Application of filamentous rhizobia in alleviating plant saline-alkali stress and improving saline-alkali land

Abstract

The present invention relates to the technical field of agricultural microorganisms, and in particular to the application of filamentous rhizobia in alleviating plant saline-alkali stress and improving saline-alkali land. The present invention finds that filamentous rhizobia (Rhizobium viscosum) ACCC 15082 has salt tolerance, drought tolerance, alkali tolerance and growth promotion characteristics, can significantly alleviate plant saline-alkali stress, can still produce IAA and EPS under high saline-alkali conditions, can promote plant growth under high saline-alkali conditions, improve soil environment, can be used as a bacterial agent or bacterial fertilizer for planting plants in saline-alkali land, or for improving saline-alkali land, and has good application value.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural microorganisms, and in particular to application of filamentous rhizobia in alleviating plant saline-alkali stress and improving saline-alkali land. Background Art

[0002] According to statistics, the total area of ​​saline soil in the world has reached 1.1 billion hectares. What is more serious is that due to improper irrigation, excessive use of fertilizers, climate warming, sea level rise and other factors, soil salinization has accelerated. Therefore, making full use of salinized arable land resources is of great significance to ensuring food security. At present, the management of salinized land has changed from the past concept of "improving soil to benefit crops" to "adapting crops to the land". It is urgent to improve the potential of crops to adapt to saline soil through biological means. Microorganisms play an important role in helping plants adapt to adverse environments: on the one hand, microorganisms can accelerate the humification and mineralization of soil organic matter, form a good microbial environment on ecologically fragile saline-alkali land, form a granular structure in the cultivated soil, block the capillary water passage, improve the permeability of saline-alkali land, and improve its nutrient status; on the other hand, microorganisms can directly or indirectly promote plant growth and improve plant salt resistance. Therefore, it is an important strategy to achieve green reduction of salinity barriers by assisting plants to adapt to saline soil, alleviate salt stress, and increase production capacity through microbial intervention. It is of great significance to develop microorganisms that can alleviate plant salt-alkali stress. Summary of the invention

[0003] The invention provides application of filamentous rhizobia in alleviating plant saline-alkali stress and improving saline-alkali land.

[0004] Rhizobia are a class of Gram-negative bacteria widely distributed in the soil. They can form nodules or stem nodules with the roots and stems of leguminous plants, fix nitrogen in the air into ammonia that can be absorbed and utilized by plants, thereby promoting crop growth and playing an important role in improving and maintaining soil fertility. With the deepening of high-throughput sequencing research on different plant microorganisms, it has been found that rhizobia are the dominant group of endophytic and rhizospheric microorganisms in many plants. At present, there are few reports on rhizobia that can alleviate salt-alkali stress and promote the growth of non-leguminous plants (such as corn).

[0005] The present invention finds that Rhizobium viscosum ACCC 15082 can not only promote seed germination of legumes such as soybean under saline-alkali stress conditions, but also effectively alleviate the saline-alkali stress of non-legumes such as corn. At the same time, it can efficiently produce IAA and EPS and dissolve inorganic phosphorus, promote the growth of non-legumes such as corn under saline-alkali conditions, improve the soil environment, and can be used as a bacterial agent or bacterial fertilizer for planting plants in saline-alkali land.

[0006] In the present invention, Rhizobium viscosum ACCC 15082 can be purchased from Agricultural Culture Collection of China (ACCC). The query website of Rhizobium viscosum ACCC 15082 is: http: / / www.accc.org.cn / Column.asp? Column_ID=34929&Model=product_detail&P_ID=10207606.

[0007] Specifically, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides use of Rhizobium viscosum ACCC 15082 or a preparation containing Rhizobium viscosum ACCC 15082 in alleviating saline-alkali stress of plants.

[0009] In the above application, alleviating plant salinity-alkali stress includes reducing the damage caused by salinity-alkali stress to the plant.

[0010] In a second aspect, the present invention provides the use of Rhizobium viscosum ACCC 15082 or a preparation containing Rhizobium viscosum ACCC 15082 in improving the salt-alkali tolerance of plants.

[0011] In the above application, improving the salt-alkali tolerance of plants includes improving the tolerance of plants to high salt and high saline-alkali conditions.

[0012] In a third aspect, the present invention provides use of Rhizobium viscosum ACCC 15082 or a preparation containing Rhizobium viscosum ACCC 15082 in promoting plant growth and / or increasing yield under saline-alkali stress conditions.

[0013] In a fourth aspect, the present invention provides the use of Rhizobium viscosum ACCC 15082 or a preparation containing Rhizobium viscosum ACCC 15082 in increasing plant biomass and / or chlorophyll content under saline-alkali stress conditions.

[0014] In a fifth aspect, the present invention provides the use of Rhizobium viscosum ACCC 15082 or a preparation containing Rhizobium viscosum ACCC 15082 in promoting plant seed germination under saline-alkali stress conditions.

[0015] In a sixth aspect, the present invention provides the use of Rhizobium viscosum ACCC 15082 or a preparation containing Rhizobium viscosum ACCC 15082 in improving saline-alkali land.

[0016] In the above application, the saline-alkali land improvement includes lowering soil pH, lowering soil salt concentration and / or increasing soil protease activity, etc.

[0017] In a seventh aspect, the present invention provides the use of Rhizobium viscosum ACCC 15082 or a preparation containing Rhizobium viscosum ACCC 15082 in promoting plant growth, increasing plant yield and / or promoting plant seed germination.

[0018] In an eighth aspect, the present invention provides the use of Rhizobium viscosum ACCC 15082 in the preparation of a preparation having any one or more of the following functions:

[0019] (1) Alleviate plant salt-alkali stress;

[0020] (2) Improve the salt and alkali tolerance of plants;

[0021] (3) Promote plant growth and / or yield under saline-alkali stress conditions;

[0022] (4) increasing plant biomass and / or chlorophyll content under saline-alkali stress conditions;

[0023] (5) Promote plant seed germination under saline-alkali stress conditions;

[0024] (6) Improvement of saline-alkali land;

[0025] (7) promoting plant growth, increasing plant yield and / or promoting plant seed germination;

[0026] (8) Produce IAA, EPS and / or dissolve inorganic phosphorus.

[0027] In the present invention, the preparation is a bacterial agent or a bacterial fertilizer.

[0028] Preferably, the active microorganisms in the preparation may contain only Rhizobium viscosum ACCC 15082, or may further contain other agricultural microorganisms.

[0029] Preferably, the preparation may further contain excipients permitted by microbial preparations.

[0030] In the present invention, the plant is a leguminous plant or a non-leguminous plant, including but not limited to corn, soybean, pea, broad bean, lentil, kidney bean, peanut, alfalfa, Arabidopsis, rice, wheat, barley, rye, oat, millet, sorghum, sesame, rapeseed, linseed, tobacco, cotton, highland barley, etc.

[0031] Preferably, the plant is soybean or corn.

[0032] The beneficial effects of the present invention include at least: the present invention finds that the linear rhizobium (Rhizobium viscosum) ACCC15082 has salt resistance, drought resistance, alkali resistance and growth-promoting characteristics, can significantly alleviate plant saline-alkali stress, can still produce IAA and EPS under high saline-alkali conditions, can effectively promote the growth of plants under high saline-alkali conditions, improve the soil environment, can be used as a bacterial agent or bacterial fertilizer for planting plants in saline-alkali land, or for improving saline-alkali land, and has good application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0034] Figure 1 The colony morphology of strain ACCC 15082 on YMA medium in Example 1 of the present invention is shown.

[0035] Figure 2 It is the glucose fermentation and milk litmus test results of strain ACCC 15082 in Example 1 of the present invention, wherein, three test tubes for closed tube fermentation 2d, 5d and 12d are sealed with paraffin oil, and the left and middle test tubes are puncture inoculation, and the right test tube is a non-inoculation control; in the three test tubes for open tube fermentation 2d, 5d and 12d, the left and middle test tubes are puncture inoculation, and the right test tube is a non-inoculation control; in the three test tubes for milk litmus test 2d, 5d and 12d, the left and middle test tubes are inoculated with bacterial liquid, and the right test tube is a non-inoculation control.

[0036] Figure 3The bromothymol blue colorimetric test results of the strain ACCC 15082 in Example 1 of the present invention, wherein (a) blank control, (b) ACCC 15082.

[0037] Figure 4 This is the phylogenetic tree of strain ACCC 15082 in Example 1 of the present invention.

[0038] Figure 5 This is the salt tolerance growth curve of strain ACCC 15082 in Example 2 of the present invention.

[0039] Figure 6 The data are shown in Table 2. The data are shown in Table 2. The data are shown in Table 2. The data are shown in Table 2. The data are shown in Table 2. The data are shown in Table 2. The data are shown in Table 2. The data are shown in Table 2. The data are shown in Table 2. The data are shown in Table 2.

[0040] Figure 7 The growth conditions of strain ACCC 15082 under different pH conditions in Example 2 of the present invention are shown.

[0041] Figure 8 These are the results of the growth-promoting ability test of strain ACCC 15082 in Example 3 of the present invention, wherein the left figure shows the production of longin (the left centrifuge tube is the positive control, the middle centrifuge tube is the negative control, and the right centrifuge tube is strain ACCC 15082), and the right figure shows the dissolution of inorganic phosphorus.

[0042] Fig. 9 This is the effect of NaCl on the EPS production of strain ACCC 15082 in Example 4 of the present invention.

[0043] Fig.10 This is the effect of NaCl on IAA production by strain ACCC 15082 in Example 4 of the present invention (P<0.05).

[0044] Fig.11 The effect of strain ACCC 15082 on soybean seed germination under salt stress in Example 5 of the present invention, and the negative control CK on the left - , the right side is the treatment with strain ACCC 15082.

[0045] Fig.12 The figure shows the growth of corn in the experimental group and the control group under salt stress conditions in Example 6 of the present invention, wherein the left side is the experimental group inoculated with ACCC15082, and the right side is the control group not inoculated with ACCC15082.

[0046] Fig.13The figures are a comparison of maize physiological and biochemical indices between the experimental group (15082) and the control group (CK) in Example 6 of the present invention, where * indicates significant difference (P < 0.05) and ** indicates extremely significant difference (P < 0.01). DETAILED DESCRIPTION

[0047] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] The culture medium formula used in the following examples is as follows:

[0049] YMA medium: mannitol / sucrose 10.0g, yeast powder 1.0g, MgSO4·7H2O0.2g, NaCl 0.1g, K2HPO40.5g, CaCO3 3.0g, 1% H3BO3 2.0mL, 1% Na2MoO4 2.0mL, agar 20.0g. Natural pH, sterilized at 115℃ for 20min. Without adding agar, it is mannitol yeast juice broth (YMB).

[0050] In the following examples, the data processing method is as follows: the original data is pre-processed by Excel, and then significance analysis and graphing are performed using R and GraphPad, and the significance level is set at α=0.05.

[0051] Example 1 Identification of Thread-like Rhizobium ACCC 15082

[0052] 1. Activation of lyophilized powder of strain ACCC 15082

[0053] In the clean bench, wipe the surface of the dry ampoule tube with a cotton ball soaked in 75% alcohol, heat the top of the ampoule tube with the outer flame of an alcohol lamp, drop cold sterile water on the top of the hot ampoule tube to break the glass, and knock the top of the ampoule tube with sterile tweezers. Use a sterile dropper to draw an appropriate amount of sterile water into the ampoule tube, and slowly blow to suspend the freeze-dried bacteria. Take another sterile dropper to draw an appropriate amount of bacterial suspension and inoculate it in mannitol yeast juice agar medium (YMA), spread it evenly, place it in a 30℃ constant temperature incubator away from light and culture for 3 to 5 days until bacterial moss appears, pick an appropriate amount of bacterial moss and purify it by streaking until a single colony appears, inoculate the pure cultured strain into the YMA slant, and store it in a 4℃ refrigerator for later use.

[0054] 2. Determination of the taxonomic status of strain ACCC 15082

[0055] (1) Morphological characteristics

[0056] The colony morphology of strain ACCC 15082 after culturing on YMA medium at 30°C for 4 days is as follows: Figure 1 The colonies were smooth and moist, with neat edges, round protrusions, mucus, and easy to pick up. The colonies on the YMA plate were milky white.

[0057] (2) Physiological and biochemical characteristics

[0058] The physiological and biochemical characteristics of strain ACCC 15082 were analyzed using traditional physiological and biochemical reactions, API 20NE and API ZYM, and Biolog identification system.

[0059] Among them, the test method for the determination of glucose oxidation fermentation is as follows: take 6 Board & Holding two-column semi-solid culture medium tubes and divide them into three groups evenly, mark the group name and date. The first two groups are inoculated with young ACCC 15082 strains by puncture method, the second group is sealed with sterile liquid paraffin (0.5-1.0cm is appropriate), and the third group is not inoculated with bacteria, one of which is sealed with liquid, and cultured at a constant temperature of 30℃, and the results are observed on the 2nd, 5th and 15th days respectively.

[0060] The litmus milk test method is as follows: take 3 tubes of litmus milk culture medium, one of which is used as a blank control, and the other two are inoculated with fresh ACCC 15082 bacterial suspension, cultured at a constant temperature of 30℃, and the results are observed on the 2nd, 5th, and 15th days respectively.

[0061] The methods for Biolog and API 20NE system identification and API ZYM enzyme activity determination are as follows: Using Biolog GENⅢ microplates, the phenotype of Gram-negative bacteria was identified by 94 biochemical tests fixed on the microplates. API 20NE system identification and API ZYM enzyme activity determination were performed according to the instructions of the reagent strips.

[0062] After analysis, ACCC 15082 in Biolog can utilize 45 carbon sources. The carbon spectrum is shown in Table 1, and the API system identification results are shown in Table 2.

[0063] Table 1 Carbon source spectrum of strain ACCC 15082

[0064]

[0065]

[0066] Note: + indicates positive, - indicates negative, and w indicates weakly positive.

[0067] Table 2 API system identification results of strain ACCC 15082

[0068]

[0069] Note: + indicates positive, - indicates negative, and w indicates weakly positive.

[0070] The results of glucose fermentation and litmus milk test of strain ACCC 15082 are as follows Figure 2 shown.

[0071] Regardless of closed-tube or open-tube fermentation, the strain can use glucose to ferment and produce acid without producing gas. Open-tube fermentation produces more acid. In the litmus milk test, as time goes by, the milk gradually coagulates and becomes clear and transparent, indicating that the strain can produce protease to decompose casein and produce acid.

[0072] The results of the colorimetric test of strain ACCC 15082 for bromothymol blue (BTB) are as follows Figure 3 As shown. With BTB as the acid-base indicator, pH 1.2-2.8 is yellow, pH 8.0-9.6 is blue, and neutral is green. According to the color reaction, ACCC15082 produces acid and is yellow.

[0073] (3) Molecular characteristics

[0074] The 16S rRNA gene sequence (SEQ ID NO.1) of strain ACCC 15082 was determined using universal primers 27F and 1492R of bacterial 16S rRNA gene (27f: 5'-GTTTGATCMTGGCTCAG-3'; 1492r: 5'-TACGGYTACCTTGTTACGACTT-3'), and compared with known model bacteria through the website (http: / / www.ezbiocloud.net / eztaxon / ), and a phylogenetic tree was constructed. The results showed ( Figure 4 ), strain ACCC 15082 and Rhizobiumviscosum were clustered into the same branch.

[0075] Example 2 Growth characteristics of strain ACCC 15082

[0076] 1. Detection of the effect of NaCl on the growth characteristics of strains

[0077] According to the concentration of salt ions in saline-alkali farmland, 1-3‰ is mild, 4-6‰ is moderate, and 6‰ or more is severe saline-alkali land. The NaCl concentration gradient was set to 0, 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 1.2%, 1.4%, and 1.6% to detect the effect of NaCl on the growth of ACCC 15082. The experiment was carried out in a 48-well plate, the culture medium was YMB liquid culture medium, the liquid volume per well was 800μL, the bacterial suspension inoculation volume was 1%, and each treatment was repeated three times. After the micropores of the non-salt treatment group became turbid, the OD was measured. 600 .

[0078] The results are as follows Figure 5 As shown, the effect of NaCl on the growth of the strain showed a trend of promotion at low concentrations and inhibition at high concentrations, with 1% concentration as the cutoff point (P=0.369). When the NaCl concentration was less than 1.0%, the growth of the strain was promoted (P<0.01), especially in the concentration range of 0.2-0.8%, the growth was very good (P<0.001), which showed that the strain was suitable for application in moderately light saline-alkali land and severely saline-alkali land below 0.8%; when the NaCl concentration was greater than 1.0%, the growth of the strain was gradually inhibited (P<0.001), and when the NaCl concentration was greater than 1.2%, there was no difference in the growth of the strain (P>0.05), and the inhibitory effect was the strongest.

[0079] 2. Drought resistance test

[0080] Use PEG-6000 to simulate different osmotic potential environments, and use the ability of the strain to grow under different osmotic potential environments as the basis for its drought resistance. Prepare 100 mL of YMB liquid culture medium with PEG-6000 content of 0 (blank control), 5% (-0.2mPa), 10% (-0.2mPa), 15% (-0.4mPa), 20% (-0.6mPa), and 25% (-0.8mPa), respectively, and place them in 250 mL conical flasks, sterilize and set aside. Inoculate ACCC 15082 fresh bacterial suspension with 1% inoculation amount, and culture at 30°C, 200r / min for 24 hours, then measure OD 600 , zeroed with uninoculated medium, and set three replicates for each concentration.

[0081] Effects of osmotic pressure on bacterial growth Figure 6 As shown in the data, when the osmotic pressure was less than -0.32 mPa (15% PEG 6000), the growth of the strain showed a downward trend. When the osmotic pressure was between -0.32 and -0.79 mPa (15% to 25% PEG 6000), the growth of the strain entered a plateau phase (P>0.1). When the osmotic pressure reached -1.10 mPa (30% PEG 6000), the growth of the strain was significantly inhibited (P<0.001), which was 65.60% lower than the blank control.

[0082] 3. Detection of the effect of pH on the growth of strains

[0083] The pH of the YMB liquid medium was set to 4.5, 5.5, 6.5, 7.5, 8.5, 9.5 and 10.5 respectively, and the effect of the initial pH of the fermentation on the growth of the strain was detected. Figure 7 As shown in the figure, with the increase of pH, the growth of the strain showed a trend of first increasing and then decreasing: when the pH was between 4.5 and 8.5, the growth of the strain gradually increased and reached the highest point at pH 8.5, which was significantly different from other treatments (P<0.001).

[0084] Example 3 Detection of the growth-promoting ability of strain ACCC 15082

[0085] The ability of strain ACCC 15082 to produce IAA, EPS and dissolve inorganic phosphorus was tested as follows:

[0086] 1. Determination of the ability to produce IAA

[0087] The fresh bacterial suspension of strain ACCC 15082 was inoculated with 1% inoculum into a 150mL conical flask containing 50mL fresh YMB liquid medium (tryptophan was added to the medium after filtration sterilization, with a final concentration of 0.2g / L), and cultured at 30°C, 200r / min for 96h. 100μL of bacterial suspension was mixed with equal volumes of Salkowski colorimetric solution, and an equal amount of uninoculated medium was used as a negative control, and an equal amount of 100mg / L IAA solution was used as a positive control. After 30min of color development in the dark, the color change was observed. If the color turned red, it indicated that the strain could produce IAA. The darker the color, the higher the yield.

[0088] 2. Determination of the ability to produce extracellular polysaccharides (EPS)

[0089] The anthrone-sulfuric acid method was used to determine the EPS yield, as follows:

[0090] (1) Glucose standard curve: Take 6 stoppered test tubes and add the corresponding reagents according to Table 3. Place the test tubes in a 100°C water bath for 10 min. After cooling to room temperature, measure the absorbance at 620 nm. Calculate the regression equation of the standard curve based on the sugar solution concentration and the corresponding absorbance value in each test tube.

[0091] Table 3 Glucose standard curve reagent content table

[0092]

[0093] (2) EPS crude extraction: Inoculate 1% of the fresh bacterial suspension of strain ACCC 15082 into a 150 mL conical flask containing 50 mL of fresh YMB liquid medium, and culture at 30°C, 200 r / min for 48 h. Take an appropriate amount of fresh bacterial suspension and centrifuge at 4°C, 12000 r / min for 10 min. The supernatant is the EPS crude extract.

[0094] (3) EPS content determination: Take 0.1 mL of EPS crude extract in a clean and dry test tube, add 0.9 mL of distilled water to obtain a ten-fold dilution of EPS, add 5.0 mL of anthrone sulfuric acid reagent, mix quickly, boil in a boiling water bath for 10 min, let stand and cool at room temperature, measure the absorbance at 620 nm, and calculate the total sugar content according to the Glu standard curve.

[0095] 3. Phosphorus dissolution test

[0096] The fresh bacterial suspension of strain ACCC 15082 was spotted onto Montgena inorganic phosphate medium and Montgena lecithin medium, 5 μL per spot, and repeated 3 times for each strain. Cultured at 30°C for 5 to 7 days, and observed whether there was a transparent circle. If so, it means that the bacteria has the ability to solubilize phosphate, otherwise it does not have the ability to solubilize phosphate.

[0097] Some of the growth-promoting ability test results of strain ACCC 15082 are as follows Figure 8 The results showed that the strain could secrete auxin (42.01±0.13 mg / L) and EPS, with a yield of up to 162.12±10.81 mg / L, and could dissolve inorganic phosphorus (phosphate dissolving circle d=1.31±0.04 cm).

[0098] Example 4 Detection of the growth-promoting ability of strain ACCC 15082 under different salt concentration conditions

[0099] Combined with the tolerance of strain ACCC 15082 to NaCl, NaCl concentration gradients of 0, 0.4%, 0.8%, and 1.2% were selected to determine the effects of different NaCl concentrations on the growth-promoting ability (production of IAA and EPS) of strain ACCC 15082. The determination method was similar to Example 3, and different concentrations of NaCl were added to the YMB liquid culture medium.

[0100] IAA and EPS production of strains under different NaCl concentrations Fig. 9 and Fig.10shown. With the increase of salt concentration (0, 0.4%, 0.8%, 1.2%), the IAA production was 42.01±0.13mg / L, 32.37±0.92mg / L, 19.99±0.28mg / L, 11.9±0.27mg / L, respectively. The IAA production was extremely significantly affected by salt (P<0.001), and showed an overall downward trend. The EPS production was 162.12±10.81mg / L, 168.23±0.99mg / L, 108.67±0.7mg / L, 129.63±0.67mg / L, respectively. NaCl had a significant effect on EPS (P<0.05), showing a bipolar trend: when the NaCl concentration was low (0, 0.4% NaCl), there was no significant difference in EPS production (P=0.634), and when the NaCl concentration was high (0.8%, 1.2% NaCl), EPS production was inhibited.

[0101] In summary, strain ACCC 15082 can tolerate 1.2% NaCl and -0.79mPa drought, produce acid in bromothymol blue reaction, produce acid in closed tube fermentation and open tube fermentation, decompose casein to make milk coagulate, ACCC 15082 can dissolve inorganic phosphorus (d = 1.31 ± 0.04cm), produce IAA (42.01 ± 0.13mg / L), secrete EPS (162.12 ± 10.81mg / L), and the IAA and EPS production in 0.4% NaCl environment are 32.37 ± 0.92mg / L and 168.23 ± 0.99mg / L, respectively. The ability to produce IAA is extremely sensitive to salt concentration. This strain is best used in the restoration of moderate and mild saline-alkali land. At the same time, it can be seen that the strain can still produce higher concentrations of EPS and IAA under high concentration NaCl conditions, indicating that even in severe saline-alkali land, the strain will play a good role in promoting growth.

[0102] Example 5 Strain ACCC 15082 alleviates the inhibition of salt on soybean seed germination

[0103] 1. Seed preparation: Soybean seeds were purchased from Harbin Tuanmao Food Co., Ltd. The sterilized seeds were soaked in sterile water for 6-8 hours, and the volume of sterile water was about 250%-300% of the total volume of the seeds. The swollen seeds were sown in an aluminum box covered with 4 layers of sterile moist gauze, and placed in a 25℃ constant temperature incubator to germinate for 24-48 hours away from light. The seed radicle elongated about 0.5-1.0 cm, and the germination bed was kept moist during this period.

[0104] 2. Preparation of bacterial suspension: After a large area of ​​bacterial moss grows on the slant of the test strain, add 5 mL of sterile water, scrape the bacterial moss with a sterile inoculating loop, and vortex for 20 seconds to make a bacterial suspension.

[0105] 3. Soybean germination test: 0.4% NaCl was selected as the salt stress concentration. After the seeds absorbed water and swelled, they were soaked in bacterial solution for 30 minutes. The amount of bacterial solution should be enough to cover the seeds. After that, they were placed on a clean bench to air dry the surface bacterial solution and placed on a saline agar germination bed containing 0.4% NaCl for germination. The negative control (CK) was used as the non-inoculated control. - ), and the positive control (CK) was not treated with salt and was not inoculated with bacteria. + ). Each group was replicated three times, with 50 seeds in each replicate, and the fresh weight and radicle length of the germinated seeds in each group were counted.

[0106] Effects of strain ACCC 15082 on soybean seed germination under salt stress Fig.11 Compared with CK - (84.46±6.32mm), ACCC 15082 (158.00±5.79mm) could significantly (P<0.001) promote the elongation of seed radicle.

[0107] Example 6 Strain ACCC 15082 alleviates the effect of salt on corn growth

[0108] The corn pot experiment was used to analyze the ability of strain ACCC 15082 in alleviating the effects of salt on corn growth, as follows:

[0109] 1. Preparation of bacterial solution

[0110] Use YMA solid medium to activate strain ACCC 15082: Use a sterile pipette to transfer 100-200 μL from the glycerol tube to YMA solid medium and spread it using the continuous streak method. Incubate at 30°C for 1-2 days. Then use YMB liquid medium to expand: Pick a single colony from the plate and transfer it to YMB liquid medium. Place the medium in a constant temperature shaker and incubate overnight at 30°C and 180 rpm until the bacterial solution becomes turbid and the OD 600 ≈1.0.

[0111] 2. Experimental process

[0112] The experiment set up two treatments: a control group (no ACCC 15082 inoculation) and an experimental group (ACCC 15082 inoculation). Vermiculite was selected as the potting medium, with a flower pot diameter of 7.5 cm and a height of 8 cm. Each pot of medium was about 70 g, and each treatment was 9 pots. Each treatment was placed in the same tray, and 9 seeds were sown in each pot. They were cultured in a 16h / 8h light incubator, and water was added regularly. When the corn grew to two leaves and one heart, the seedlings were thinned to 3-4 plants, and 15 mL of ACCC 15082 bacterial solution prepared in step 1 was inoculated in each pot. During the period, tap water containing 0.2%, 0.4%, 0.8%, and 1.0% NaCl was used for irrigation, with 500 mL of salt solution per tray each time, and the final salt concentration in the matrix was about 74.16 mS / cm. The growth of corn was observed and recorded, and the plant traits were investigated after the experimental group and the control group showed obvious differences.

[0113] 3. Experimental results

[0114] After salt stress treatment in potted culture, it was found that there were some differences between the treatment with strain ACCC 15082 and the control group, and the growth of the experimental group was better than that of the control group ( Fig.12 The plant height, fresh weight, dry weight, chlorophyll and salt stress-related indexes (reduced glutathione content (GSH)) were measured. The results are as follows: Fig.13 As shown. The results showed that the fresh weight of the aboveground and underground parts of the treatment group with ACCC 15082 solution was 2.22±1.06g and 2.15±0.89g, respectively, while the fresh weight of the aboveground and underground parts of the control group was 1.41±0.55g and 1.38±0.75g, respectively. The fresh weight of the experimental group with ACCC15082 solution was significantly higher than that of the control group (P<0.05); the dry weight of the aboveground and underground parts of the experimental group with ACCC 15082 solution was 0.27±0.12g and 0.19±0.07g, respectively, while the dry weight of the control group was 0.18±0.08g and 0.16±0.06g, respectively. The dry weight of the aboveground part of the experimental group was significantly higher than that of the control group (P<0.05), but the difference in the underground part was not significant (P>0.05); The plant height of the 15082 bacterial solution experimental group was 41.88±5.70cm, which was significantly different from the plant height of 33.31±6.20cm in the control group (P<0.01); at the same time, the chlorophyll content of the ACCC 15082 bacterial solution experimental group was 1.37±0.25SPAD, significantly higher than that of the control group (P<0.05), and the GSH content was 606.15±51.04μg / g, lower than that of the control group (782.65±35.14μg / g) and the difference was significant (P<0.05).

[0115] The results of the test on the physical and chemical properties of rhizosphere soil (Table 4) showed that the pH value of rhizosphere soil (8.19) of the experimental group treated with ACCC 15082 solution was significantly lower than that of the control group (8.75) (reduced by 0.56). The electrical conductivity of rhizosphere soil was also significantly reduced, indicating that a microenvironment with reduced salt and alkali concentration was formed in the rhizosphere of plants, which was more suitable for crop growth. The activity of soil protease was also increased, indicating that microorganisms were more active in the rhizosphere environment.

[0116] Table 4 Effects of ACCC 15082 inoculation on the physical and chemical parameters of maize rhizosphere soil (P<0.05)

[0117]

[0118] The above results show that the application of ACCC 15082 bacterial solution increases the fresh weight, plant height and chlorophyll content of the aboveground and underground parts of corn seedlings under NaCl stress, and reduces the content of GSH. Since a large amount of reactive oxygen accumulates when plants are subjected to adverse stress, GSH plays an important role in removing reactive oxygen damage. The treatment of ACCC 15082 bacterial solution in the present invention makes the GSH content of corn significantly lower than that of the control group, and the rhizosphere pH and salt concentration are significantly reduced, indicating that the strain ACCC 15082 alleviates the stress state of corn under NaCl treatment to a certain extent. Combining other physiological and biochemical indicators, the results show that the application of strain ACCC 15082 has a positive effect on alleviating NaCl stress in corn seedlings and promoting the growth of corn, and the effect is significant.

[0119] 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 aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of Rhizobium viscosum ACCC 15082 or a preparation containing Rhizobium viscosum ACCC 15082 in alleviating saline-alkali stress in plants.

2. Use of Rhizobium viscosum ACCC 15082 or a preparation containing Rhizobium viscosum ACCC 15082 in improving the salt-alkali tolerance of plants.

3. Use of Rhizobium viscosum ACCC 15082 or a preparation containing Rhizobium viscosum ACCC 15082 in promoting plant growth and / or increasing yield under saline-alkali stress conditions.

4. Use of Rhizobium viscosum ACCC 15082 or a preparation containing Rhizobium viscosum ACCC 15082 in increasing plant biomass and / or chlorophyll content under saline-alkali stress conditions.

5. Use of Rhizobium viscosum ACCC 15082 or a preparation containing Rhizobium viscosum ACCC 15082 in promoting plant seed germination under saline-alkali stress conditions.

6. Use of Rhizobium viscosum ACCC 15082 or a preparation containing Rhizobium viscosum ACCC 15082 in improving saline-alkali land.

7. Use of Rhizobium viscosum ACCC 15082 or a preparation containing Rhizobium viscosum ACCC 15082 in promoting plant seed germination.

8. Use of Rhizobium viscosum ACCC 15082 in the preparation of a preparation having any one or more of the following functions: (1) Alleviate plant salt-alkali stress; (2) Improve the salt and alkali tolerance of plants; (3) Promote plant growth and / or yield under saline-alkali stress conditions; (4) increasing plant biomass and / or chlorophyll content under saline-alkali stress conditions; (5) Promote plant seed germination under saline-alkali stress conditions; (6) Improvement of saline-alkali land; (7) Promote plant seed germination; (8) Produce IAA, EPS and / or dissolve inorganic phosphorus.

9. The use according to any one of claims 1 to 8, characterized in that: The preparation is a bacterial agent or a bacterial fertilizer.

10. The use according to any one of claims 1 to 8, characterized in that: The plant is soybean or corn.

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