rhizosphere growth-promoting bacterium of Elaeagnus angustifolia, and the salt-tolerant strain SZ1NY of SZ1, a rhizosphere growth-promoting bacterium of Elaeagnus angustifolia, to degrade organic phosphorus.

[0030] Figure 5 The results of the inorganic phosphorus decomposition ability test of the rhizosphere growth-promoting bacteria SZ1 and the salt-tolerant strain SZ1NY of the rhizosphere growth-promoting bacteria of the angustifolia.

[0031] Figure 6 The results of the IAA production ability test of the rhizosphere growth-promoting bacteria SZ1 and the salt-tolerant strain SZ1NY of the rhizosphere growth-promoting bacteria of Elaeagnus angustifolia.

[0032] Figure 7The results of the NH3 production ability test of the rhizosphere growth-promoting bacteria SZ1 and the salt-tolerant strain SZ1NY of the rhizosphere growth-promoting bacteria of Elaeagnus angustifolia.

[0033] Figure 8 The results of the citrate utilization test of the rhizosphere growth-promoting bacteria SZ1 and the salt-tolerant strain SZ1NY of the rhizosphere growth-promoting bacteria of Elaeagnus angustifolia.

[0034] Fig. 9 The results of MR test of the rhizosphere growth-promoting bacteria SZ1 and the salt-tolerant strain SZ1NY of the rhizosphere growth-promoting bacteria of Elaeagnus angustifolia.

[0035] Fig.10 The results of H2S test on the rhizosphere growth-promoting bacteria SZ1 and the salt-tolerant strain SZ1NY of the rhizosphere growth-promoting bacteria of Elaeagnus angustifolia.

[0036] Fig.11 The results of VP test of the rhizosphere growth-promoting bacteria SZ1 and the salt-tolerant strain SZ1NY of the rhizosphere growth-promoting bacteria of Elaeagnus angustifolia.

[0037] Fig.12 The results of the starch hydrolyzing enzyme assay of the rhizosphere growth-promoting bacteria SZ1 and the salt-tolerant strain SZ1NY of the rhizosphere growth-promoting bacteria of the angustifolia.

[0038] Fig.13 The results of the protease assay test of the rhizosphere growth-promoting bacteria SZ1 and the salt-tolerant strain SZ1NY of the rhizosphere growth-promoting bacteria of the angustifolia.

[0039] Fig.14 The results of the cellulase assay of the rhizosphere growth-promoting bacteria SZ1 and the salt-tolerant strain SZ1NY of the rhizosphere growth-promoting bacteria of the angustifolia.

[0040] Fig.15 The results of the glucanase assay test of the rhizosphere growth-promoting bacteria SZ1 and the salt-tolerant strain SZ1NY of the rhizosphere growth-promoting bacteria of the angustifolia.

[0041] Fig.16 The chitinase assay results of the rhizosphere growth-promoting bacteria SZ1 and the salt-tolerant strain SZ1NY of the rhizosphere growth-promoting bacteria of the angustifolia.

[0042] Fig.17 This is the phylogenetic tree of the growth-promoting rhizobacteria SZ1 from Elaeagnus angustifolia. DETAILED DESCRIPTION

[0043] Now, various exemplary embodiments of the present invention are described in detail, and this detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention. It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention.

[0044] In addition, for the numerical range in the present invention, it is understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0045] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0046] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0047] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0048] In the following examples, a strain of rhizosphere growth-promoting bacteria SZ1 (Pantoea rodasii) of Elaeagnus angustifolia was isolated and screened from saline-alkali soil in Yuepu Lake County. It was preliminarily identified as dispersed Pantoea based on morphological characteristics and 16SrRNA sequence determination. It was deposited in the General Microbiological Center of China National Microbiological Culture Collection on October 21, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.32296.

[0049] Example 1

[0050] Induction of Salt Tolerance by Growth-Promoting Rhizobacteria SZ1 in Elaeagnus angustifolia

[0051] 1. Preparation of fermentation liquid of rhizosphere growth-promoting bacteria SZ1 of Elaeagnus angustifolia:

[0052] The rhizosphere growth-promoting bacteria SZ1 of Elaeagnus angustifolia was inoculated into 100 mL of NB medium, and cultured in a constant temperature shaker at 37° C. and 180 r / min for 18 hours to obtain the fermentation liquid of the rhizosphere growth-promoting bacteria SZ1 of Elaeagnus angustifolia;

[0053] 2. Salt tolerance induction of rhizosphere growth-promoting bacteria SZ1 in Elaeagnus angustifolia:

[0054] 1) Prepare NB broth shake flasks with different NaCl concentration gradients;

[0055] Among them, the concentration gradient of NaCl is 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 6.0%, 7.0%, 8.0%, 9%, 10%;

[0056] 2) The fermentation liquid of the rhizosphere growth-promoting bacteria SZ1 prepared in step 1 was inoculated into the NB broth shake flask containing 0.5%, 1.0% or 1.5% NaCl prepared in step 1) at an inoculation rate of 1%, and the mixture was shaken and cultured in a constant temperature shaker at 37° C. and 180 r / min for 48 hours to obtain a bacterial liquid;

[0057] 3) Determine the concentration of the bacterial solution obtained in step 2) and select OD 600 When the bacterial solution reaches 1.0, confirm the NaCl concentration, and inoculate it into the NB broth shake flask of the next concentration gradient prepared in step 1) according to the inoculum amount of 1%, and shake and culture it in a constant temperature shaker at 37°C and 180r / min for 48h to obtain the induced bacterial solution;

[0058] 4) Repeat the salt tolerance induction of step 3) for the induced bacterial solution obtained in step 3) until it is cultured in a NB broth shake flask containing 10% NaCl to obtain the salt-tolerant strain SZ1NY of Elaeagnus angustifolia rhizosphere growth-promoting bacteria with a NaCl concentration tolerance of 10.0%.

[0059] Plate images of the rhizosphere growth-promoting bacteria SZ1 and the salt-tolerant strain SZ1NY of the rhizosphere growth-promoting bacteria of Elaeagnus angustifolia Figure 1 As shown, A is the plate image of the rhizosphere growth-promoting bacteria SZ1 of Elaeagnus angustifolia in NA nutrient agar plate, and B is the plate image of the salt-tolerant strain SZ1NY of the rhizosphere growth-promoting bacteria of Elaeagnus angustifolia in NA nutrient agar plate;

[0060] Example 2

[0061] Growth-promoting performance of rhizosphere growth-promoting bacteria SZ1 and salt-tolerant strain SZ1NY of rhizosphere growth-promoting bacteria of Elaeagnus angustifolia

[0062] The growth-promoting performance of the rhizosphere growth-promoting bacteria SZ1 and the salt-tolerant strain SZ1NY of the rhizosphere growth-promoting bacteria of the angustifolia was tested as follows:

[0063] 1. Nitrogen fixation capacity test

[0064] The strain was inoculated into NB liquid culture medium, shaken and cultured in a constant temperature shaker at 37°C and 180r / min for 24h, then 5μL of bacterial solution was inoculated into the center of the Axubei nitrogen-fixing culture medium, and each strain was inoculated 3 times, and cultured at 37°C for 3d, and the growth of the strain was observed. If the strain grew normally, it was judged to have the ability to fix nitrogen;

[0065] The results of nitrogen fixation test are as follows: Figure 2 As shown;

[0066] Depend on Figure 2 It can be seen that strains SZ1 and SZ1NY both grew normally and had the ability to fix nitrogen;

[0067] 2. Siderophore production capacity test

[0068] The strain was inoculated on MKB-CAS solid plates and cultured in a constant temperature incubator at 30°C for 3 days. Each treatment was repeated 3 times. If an orange-yellow halo appeared around the colony, it was judged to have the ability to produce siderophores.

[0069] The results of the iron carrier production capacity test are as follows Figure 3 As shown;

[0070] Depend on Figure 3 It can be seen that strains SZ1 and SZ1NY both produced orange-yellow halos around them, indicating that they had the ability to produce siderophores;

[0071] 3. Detection of the ability to decompose organic phosphorus

[0072] Inoculate the strain in the center of the organic phosphorus culture medium and incubate it upside down at 37℃ for 5 days. If a transparent circle is formed around the colony, it is judged to have the ability to solubilize phosphorus.

[0073] The results of the test on the ability to decompose organic phosphorus are as follows: Figure 4 As shown;

[0074] Depend on Figure 4 It can be seen that the strains SZ1 and SZ1NY have transparent circles around them, which indicate that they have the ability to degrade organic phosphorus.

[0075] 4. Inorganic phosphorus decomposition ability test

[0076] Inoculate the strain in the center of the inorganic phosphorus culture medium and incubate it upside down at 37℃ for 5 days. If a transparent circle is formed around the colony, it is judged to have the ability to solubilize phosphorus.

[0077] The results of the inorganic phosphorus decomposition ability test are as follows Figure 5 As shown;

[0078] Depend on Figure 5 It can be seen that the strains SZ1 and SZ1NY have transparent circles around them, which indicate that they have the ability to decompose inorganic phosphorus.

[0079] 5. IAA production capacity test

[0080] The strain was adjusted to OD 600The bacterial suspension was inoculated into LB liquid medium containing L-tryptophan (100 mg / L) at a 1% (V / V) inoculation amount, and cultured in a constant temperature shaker at 30°C and 180 r / min for 1 day. After that, 50 μL of the bacterial suspension was dropped onto a white porcelain plate, and 50 μL of 50 mg / L Salkowski colorimetric solution (50 mL 35% HClO4 + 1 mL 0.5 mol / LFeCl3) was added. The uninoculated LB liquid medium was added with an equal amount of colorimetric solution as a positive control, and the white porcelain plate was placed in a light-proof environment for 30 minutes and then observed. If the solution turned red, it indicated that the strain was able to secrete IAA.

[0081] The results of IAA production capacity test are as follows Figure 6 As shown;

[0082] Depend on Figure 6 It can be seen that after the bacterial solution SZ1 and SZ1NY were reacted, the solution color turned red, and both had the ability to produce IAA;

[0083] 6. NH3 production capacity test

[0084] The strain was inoculated into a test tube containing 10 mL of peptone water liquid culture medium and incubated at 30 °C and 200 r·min －1 The culture was shaken in a constant temperature shaker for 48 h, and then 0.5 mL of Nessler reagent was added. If a yellow-brown precipitate appeared, it indicated that it had the ability to produce NH3.

[0085] The results of the NH3 production capacity test are as follows: Figure 7 As shown;

[0086] Depend on Figure 7 It can be seen that after the reaction, the bacterial liquids SZ1 and SZ1NY both produced yellow-brown precipitates and had the ability to produce NH3.

[0087] Example 3

[0088] Physicochemical properties of rhizosphere growth-promoting bacteria SZ1 and salt-tolerant strain SZ1NY of rhizosphere growth-promoting bacteria of Elaeagnus angustifolia

[0089] The physical and chemical properties of the rhizosphere growth-promoting bacteria SZ1 and the salt-tolerant strain SZ1NY of the rhizosphere growth-promoting bacteria of Elaeagnus angustifolia were tested, as follows:

[0090] 1. pH tolerance test

[0091] The pH of NB liquid medium (1 g of peptone, 0.5 g of yeast extract powder, 0.5 g of sodium chloride, 0.5 g of glucose, and 100 mL of distilled water) was adjusted to 3, 5, 7, 9, or 11 using 1 mol / L NaOH or HCl. Then, the bacterial solution to be tested was inoculated into the above NB liquid medium at an inoculum rate of 1%, and cultured in a constant temperature shaker at 37°C and 180 r / min for 48 h. The growth of the strain was observed, and tolerance was positive, and intolerance was negative;

[0092] The results of the pH tolerance test are shown in Table 1;

[0093] 2. Temperature tolerance test

[0094] Inoculate the test bacteria into NB liquid culture medium at a 1% inoculum volume, and culture in a constant temperature shaker at 25°C, 28°C, 30°C, 33°C, 35°C, 37°C, 40°C, 45°C or 50°C at 180 r / min for 48 h, and observe the growth of the strain. Tolerance is positive, and intolerance is negative.

[0095] The results of the temperature tolerance test are shown in Table 1;

[0096] 3. Sodium chloride tolerance test

[0097] The test bacterial suspension was inoculated into NB liquid culture medium containing 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 8.5%, 9.0%, 9.5% or 10% NaCl at an inoculum size of 1%, and cultured in a constant temperature shaker at 37°C and 180 r / min for 48 h. The growth of the strain was observed. Tolerance was positive, and intolerance was negative.

[0098] The results of the sodium chloride tolerance test are shown in Table 1;

[0099] 4. Citrate Utilization Test

[0100] Inoculate the test bacterial solution into citrate medium (NaCl 0.5g, MgSO4 0.02g, NH4H2PO4 0.1g, K2HPO4 0.1g, sodium citrate 0.5g, 0.2% bromothymol blue 4mL, agar powder 2g, distilled water 100mL) at an inoculum volume of 1%, and culture at 28°C for 48h. Observe the strain. If the strain changes from light green to blue, the test result is positive, otherwise the test result is negative.

[0101] The results of the citrate utilization test are shown in Table 1 and Figure 8 As shown;

[0102] 5. MR test

[0103] The bacterial solution to be tested was inoculated into a glucose-peptone culture medium (0.5 g glucose, 0.5 g peptone, 0.2 g K2HPO4, 100 mL distilled water) at an inoculum volume of 1%, and cultured at a constant temperature of 28°C for 48 h. Then, methyl red reagent was added dropwise to the glucose-peptone culture medium and mixed well. The glucose-peptone culture medium without bacterial solution inoculation was used as a control. The culture medium was observed. If the culture medium was red, the test result was positive, and if it was yellow, the test result was negative.

[0104] The results of MR test are shown in Table 1 and Fig. 9 As shown;

[0105] 6. Starch hydrolysis test

[0106] The bacterial solution to be tested was inoculated onto a starch agar plate (soluble starch 0.2g, beef extract 0.5g, peptone 1g, NaCl 0.5g, agar 2g, distilled water 100mL) according to an inoculum volume of 1%, and cultured at 28°C for 48h. After the colonies were formed, iodine solution was dripped onto the surface of the plate and the plate was observed. If the plate was blue-black with a colorless transparent circle (starch had been hydrolyzed), the test result was positive, otherwise the test result was negative;

[0107] The test results of starch hydrolysis are shown in Table 1;

[0108] 7. H2S test

[0109] The test bacteria liquid was inoculated into Chessner medium (1 g of peptone, 0.05 g of ferric citrate, 1.8 g of agar, and 100 mL of distilled water) at an inoculum volume of 1%, and cultured at 28°C for 48 h to observe the production of melanin (hydrogen sulfide). If melanin was produced, the test result was positive, otherwise the test result was negative.

[0110] The results of H2S test are shown in Table 1 and Fig.10 As shown;

[0111] 8. VP test

[0112] The bacterial liquid to be tested was inoculated into a glucose-peptone culture medium (0.5 g glucose, 0.5 g peptone, 0.2 g K2HPO4, 100 mL distilled water) at an inoculum volume of 1%, and cultured at a constant temperature of 28°C for 48 h. Afterwards, the glucose-peptone culture medium and NaOH were mixed according to 60% glucose-peptone culture medium and 40% NaOH, and a small amount of creatine was added. The glucose-peptone culture medium without bacterial liquid inoculation was used as a control. The culture medium was observed. If the culture medium was red, the test result was positive, otherwise the test result was negative.

[0113] The VP test results are shown in Table 1 and Fig.11 As shown;

[0114] 9. Carbon source utilization

[0115] 10 mL of carbon source utilization basal culture medium ((NH4)2SO4 0.2 g, MgSO4 0.02 g, NaH2PO4 0.05 g, CaCl2 0.01 g, K2HPO4 0.05 g, distilled water 100 mL) was placed in a centrifuge tube, and carbon source substrates (glucose, lactose, glycerol, soluble starch, sucrose, fructose, maltose, chitosan or rhamnose, wherein sugar alcohol carbon source substrates were added at a final concentration of 1%, and other carbon source substrates were added at a final concentration of 0.2%), and sterilized by high pressure steam; thereafter, the bacterial liquid to be tested was inoculated into the above-mentioned carbon source utilization basal culture medium containing the carbon source substrate according to an inoculation amount of 1%, and cultured in a constant temperature shaker at 28°C and 180 r / min for 48 h, and the culture medium was observed. If the culture medium became turbid, the experimental result was positive, otherwise the experimental result was negative;

[0116] The results of carbon source utilization detection are shown in Table 1;

[0117] 10. Nitrogen source utilization

[0118] 10 mL of nitrogen source utilization basal medium (glucose 1 g, KH2PO4 0.136 g, CaCl2 0.01 g, Na2HPO4 0.213 g, MgSO4 0.02 g, FeSO4 0.05 g, distilled water 100 mL) was placed in a centrifuge tube, and nitrogen source substrates (ammonium sulfate, diammonium phosphate, ammonium oxalate, L-tyrosine, diammonium citrate, glutamic acid, added at a final concentration of 0.1%) were added, and sterilized with high pressure steam; thereafter, the bacterial liquid to be tested was inoculated into the nitrogen source utilization basal medium containing the nitrogen source substrate according to an inoculation amount of 1%, and cultured in a constant temperature shaker at 28°C and 180 r / min for 48 h, and the condition of the culture medium was observed. If the culture medium became turbid, the experimental result was positive, otherwise the experimental result was negative;

[0119] The results of nitrogen source utilization test are shown in Table 1;

[0120] Table 1 Physical and chemical properties test results

[0121]

[0122]

[0123] Note: "+" in the table indicates positive, "-" indicates negative;

[0124] The results of the pH tolerance test showed that the pH tolerance range of strain SZ1 was 5-9; the results of the temperature tolerance test showed that the optimal growth temperature range of strains SZ1 and SZ1NY was 28-37°C; the results of the sodium chloride tolerance test showed that the maximum salt concentration tolerance of strain SZ1 was 3%. As the salt concentration gradually increased, the growth of strain SZ1 was inhibited and eventually stopped growing. After salt tolerance induction, the maximum salt concentration tolerance of strain SZ1NY was 10%; the results of the citrate utilization test showed that strains SZ1 and SZ1NY were both positive; the results of the MR test showed that strains SZ1 and SZ1NY were both positive; the results of the starch hydrolysis test showed that strains SZ1 and SZ1NY were both positive; the results of the H2S test showed that strains SZ1 and SZ1NY were both positive; the results of the VP test showed that strains SZ1 and SZ1N Y were all negative; the results of carbon source utilization test showed that strains SZ1 and SZ1NY could effectively utilize a variety of sugars such as glucose, glycerol, sucrose, maltose, lactose, soluble starch and fructose as carbon sources for growth and metabolism, but did not have the ability to utilize rhamnose and chitosan; the results of nitrogen source utilization test showed that strains SZ1 and SZ1NY both showed a certain selectivity and could not effectively utilize ammonium sulfate, but had a strong ability to utilize ammonium oxalate, diammonium citrate, L-tyrosine, diammonium phosphate and glutamic acid.

[0125] Example 4

[0126] Enzyme activities of growth-promoting rhizobacteria SZ1 and salt-tolerant strain SZ1NY of rhizobacteria promoting growth in Elaeagnus angustifolia

[0127] The enzyme activities of the rhizosphere growth-promoting bacteria SZ1 and the salt-tolerant strain SZ1NY of the rhizosphere growth-promoting bacteria of Elaeagnus angustifolia were detected as follows:

[0128] 1. Starch hydrolyzing enzyme assay

[0129] The strain to be tested was inoculated into the starch hydrolyzing enzyme detection medium plate, cultured at 37°C for 72 hours, the medium plate was taken out and iodine solution was added, and the iodine solution in the culture dish was discarded after color development, and the result of the test was observed to see whether a transparent circle was produced. If a transparent circle was produced, the test result was positive, otherwise the test result was negative;

[0130] The results of the starch hydrolyzing enzyme assay are shown in Table 2 and Fig.12 As shown;

[0131] 2. Protease assay

[0132] The strain to be tested was inoculated into a protease detection medium plate and cultured at 37°C for 72 hours to observe whether a transparent circle was produced. If a transparent circle was produced, the test result was positive, otherwise the test result was negative;

[0133] The results of the protease assay are shown in Table 2 and Fig.13 As shown;

[0134] 3. Cellulase assay

[0135] The strain to be tested was inoculated into a cellulase detection medium plate, cultured at 37°C for 72 hours, the medium plate was taken out and 1 mg / mL Congo red dye solution was added, dyed for 1 hour, the dye solution was discarded, 1 mol / L NaCl solution was added, decolorized for 30 minutes, the decolorization solution was discarded, and whether a transparent circle was produced was observed. If a transparent circle was produced, the test result was positive, otherwise the test result was negative;

[0136] The results of the cellulase assay are shown in Table 2 and Fig.14 As shown;

[0137] 4. Glucanase assay

[0138] The strain to be tested was inoculated into a glucanase detection medium plate and cultured at 37°C for 72 hours to observe whether a transparent circle was produced. If a transparent circle was produced, the test result was positive, otherwise the test result was negative;

[0139] The results of the glucanase assay are shown in Table 2 and Fig.15 As shown;

[0140] 5. Chitinase assay

[0141] The strain to be tested was inoculated into a chitinase detection medium plate and cultured at 37°C for 72 hours to observe whether a transparent circle was produced. If a transparent circle was produced, the test result was positive, otherwise the test result was negative;

[0142] The chitinase assay test results are shown in Table 2 and Fig.16 As shown;

[0143] Table 2 Enzyme activity test results

[0144]

[0145] Note: "+" in the table indicates positive, "-" indicates negative;

[0146] From the results of the starch hydrolase assay test, it can be seen that strains SZ1 and SZ1NY are both positive, indicating that the strains can produce amylase and have the ability to decompose starch; from the results of the protease assay test, it can be seen that strains SZ1 and SZ1NY are both positive, indicating that the strains can secrete protease and have the ability to decompose protein; from the results of the cellulase assay test, it can be seen that strains SZ1 and SZ1NY are both positive, indicating that the strains can secrete cellulase and have the ability to decompose cellulose; from the results of the glucanase assay test, it can be seen that strains SZ1 and SZ1NY are both positive, indicating that the strains can secrete glucanase and have the ability to decompose glucan; from the results of the chitinase assay test, it can be seen that strains SZ1 and SZ1NY are both negative, indicating that the strains do not have the ability to decompose chitin.

[0147] Example 5

[0148] Optimization of fermentation technology of salt-tolerant strain SZ1NY of rhizosphere growth-promoting bacteria of Elaeagnus angustifolia

[0149] 1. Preparation of fermentation liquid of salt-tolerant strain SZ1NY of rhizosphere growth-promoting bacteria of Elaeagnus angustifolia:

[0150] The salt-tolerant strain SZ1NY of the rhizosphere growth-promoting bacteria of the angustifolia was inoculated into 100 mL of NB medium, and cultured in a constant temperature shaker at 37° C. and 180 r / min for 18 hours to obtain a fermentation liquid of the salt-tolerant strain SZ1NY of the rhizosphere growth-promoting bacteria of the angustifolia;

[0151] 2. Single factor screening of carbon source

[0152] 10 g / L of carbon source (glucose, sucrose, molasses or white sugar) was added to the NB medium, and sterilized with high-pressure steam. Then, the fermentation liquid of the salt-tolerant strain SZ1NY of the rhizosphere growth-promoting bacteria of Elaeagnus angustifolia was inoculated into the above medium at an inoculation rate of 1%, and cultured in a constant temperature shaker at 28°C and 180 r / min for 2 days, and the concentration of the fermentation liquid was measured;

[0153] The results of single factor screening of carbon sources are shown in Table 3;

[0154] Table 3 Carbon source single factor screening results

[0155]

[0156] From the data in Table 3, we can see that the concentration of fermentation broth in the molasses group is significantly higher than that in other carbon source groups. The concentrations of fermentation broth in other carbon source groups are white sugar > glucose > sucrose > CK. Molasses was selected as the carbon source in subsequent experiments.

[0157] 3. Single factor screening of nitrogen source

[0158] 10 g / L of nitrogen source (yeast extract, beef extract powder, peptone or soy peptone) was added to the NB medium, and sterilized with high-pressure steam. Then, the fermentation liquid of the salt-tolerant strain SZ1NY of the rhizosphere growth-promoting bacteria of Elaeagnus angustifolia was inoculated into the above medium at an inoculation rate of 1%, and cultured in a constant temperature shaker at 28°C and 180 r / min for 2 days, and the concentration of the fermentation liquid was measured;

[0159] The results of single factor screening of nitrogen sources are shown in Table 4;

[0160] Table 4 Single factor screening results of nitrogen source

[0161]

[0162] From the data in Table 4, we can see that the concentration of fermentation broth in yeast extract group is significantly higher than that in other nitrogen source groups. The concentration of fermentation broth in other nitrogen source groups is peptone>soybean peptone>beef extract powder>CK from high to low. Yeast extract is selected as nitrogen source in subsequent experiments.

[0163] 4. Carbon-nitrogen ratio screening

[0164] The carbon source and nitrogen source obtained by the above screening were added to the NB medium according to the carbon-nitrogen ratio of CN1 to CN16, and sterilized by high-pressure steam. Then, the fermentation liquid of the salt-tolerant strain SZ1NY of the rhizosphere growth-promoting bacteria of Elaeagnus angustifolia was inoculated into the above medium at an inoculation rate of 1%, and cultured in a constant temperature shaker at 28°C and 180r / min for 2 days, and the concentration of the fermentation liquid was determined;

[0165] The carbon-nitrogen ratio screening results are shown in Table 5;

[0166] Table 5 Carbon-nitrogen ratio screening results

[0167]

[0168]

[0169] From the data in Table 5, it can be seen that the concentration of the fermentation liquid in the CN11 group was significantly higher than that in the other carbon-nitrogen ratio groups. In the subsequent experiments, 1.5% molasses and 1.5% yeast extract were selected for the experiments;

[0170] 5. Single factor screening of inorganic salts

[0171] 1.5% molasses and 1.5% yeast extract were added to NB medium, and 0.5 g / L of inorganic salts (MnCl2, MnSO4·7H2O, (NH4)2SO4, NaCl, K2HPO4, MgSO4, KCl, FeCl2, NaH2PO4, FeSO4, Na2HPO4, KH2PO4) were added, and sterilized with high-pressure steam. Then, the fermentation liquid of the salt-tolerant strain SZ1NY of the rhizosphere growth-promoting bacteria of Elaeagnus angustifolia was inoculated into the above medium at an inoculation rate of 1%, and the culture was shaken in a constant temperature shaker at 28°C and 180 r / min for 2 days, and the concentration of the fermentation liquid was measured;

[0172] The results of single factor screening of inorganic salts are shown in Table 6;

[0173] Table 6 Inorganic salt single factor screening results

[0174]

[0175]

[0176] From the data in Table 6, it can be seen that the concentration of the fermentation broth in the FeCl2 group is significantly higher than that in the other inorganic salt groups, followed by (NH4)2SO4>MgSO4·7H2O>K2HPO4>FeSO4>Na2HPO4>NaH2PO4>KCl>KH2PO4>NaCl>MnCl2>MgSO4. The first four were selected for subsequent experiments;

[0177] The final fermentation formula was: peptone 5 g / L (derived from NB medium), NaCl 1 g / L (derived from NB medium), molasses 15 g / L, yeast extract 15 g / L (containing 5 g of yeast extract in NB medium), FeCl2 0.5 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.5 g / L, (NH4)2SO4 0.5 g / L;

[0178] 6. pH single factor screening

[0179] 1.5% molasses and 1.5% yeast extract were added to NB medium, and FeCl2 0.5 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.5 g / L and (NH4)2SO4 0.5 g / L were added, and the pH of the medium was adjusted to 3, 5, 7, 9 or 11, and sterilized with high-pressure steam. Then, the fermentation liquid of the salt-tolerant strain SZ1NY of the rhizosphere growth-promoting bacteria of Elaeagnus angustifolia was inoculated into the above medium at an inoculation rate of 1%, and cultured in a constant temperature shaker at 28°C and 180 r / min for 2 days, and the concentration of the fermentation liquid was determined;

[0180] The results of single factor screening with pH are shown in Table 7;

[0181] 7. Temperature single factor screening

[0182] 1.5% molasses and 1.5% yeast extract were added to NB medium, and FeCl2 0.5 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.5 g / L and (NH4)2SO4 0.5 g / L were added, the pH of the medium was adjusted to 7, and high-pressure steam sterilization was performed, and then the fermentation liquid of the salt-tolerant strain SZ1NY of the rhizosphere growth-promoting bacteria of Elaeagnus angustifolia was inoculated into the above medium at an inoculation rate of 1%, and the culture was shaken in a constant temperature shaker at 28°C, 30°C, 33°C, 35°C, 37°C, 40°C, 45°C or 50°C and 180 r / min for 2 days, and the concentration of the fermentation liquid was determined;

[0183] The results of single factor screening of temperature are shown in Table 7;

[0184] 8. Speed ​​single factor screening

[0185] 1.5% molasses and 1.5% yeast extract were added to NB medium, and FeCl2 0.5 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.5 g / L and (NH4)2SO4 0.5 g / L were added, the pH of the medium was adjusted to 7, and high-pressure steam sterilization was performed, and then the fermentation liquid of the salt-tolerant strain SZ1NY of the rhizosphere growth-promoting bacteria of Elaeagnus angustifolia was inoculated into the above medium at an inoculation rate of 1%, and the culture was shaken and cultured in a constant temperature shaker at 37°C, 100 r / min, 120 r / min, 140 r / min, 160 r / min, 180 r / min or 200 r / min for 2 days, and the concentration of the fermentation liquid was determined;

[0186] The single factor screening results of speed are shown in Table 7;

[0187] Table 7 Fermentation condition single factor screening results

[0188]

[0189] From the data in Table 7, it can be seen that the fermentation broth concentration of the pH 7.0 group was significantly higher than that of the other pH groups, the fermentation broth concentration of the temperature 37°C group was significantly higher than that of the other temperature groups, and the fermentation broth concentration of the speed 180r / min and 200r / min groups was significantly higher than that of the other speed groups, and there was no significant difference between the two.

[0190] The final fermentation conditions were: pH 7.0, 37°C, 180 r / min;

[0191] In summary, the optimal fermentation formula and conditions are shown in Table 8;

[0192]

[0193]

[0194] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

[0195]

[0196]

[0197]

Claims

1. A rhizosphere growth-promoting bacterium SZ1 (Pantoea rodasii), characterized in that: The rhizosphere growth-promoting bacteria SZ1 of Elaeagnus angustifolia were deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 21, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.32296.

2. An application of the rhizosphere growth-promoting bacteria SZ1 of Elaeagnus angustifolia as described in claim 1 in promoting plant growth and ecological restoration in saline-alkali land.

3. A method for inducing salt tolerance of the rhizosphere growth-promoting bacteria SZ1 of Elaeagnus angustifolia as claimed in claim 1, characterized in that: The following steps are involved: 1) Prepare NB broth shake flasks with different NaCl concentration gradients; 2) inoculating the fermentation liquid of the rhizosphere growth-promoting bacteria SZ1 of Elaeagnus angustifolia into the shaking bottle of the low-concentration NaCl broth prepared in step 1), and culturing by shaking to obtain a bacterial liquid; 3) Determine the concentration of the bacterial solution obtained in step 2) and select OD 600 When the bacterial solution reaches 1.0, confirm the NaCl concentration and inoculate it into the NB broth shake flask with the next concentration gradient prepared in step 1), shake and culture to obtain the induced bacterial solution; 4) Repeat the salt tolerance induction of step 3) for the induced bacterial solution obtained in step 3) until it is cultured in a NB broth shake flask containing 10% NaCl to obtain a salt-tolerant strain of Elaeagnus angustifolia rhizosphere growth-promoting bacteria that tolerates a NaCl concentration of 10.0%.

4. The salt tolerance inducing method according to claim 3, characterized in that: In step 1), the different NaCl concentration gradients are specifically: 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 6.0%, 7.0%, 8.0%, 9% and 10%.

5. The salt tolerance inducing method according to claim 3, characterized in that: In step 2), the preparation of the fermentation liquid of the rhizosphere growth-promoting bacteria SZ1 of Elaeagnus angustifolia comprises the following steps: The rhizosphere growth-promoting bacteria SZ1 of Elaeagnus angustifolia was inoculated into 100 mL of NB culture medium, and cultured in a constant temperature shaker at 37° C. and 180 r / min for 18 hours to obtain the fermentation liquid of the rhizosphere growth-promoting bacteria SZ1 of Elaeagnus angustifolia.

6. The salt tolerance inducing method according to claim 3, characterized in that: In step 2), the inoculation amount of the fermentation liquid of the Elaeagnus angustifolia rhizosphere growth-promoting bacteria SZ1 is 1%.

7. The salt tolerance inducing method according to claim 3, characterized in that: In step 2), the concentration of NaCl in the NaCl low-concentration NB broth shake flask is 0.5%, 1.0% or 1.5%.

8. The salt tolerance inducing method according to claim 3, characterized in that: In step 3), the inoculation amount of the bacterial solution is 1%.

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