Agrobacterium strain EC17 for improving salt tolerance of alfalfa and application of agrobacterium strain EC17

Abstract

The invention discloses an agrobacterium strain EC17 for improving salt tolerance of alfalfa and application of the agrobacterium strain EC17. Wherein the agrobacterium strain EC17 is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, the preservation date is December 25, 2024, and the preservation number is CGMCC NO.33194. The agrobacterium strain EC17 for improving salt tolerance of alfalfa has the functions of dissolving organic phosphorus and inorganic phosphorus, dissolving potassium, producing a homogeneous membrane, producing IAA and producing ACC deaminase. Under the stress of 200mmol / L and 250mmol / L NaCl, the germination index of the alfalfa seeds can be obviously improved. Under the stress of 200mmol / L NaCl, the growth index of alfalfa seedlings can be obviously improved.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to an agrobacterium that enhances salt tolerance in alfalfa. Agrobacterium sp. Strain EC17 and its applications. Background Technology

[0002] Soil salinization is a global ecological and agricultural challenge. It severely damages over 20% of the world's arable land, becoming one of the core causes of soil degradation. In my country, the total area of ​​saline-alkali land reaches 100 million hectares, accounting for 10% of the country's total land area, posing a serious challenge to sustainable agricultural development. However, saline-alkali land is not "wasteland," but rather a potential exploitable land resource. Its improvement and rational utilization can effectively alleviate the contradiction between arable land supply and demand in my country, providing important land security for the long-term development of agriculture.

[0003] To achieve the goal of cultivating alfalfa and other economic crops on saline-alkali land, two fundamental approaches are stress-resistance breeding to enhance plant salt tolerance and utilizing soil microorganisms to improve saline-alkali land. Currently, related research largely focuses on enhancing the plant's own salt tolerance, i.e., cultivating salt-tolerant crops and forage varieties. With the deepening of microbial research, utilizing soil microorganisms to enhance plant stress tolerance has become a new research hotspot—soil microorganisms can regulate multiple physiological functions of host plants, not only promoting plant growth and enhancing the efficiency of root absorption of soil nutrients, but also improving root resistance to biotic and abiotic stresses (such as salt-alkali stress), showing great potential for agricultural applications. Therefore, by creating a cumulative effect between soil microorganisms and the plant's own salt tolerance, target plants can grow in saline-alkali land with higher concentrations, providing a better solution for the efficient utilization of idle saline-alkali land. Therefore, there is an urgent need in existing technologies for a new strain that can improve the salt tolerance of alfalfa. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the existing technology and provide an Agrobacterium strain EC17 that enhances the salt tolerance of alfalfa and its application.

[0005] The objective of this invention is achieved through the following technical solution: an Agrobacterium strain EC17 that enhances the salt tolerance of alfalfa, wherein the Agrobacterium strain EC17 is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on December 25, 2024, with accession number CGMCCNO.33194.

[0006] This invention also provides the application of the Agrobacterium strain EC17, which enhances salt tolerance in alfalfa, to improve alfalfa seed germination indices under salt stress.

[0007] Furthermore, the germination indicators include germination potential, germination rate, germination index, radicle length, plumule length, and fresh weight of the seedling population.

[0008] Furthermore, the salt stress is 200 mmol / L NaCl stress.

[0009] Furthermore, the salt stress is 250 mmol / L NaCl stress.

[0010] The present invention also provides the application of the soil bacterium strain EC17, which enhances the salt tolerance of alfalfa, to improve the growth of alfalfa seedlings under salt stress.

[0011] Furthermore, the seedling growth status includes plant height, stem diameter, leaf area, above-ground fresh weight, and above-ground dry weight.

[0012] Furthermore, the salt stress is 200 mmol / L NaCl stress.

[0013] The beneficial effects of this invention are as follows: The *Agrobacterium* strain EC17, which enhances the salt tolerance of alfalfa, possesses the functions of dissolving organic and inorganic phosphorus, potassium solubilizing, producing bacterial films, producing IAA (inorganic acid oxidase), and producing ACC (anammase-dependent amino acids). Under stress of 200 mmol / L and 250 mmol / L NaCl, it can significantly improve alfalfa seed germination potential, germination rate, germination index, radicle length, plumule length, and fresh weight of seedlings. At 200 mmol / L… NaCl stress can significantly improve the growth indicators of alfalfa seedlings. Attached Figure Description

[0014] Figure 1 This describes the colony morphology of strain EC17; Figure 2 The results are the culture results of strain EC17 in solid medium containing inorganic phosphorus and solid medium containing organic phosphorus; where A is the culture result of strain EC17 in solid medium containing inorganic phosphorus and B is the culture result of strain EC17 in solid medium containing organic phosphorus. Figure 3 This is the result of culturing strain EC17 in a potassium-containing solid medium; Figure 4 This shows the crystal violet staining of strain EC17 in a 96-well plate. Figure 5 These are the results of the culture of strain EC17 in a siderophore-selective solid medium; Figure 6 This shows the staining results of Sachs reagent on 96-well plates of strain EC17; where CK is the control group and EC17 is the experimental group. Figure 7This is a bar chart of various germination indicators of alfalfa seeds under 200 mmol / L NaCl stress; where A is the germination potential bar chart, B is the germination rate bar chart, C is the germination index bar chart, D is the radicle length bar chart, E is the plumule length bar chart, and F is the fresh weight of the seedling population bar chart. Figure 8 This is a bar chart of various germination indicators of alfalfa seeds under 250 mmol / L NaCl stress; where A is the germination potential bar chart, B is the germination rate bar chart, C is the germination index bar chart, D is the radicle length bar chart, E is the plumule length bar chart, and F is the fresh weight of the seedling population bar chart. Figure 9 It is 200 mmol / L Bar charts showing the growth indicators of alfalfa seedlings under NaCl stress; where A is the plant height bar chart; B is the stem diameter bar chart; C is the leaf area bar chart; D is the aboveground fresh weight bar chart; and E is the aboveground dry weight bar chart. Detailed Implementation

[0015] The present invention will now be described in detail with reference to the accompanying drawings.

[0016] The chemical reagents used in the following examples are all commercially available conventional reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0017] The TSA solid culture medium used in the examples included: 15 g / L tryptone, 5 g / L soybean peptone, 5 g / L NaCl and 15 g / L agar.

[0018] The TSB liquid culture medium used in the examples included: 8.5 g / L tryptone, 1.5 g / L plant peptone, 2.5 g / L NaCl, 1.25 g / L K2HPO4, 1.25 g / L glucose, and distilled water.

[0019] The inorganic phosphorus-containing solid culture medium used in the examples included: KCl 0.2 g / L, glucose 10 g / L, Ca3(PO4) 5 g / L, MgCl2·6H2O 0.5 g / L, MgSO4·7H2O 0.25 g / L, (NH4)2SO4 0.1 g / L and agar 20 g / L.

[0020] The solid culture medium containing organophosphorus compounds used in the examples included: 10 g / L glucose, 0.5 g / L (NH4)2SO4, 0.3 g / L NaCl, 0.3 g / L KCl, 0.03 g / L FeSO4·7H2O, 0.03 g / L SO4Mn4·H2O, 0.2 g / L lecithin, 0.4 g / L yeast extract (Beijing Cooler Master Technology Co., Ltd., model: CY12011-500g), and 20 g / L agar, with a pH of 7.0-7.5.

[0021] The potassium-containing solid culture medium used in the examples included: sucrose 5 g / L, Na2HPO4 2 g / L, MgSO4·7H2O 0.005 g / L, FeCl3 0.005 g / L, CaCO3 0.1 g / L, potassium feldspar powder 0.1 g / L, and agar 20 g / L.

[0022] The siderophore-selective solid culture medium used in the examples included: CAS (Crazinium S) 60.5 mg / L, distilled water 50 ml / L, HCl 8 μL / L, 2 mol / L FeCl3·6H2O 5 μL / L, HDTMA (hexadecyltrimethylammonium bromide) 72.9 mg / L, proteose peptone 20 g / L, MgSO4·7H2O 1.5 g / L, KH2PO4 1.2 g / L, glycerol 10 g / L, agar 13 g / L, pipes (piperazine-1,4-diethanesulfonic acid) 30 g / L, and 50% NaOH 12 ml / L.

[0023] Example 1: Isolation, screening and identification of strain EC17 (a) Separation and screening This strain was derived from the roots of alfalfa planted in saline soil at the Hailiutu Experimental Base of Inner Mongolia Agricultural University. Alfalfa seedlings were planted in saline soil from the Hailiutu Experimental Base and then subjected to salt stress treatment (the seedling pots were immersed in 200 mmol / L NaCl solution for 30 min). After culturing the alfalfa seedlings for 4 weeks, samples were collected for subsequent analysis.

[0024] Preparation of plant root samples: Using sterile forceps, remove alfalfa roots and place them in a test tube containing 25 ml of phosphate buffer. Vortex for 15 seconds. Then, using sterile forceps, adhere the roots to the inner wall of the test tube, slowly pour out the phosphate buffer, and add 25 ml of fresh phosphate buffer. Repeat this step until there is no rhizosphere soil in the phosphate buffer. Place the alfalfa roots in a 15 ml test tube and add 12 ml of phosphate buffer. Place the test tube in an ultrasonic sample homogenizer and sonicate for 5 minutes, followed by vortexing for 5 seconds. Rinse the roots with phosphate buffer. Dry the roots on filter paper, flash-freeze in liquid nitrogen, and store at -80°C.

[0025] Plant root samples were taken and cultured in TSA solid medium at 25°C. Colonies were picked based on their color (yellow) and morphology (irregular edges) and cloned. The colonies produced from the plant root samples were streaked onto fresh TSA solid medium for purification. The purified bacteria were then transferred to TSB liquid medium, 40% (v / v) glycerol was added, and the medium was stored at -80°C for later use.

[0026] (II) Classification and Identification 1. Activated strains: The strains preserved using the above method were taken out of the -80℃ ultra-low temperature freezer, thawed at room temperature, activated and cultured on TSA solid medium, and the colony morphology was observed.

[0027] 2. Morphological characteristics of the strain: The cytological characteristics of the strain are: colonies are pale yellow to translucent, moist, and have smooth edges (see...). Figure 1 ).

[0028] 2.16S rRNA identification: The bacterial strain was taken out from the -80℃ ultra-low temperature freezer and inoculated into TSB liquid medium. It was cultured in a shaker at 25℃ and 220r / min for 3 days to obtain bacterial suspension. Then, 1mL of bacterial suspension was taken, centrifuged to collect bacterial cells, and genomic DNA was extracted from the sample using the MoBio Power Soil DNA Isolation Kit (MoBio Corporation, USA) according to the standard procedure in the kit instructions. The sample was stored at -20℃ for later use.

[0029] The 16S rRNA gene in the extracted genomic DNA was amplified by PCR using the following specific primer pairs: Forward primer 16S-63F: AGTTTGATCMTGGCTCAG (SEQ ID NO:2); Reverse primer 16S-1389R: GGTTACCTTGTTACGACTT (SEQ ID NO:3).

[0030] Amplification system (total 50 μL): 10×Buffer 2.5 μL, 2 mmol / L dNTP 2.5 μL, forward primer 16S-63F 1 μL, reverse primer 16S-1389R 1 μL, dNTP (2 mmol / L) 2.5 μL, DNA 2 μL, Taq polymerase 0.1 μL and ddH2O 15.9 μL.

[0031] The PCR reaction procedure was as follows: denaturation at 94℃ for 5 min; 30 cycles of 94℃ for 1 min, 55℃ for 1 min, and 72℃ for 1 min; extension at 72℃ for 10 min. PCR products were detected by 1.5% agarose gel electrophoresis. After the extracted DNA passed purity and integrity tests, it was sent to a sequencing company (Beijing Novogene Technology Co., Ltd.) for sequencing.

[0032] After comparing the bacterial morphology, 16S gene sequence, and the NCBI (National Center for Biotechnology Information) database, the strain was identified as *Agrobacterium*. Agrobacterium sp. The strain was named EC17. EC17 was deposited on December 25, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNO.33194.

[0033] Example 2: Biological Function Test of the Strains 1. Strain pretreatment: The test strain was inoculated into 20 ml of TSB liquid medium and cultured in a shaker at 25°C and 220 r / min for 3 days. Afterwards, the culture was centrifuged at 4°C and 8000 r / min for 10 min, and the bacterial pellet was collected. The bacterial concentration was then adjusted to OD using a spectrophotometer. 600 =1, used for subsequent experiments.

[0034] 2. Phosphorus solubility test: Take 2 μL of bacterial culture and inoculate it onto the surface of solid culture medium containing organic phosphorus and solid culture medium containing inorganic phosphorus, respectively. Incubate at 25℃, with 3 biological replicates for each treatment. After 3-5 days, observe whether a phosphate-solubilizing zone appears in the culture medium. The presence or absence of a phosphate-solubilizing zone indicates whether the treatment has the function of dissolving insoluble organic and inorganic phosphorus.

[0035] The results are as follows Figure 2 A, Figure 2 As shown in Figure B, the produced phosphorus-solubilizing zone is relatively small, indicating that strain EC17 has the ability to dissolve both inorganic and organic phosphorus, but it is weak.

[0036] 3. Determination of potassium solubilization capacity: Take 2 μL of bacterial suspension and inoculate it onto the surface of a potassium-containing solid culture medium. Incubate at 25°C with three biological replicates. After 3-5 days, observe the growth of colonies on the surface of the culture medium and record whether transparent oil droplet-like colonies appear. The appearance of transparent oil droplet-like colonies indicates potassium-solubilizing ability.

[0037] The results are as follows Figure 3 As shown, oil droplet-like colonies appeared on the surface of the culture medium, proving that strain EC17 has a strong potassium-solubilizing ability.

[0038] 4. Determination of biofilm production capacity: Take 20 μL of bacterial culture and add it to a 96-well plate pre-filled with 180 μL of TSB liquid medium. Incubate at 25℃ and 220 r / min for 2-3 days. After standing, add 0.1 μL of crystal violet dye (containing 0.1% Crytorl) to each well and incubate for 15 min. Wash three times with sterile water. Add 200 μL of 96% ethanol to dissolve the dye remaining on the tube wall for 2 min. Observe whether there is purple staining in the 96-well plate. Determine whether strain EC17 has the ability to produce biofilm and the strength of the biofilm production ability based on the presence and degree of purple staining.

[0039] The results are as follows Figure 4 As shown, a purple-colored bacterial film appeared at the bottom of the 96-well plate, and the color was relatively dark, proving that strain EC17 has a strong ability to produce bacterial film.

[0040] 5. Determination of iron production capacity: Take 2 μL of bacterial culture and inoculate it onto the surface of siderophore-selective solid medium. Incubate at 25°C with three biological replicates. After 3-5 days, observe whether there is an orange-yellow secretion zone in the siderophore-selective solid medium. The presence of an orange-yellow secretion zone indicates the ability to produce siderophores.

[0041] The results are as follows Figure 5 As shown, the colonies in the culture medium did not have an orange-yellow secretion zone, proving that strain EC17 has no ability to produce siderophores.

[0042] 6. Assay for ACC deaminase production capacity: Take 10 ml of bacterial suspension into a 15 ml centrifuge tube, centrifuge at 4℃ and 8000 r / min for 10 min, discard the supernatant, leaving the bacterial precipitate, add 7.5 ml of nitrogen-free DF medium (Beijing Cooler Technology Co., Ltd., model: MM6150-2) to the centrifuge tube, and add 45 μL of filtered and sterilized 0.5 mol / L ACC (1-aminocyclopropane-1-carboxylic acid) solution to make the ACC solution concentration 3 mmol / L. Then, culture at 25℃ and 220 r / min with shaking for 24 h (to induce the ACC deaminase activity of bacteria) to obtain the bacterial suspension. Centrifuge the above bacterial suspension at 4°C and 8000 rpm for 10 min, remove the supernatant, collect the bacterial pellet, add 5 ml of Tris-HCl buffer (0.1 mol / L, pH 7.6) to the pellet to resuspend the bacteria, centrifuge at 4°C and 8000 rpm for 10 min, and collect the pellet. Repeat this step 3 times to completely remove the nitrogen-free DF medium. Add 1 ml of Tris-HCl buffer (0.1 mol / L, pH 7.6) to the pellet to resuspend the bacteria, then transfer to a 1.5 ml centrifuge tube, centrifuge at 15000 rpm for 5 min, remove the supernatant, and collect the bacterial pellet. Then add 600 μL of Tris-HCl buffer (0.1 mol / L, pH 8.5) to the pellet again, add 30 μL of toluene, and vortex for 30 s to disrupt the bacteria. Take 200 μL of the lysed bacterial suspension, add 20 μL of ACC solution (0.5 mol / L), mix well, and incubate at 30°C for 15 min. Then add 1 mL of HCl solution (0.56 mol / L), mix well, and centrifuge at 15000 rpm for 5 min at room temperature to obtain the supernatant. Take 1 mL of the supernatant, add 800 μL of HCl solution (0.56 mol / L), mix well, and then add 300 μL of 2,4-dinitrophenylhydrazine (2 g / L, dissolved in 2 mol / L HCl), and incubate at 30°C for 30 min. Subsequently, measure the absorbance at 540 nm using a UV-Vis spectrophotometer, with distilled water as a control. Determine whether strain EC17 can produce ACC deaminase based on the OD value.

[0043] The results are shown in Table 1. The OD value of ACC deaminase of strain EC17 was 0.027, which proves that strain EC17 has a very strong ability to produce ACC deaminase.

[0044] Table 1. OD values ​​of ACC deaminase and IAA in strain EC17 deal with <![CDATA[ACC deaminase (OD 450 )]]> <![CDATA[IAA(OD 600 )]]> CK 0 0 EC17 0.027 0.262 7. IAA production capacity determination: In a 96-well plate, pre-add 180 μL of TSB liquid medium and 1.5 μL of tryptophan solution (0.11 g / L, sterilized by filtration). Add 20 μL of bacterial culture to the 96-well plate and incubate at 25℃ and 220 r / min for 2-3 days. Then add Sachs' reagent (3.51 mL of 35% HClO4 + 2 μL of 0.5 mol / L FeCl3) and incubate in the dark for 30 min. Observe whether the 96-well plate becomes turbid. [The last sentence appears to be incomplete and possibly refers to a measurement based on OD...] 600 The absorbance of strain EC17 was measured under certain conditions, with distilled water as a control, to determine whether strain EC17 has the function of producing IAA.

[0045] The results are as follows Figure 6 As shown, the 96-well plate of strain EC17 was more turbid than that of CK, and the OD value of IAA of strain EC17 was 0.262 (see Table 1), which proves that strain EC17 has the ability to produce IAA, but it is weak.

[0046] Example 3: Germination of alfalfa seeds after soaking in 200 mmol / L NaCl stress Remove the bacterial strain from the refrigerator, streak it onto fresh TSA solid medium, and incubate statically at 25°C for 3-5 days. After colonies have grown, elute them using sterile 0.9% NaCl solution in a laminar flow hood, and then adjust the OD of the bacterial culture using a spectrophotometer. 600 =0.1, prepare EC17 bacterial solution for later use.

[0047] Plump and intact alfalfa seeds (“Prairie No. 3” hybrid alfalfa) were selected and soaked in EC17 bacterial solution for 8 hours. Sterile water was used as a control. Germination tests were conducted on a paper bed containing 200 mmol / L NaCl solution. Fifty seeds were placed in each petri dish, with three replicates. The petri dishes were pre-sealed with two layers of sealing film to prevent moisture evaporation, and water was replenished daily to maintain a stable salt concentration. The number of germinated seeds in each dish was recorded daily until day 7. Germination potential, germination rate, and germination index were calculated. At the end of the experiment, 10 seedlings were randomly selected from each petri dish to measure the radicle length and plumule length, and the fresh weight of all seedlings in the dish was determined.

[0048] The results are as follows Figure 7 As shown, after soaking in EC17 bacterial solution, all germination indicators of alfalfa seeds were higher than those of the CK control. Germination rate, germination index, plumule length, and seedling fresh weight were all significantly higher than those of the CK control (see...). Figure 7 B, 7C, 7E, and 7F) showed significantly longer radicles than the control (CK) (see [reference needed]). Figure 7 (D) Germination rate, germination index, plumule length, and seedling fresh weight were 52.17%, 42.67%, 11.02%, and 63.19% higher than the control, respectively. P< 0.05), the radicle length was 41.67 mm longer than the control.P< 0.01).

[0049] Example 4 250 mmol / L Alfalfa seeds germinated after soaking under NaCl stress The experimental steps in this embodiment are the same as in Example 3, except that the 200 mmol / L NaCl solution is replaced with a 250 mmol / L NaCl solution.

[0050] The results are as follows Figure 8 As shown, after soaking in EC17 bacterial solution, all germination indicators of alfalfa seeds were higher than those of the CK control. Germination potential, germination rate, germination index, radicle length, and fresh weight of seedlings were all significantly higher than those of the CK control (see...). Figure 8 A, 8B, 8C, 8D, and 8F were 23.33%, 116.22%, 41.83%, 33.09%, and 184.20% higher than the control, respectively. P< 0.05), and the plumule length was significantly higher than that of the CK control (see 0.05). Figure 8 E), which was 14.33% higher than the control (E). P< 0.01).

[0051] Example 5 200 mmol / L Alfalfa seedling growth under NaCl stress Select plump and intact alfalfa seeds (“Prairie No. 3” mixed alfalfa) and germinate them in sterile petri dishes. After 6-7 days, select seedlings with uniform growth and transplant them into pots (pot dimensions: 0cm×10cm×8cm; the substrate in the pot consists of sterilized vermiculite and volcanic rock, with the weight of the volcanic rock being twice the weight of the vermiculite). Three days after transplanting, inoculate with EC17 strain (first prepare the bacterial solution using the same method as in Example 3, the difference being that OD is prepared). 600 =1 EC17 bacterial solution; after preparing the EC17 bacterial solution, use a pipette to inject 1 ml of EC17 bacterial solution into the soil around the roots of the alfalfa to complete the inoculation (with no EC17 bacterial solution as the control). After 2 weeks, salt stress treatment is carried out (the potted plants are soaked in 200 mM / L NaCl solution for 30 min). After 4 weeks, the seedlings are harvested and the plant height, stem diameter, and fresh and dry weight of the above-ground parts are measured.

[0052] Depend on Figure 9 As shown in A-9E, after salt stress, the seedlings inoculated with EC17 strain had significantly higher plant height, stem diameter, leaf area, and fresh and dry weight of the above-ground parts than the CK control, indicating that salt stress can significantly improve the growth indicators of alfalfa seedlings.

[0053] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A soil bacterium (Agrobacterium) strain EC17, characterized by: Agrobacterium sp. The Agrobacterium strain EC17 is preserved in the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Yard 3, Chaoyang District, Beijing, on December 25, 2024, and the preservation number is CGMCC NO. 33194. ​ 2. The application of the Agrobacterium strain EC17 for improving the salt tolerance of alfalfa to improve the seed germination index of alfalfa under salt stress according to claim 1.

3. Use according to claim 2, characterized in that: The germination index includes germination energy, germination rate, germination index, radicle length, plumule length and fresh weight of group seedlings.

4. Use according to claim 2, characterized in that: The salt stress is 200 mmol / L NaCl stress.

5. Use according to claim 2, characterized in that: The salt stress is 250 mmol / L NaCl stress.

6. The application of the Agrobacterium strain EC17 for improving the salt tolerance of alfalfa to improve the seed germination index of alfalfa under salt stress according to claim 1.

7. Use according to claim 6, characterized in that: The seed germination index includes germination energy, germination rate, germination index, radicle length, plumule length and fresh weight of group seedlings.

8. Use according to claim 6, characterized in that: The salt stress is 200 mmol / L NaCl stress. The salt stress is 250 mmol / L NaCl stress.

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