CN115838655A - A Strain of Bradyrhizobium Ercani and Its Application - Google Patents

Abstract

The invention discloses a strain of Bradyrhizobium ercanii and its application. The RY8 strain of Bradyrhizobium elkanii was deposited in the China Center for Type Culture Collection on April 28, 2021, with the preservation number CCTCC NO: M 2021480. The research of the present invention shows that rhizobium RY8 can significantly increase the biomass and yield of plants after being reattached to soybeans; at the same time, it also has the ability to secrete auxin, and the amount of IAA secreted is significantly better than that of other growth-promoting rhizobia. The effect is remarkable. The RY8 strain has a strong ability to infiltrate root nodules, and soybeans can significantly promote plant growth after inoculation, and has strong acid resistance. Harmless, will not pollute the environment.

Description

A Strain of Bradyrhizobium Ercani and Its Application

technical field

The invention belongs to the technical field of agricultural microorganisms. More specifically, it relates to a strain of Bradyrhizobium ercanii and its application.

Background technique

Nitrogen is an indispensable element that constitutes proteins, nucleic acids, chlorophyll, enzymes, etc. in plants, and is a key factor in the formation of crop yields. The sources of nitrogen in soil are mainly nitrogen fertilizer application and biological nitrogen fixation by soil microorganisms. The production of chemical nitrogen fertilizers requires high energy consumption, and long-term use brings serious economic and environmental pressures. Excessive use of chemical fertilizers also leads to acidification and compaction of farmland soil, reduces the ability of soil to absorb water and retain moisture, and aggravates nitrogen deposition to cause air pollution.

Biological nitrogen fixation is the transformation of nitrogen _- fixing microorganisms in the soil into NH ⁴⁺ in the atmosphere. This process is efficient and environmentally friendly. According to the relationship between nitrogen-fixing microorganisms and higher plants, biological nitrogen fixation can be divided into three systems: authigenic nitrogen fixation, symbiotic nitrogen fixation, and combined nitrogen fixation, among which symbiotic nitrogen fixation is the most important one. Among them, the most common one is the symbiotic nitrogen fixation between rhizobia and leguminous plants. Due to the common influence of legumes, rhizobia and soil physical and chemical properties, only highly efficient rhizobia strains screened under specific circumstances can be inoculated on corresponding legumes. in order to maximize nitrogen fixation efficiency. However, due to different regions or soil properties, the main leguminous crops in some areas cannot be inoculated with rhizobia, or the inoculated rhizobia are not suitable for specific soil conditions, so efficient nitrogen fixation cannot be achieved.

Soybean (Glycine max (Linn.) Merr.) is the main grain, oil and feed crop in my country. The whole island of Hainan is suitable for growing soybeans. The soybeans grown in Hainan are mainly fresh soybeans. According to statistics, the annual planting area of Hainan Island reaches 2,481 hectares, with an output of 7,303 tons. The planting area has been increasing year by year in recent years. The amount of nitrogen fixation by soybean rhizobia under natural conditions can account for up to 95% of the total nitrogen required by soybeans. Inoculation of rhizobia can increase the nodulation rate of soybeans, increase the number and yield of nodules, and increase soil organic matter and available nitrogen content content. According to preliminary investigations, the cultivation of soybeans in Hainan relies entirely on the application of urea to provide nitrogen sources, and there is no cultivation technique for inoculating rhizobia. In the soybean planting areas of northern my country, there are production and sales of bacterial agents for soybean nitrogen fixation inoculation, but the effect is not significant after inoculation in Hainan, mainly due to the lack of strains suitable for Hainan's soil and climatic conditions. For example, a bradyrhizobium strain suitable for stress nitrogen fixation in the Northeast region and its application disclosed in the prior art has developed a strain of Bradyrhizobium DBPB that is specially adapted to the Northeast region, but there is still a shortage of bradyrhizobium strains suitable for different soils and Climatic nitrogen-fixing rhizobia, especially soybean rhizobia strains suitable for the climate and soil conditions in Hainan, so it is of great practical significance to screen the high-efficiency nitrogen-fixing rhizobia suitable for specific areas from the indigenous bacteria in different types of soils where soybeans are grown in Hainan .

Contents of the invention

The technical problem to be solved by the present invention is to overcome the above-mentioned defects and deficiencies, and provide a strain of Bradyrhizobium ercanii and its application.

The object of the present invention is to provide a strain of Bradyrhizobium ercanii.

Another object of the present invention is to provide the application of the strain.

Another object of the present invention is to provide a bacterial agent for growth promotion and/or nodulation and nitrogen fixation.

Another object of the present invention is to provide a method for promoting soybean growth and/or nodulation and nitrogen fixation in acidic soil.

The above object of the present invention is achieved through the following technical solutions:

The present invention isolates and obtains a bradyrhizobium strain from soybean plants, and the colonies are water-stained translucent dots in the early stage of cultivation, and the colonies in the later stage are round, dot-shaped, translucent, with neat edges and less mucus. After 4 days of culture, the colony is about 1 mm long, and after 8 days of culture, the culture dish can be covered with a single colony; the rod is a shape, which belongs to Gram-negative bacteria. The optimum growth conditions are: temperature 28°C, pH=5.5, rotation speed 180r/min. The nodules were obvious after backgrafting of soybeans, and after purification and identification, it was Bradyrhizobium elkanii, named Bradyrhizobium elkanii strain RY8, and was preserved on April 28, 2021 in China Center for Type Culture Collection, the collection number is CCTCC NO: M 2021480, and its 16S DNA sequence is shown in SEQ ID NO: 1.

The research of the present invention shows that the rhizobia RY8 has strong acid resistance, is sensitive to salt, and is not suitable for inoculation in saline-alkali land environment, and has a pH adaptation range of 4.5-7.0, and grows best when the pH value is 5.5. RY8 rhizobia has a strong ability to secrete auxin, and its effect is significantly higher than that of other Hainan soybean rhizobia control strains; after soybean inoculation with rhizobia RY8, it can secrete auxin, increase soybean yield, increase plant nitrogen content, and significantly promote plant growth. growth.

Therefore, the present invention provides Bradyrhizobium ercanii as nodulation nitrogen-fixing bacteria in nodulation and nitrogen fixation, in promoting soybean growth or in acidic soil to promote soybean growth or in the preparation of growth-promoting and/or nodulation nitrogen-fixing bacterial agents application.

The invention provides a bacterial agent for promoting growth and/or nodulation and nitrogen fixation, which contains Bradyrhizobium ercanii or its bacterial liquid.

Preferably, the Bradyrhizobium ercanii or its bacterial solution concentration is not less than 1.0×10 ⁹ CFU/mL.

The invention provides a method for promoting soybean growth and/or nodulation and nitrogen fixation in acidic soil. Bradyrhizobium ercanii or its bacterial solution is used for seed dressing treatment, or directly applied to soybean roots.

Further, Bradyrhizobium ercanii or its bacterium solution and the mixed bacterium adsorption material are prepared into a bacterium agent and then mixed and applied to the crop soil.

Preferably, the mixed bacterium adsorption material is one or more of peat, coconut peat, vermiculite or perlite.

More preferably, the mixed bacterial cell adsorption material is peat and coconut bran, and the peat and coconut bran are mixed at a ratio of 1:1, and the mixing ratio is 40mL bacterial liquid/100g adsorbent material.

Further, the pH of the bacterial solution is: 4.5-7.0.

Preferably, the pH of the bacterial solution is 5.5.

The present invention has the following beneficial effects:

The invention provides a strain of Bradyrhizobium elkanii RY8 strain, which can increase the fresh weight of soybean plants by more than 2.42 times and increase the yield of soybeans by more than 2.83 times after the RY8 strain is reattached to soybeans. The nitrogen content increased by 1.36 times, which can significantly increase the biomass and yield of plants; the RY8 strain also has the ability to secrete auxin, and the amount of IAA secreted is significantly better than other rhizobia, reaching 12.78mg.L ^-1 . Significant effect. At the same time, the inoculum prepared by back-inoculation with bacterial solution in sand culture and soil culture with RY8 and bacterial adsorption material also had such yield-increasing effect. The RY8 strain has a strong ability to infiltrate root nodules, can significantly promote plant growth after soybean inoculation, and has strong acid resistance, which is suitable for inoculation in acidic soil. When the bacteria are released into the natural environment, they are harmless to humans, animals and plants, and will not pollute the environment. Instead, they increase the population of rhizobia in the soil and promote the nodulation and nitrogen fixation of soybeans.

Description of drawings

Fig. 1 is the check diagram of the rhizobia RY8 back connection;

Fig. 2 is a colony diagram of rhizobia RY8;

Fig. 3 is the Gram stain picture of Rhizobium RY8;

Fig. 4 is RY8 agarose gel electrophoresis figure;

Figure 5 is the comparison chart of NCBI;

Figure 6 is a cluster analysis diagram of RY8 rhizobia;

Fig. 7 is a comparison chart of soybean growth after inoculation with RY8 (wherein, the left is the control (CK) inoculated with root nodules, the middle plant is treated with the same batch of isolated rhizobia 20190721004-2, and the right is treated with inoculated with RY8);

Fig. 8 is the map of the fresh weight of the above-ground part of soybean after being inoculated with different strains;

Fig. 9 is the yield figure of beans after soybean inoculation with different strains;

Figure 10 is a diagram of the number of nodules after soybean inoculation with different strains;

Fig. 11 is a diagram of the nitrogen content of soybean plants after inoculation with RY8;

Figure 12 is a diagram of the ability of RY8 to secrete auxin;

Figure 13 is a graph showing the growth of RY8 in media with different pH values;

Figure 14 is a graph showing the growth of RY8 on different concentrations of NaCl medium;

Figure 15 is the effect diagram after inoculating RY8 in production;

Fig. 16 is a graph showing the effect on yield after the application of RY8 bacterial agent.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

The culture medium that the present invention adopts is as follows:

YMA solid medium formula (L ^-1 ): mannitol 10g, MgSO4.7H ₂ O 0.2g, NaCl 0.1g, yeast powder 3g, K ₂ HPO ₄ 0.25g, KH ₂ PO ₄ 0.25g, CaCO ₃ 3g (Add when saving), agar 15g.

The isolation and cultivation of embodiment 1 bacterial strain

1. Collection of samples

The present invention collects a soybean plant that grows vigorously, has many nodules, and has large nodules from Tuyan Village, Fushan Town, Chengmai County, Hainan Province (E 109.96, N 19.77, H 55.9 meters). The whole root system is cut off, and after removing the soil Put it in a fresh-keeping bag and store it in an ice pack at low temperature. Take out the soybean root system and rinse it under tap water for 2 to 3 times to clean the root system. Use scissors to cut off the fresh, complete, large and dark red root nodules in a petri dish and rinse the surface with grade 2 water; in this process, ensure that The integrity of the root nodule surface.

2. Culture of strains

Transfer the rinsed root nodules to a sterilized petri dish, add 95% alcohol to soak for 3 minutes, pour out the alcohol, rinse with sterile water 4-5 times, add 30% hydrogen peroxide to sterilize 2 ~3 minutes, quickly pour out the hydrogen peroxide and add sterile water, transfer to the ultra-clean workbench for operation, rinse with sterile water for more than 6 times, pour out the sterile water, and transfer the selected root nodules to the sterilized culture In a dish (can be sterilized on flame), cut the nodules in half with a sterilized scalpel, mash the nodules with sterilized tweezers and an inoculation loop, dip the milky white juice in the prepared YMA solid culture Baseline. Put the scratched plate into a fresh-keeping bag and place it upside down in an incubator (28±1°C, dark) for cultivation. On the 3rd day of culture, check whether there are colonies growing on the medium, until about the 15th day, mark the colonies and record the shape and gloss.

During the cultivation process, determine whether it is a rhizobia from the following three points:

a. Colony morphology: translucent or white opaque dots in the water samples of rhizobia colonies in the early stage of growth, and later colonies are round, milky white, translucent, with neat edges and more or less sticky substance. After 2-4 days of cultivation, the colonies with a diameter of 2-4 mm are fast-growing rhizobia, and those with a colony diameter of 1 mm after 5-10 days are brady-growing rhizobia.

b. Mark the preliminarily confirmed rhizobia colony, pick a smear of the bacterial lawn from it, carry out Gram staining, and observe under a 100-fold microscope. Rhizobia are small bacilli with a diameter of (0.5-0.9)×(1.2-0.3) μm. It often contains β-hydroxybutyric acid, that is, strong refraction-like hollow particles or ring-shaped bacteria, Gram-negative (G-), no spores, and the bacteria are single or in pairs.

c. By returning the isolated rhizobia to soybeans under aseptic conditions, those capable of nodulation are rhizobia.

4. Morphological characteristics of the strain

A slow-growing rhizobia strain was identified through the cultivation process, named Reyan No. 8 (RY8). In the early stage of cultivation, the colonies were water-soaked and translucent dots, and in the later stage, the colonies were round, dot-shaped, translucent, and the edges were neat. , less viscous. After 4 days of cultivation, the colony size is about 1 mm, and after 8 days of cultivation, the culture dish can be covered with a single colony. The colony characteristics of RY8 rhizobia are shown in Figure 1.

5. Optimal growth conditions for RY8

After culturing the strain, it was determined that the optimum growth conditions for RY8 were: temperature 28°C, pH=5.5, rotational speed 180r/min. It can widely use carbon and nitrogen, and can grow well on the comprehensive extracts of various plant sources, and grow better on YMA medium than on peptone medium.

6. Physiological characteristics of RY8

Gram staining of Rhizobium RY8 is shown in Figure 2, Rhizobium RY8 is purple under the microscope after Gram staining, and the shape is a rod-shaped body, which is a Gram-negative bacterium. Bacterial cells often stain unevenly or form rings due to the accumulation of non-staining and highly refractive poly-β-hydroxybutyric acid particles.

Excluding non-rhizobium colonies, according to the above-mentioned marked colonies, the colonies with a difference of more than 3 days in the growth time of a single colony and the growth shape, colony size, transparency of the colony, and refraction under the light, and colonies with inconsistent colors were picked out. The new medium was drawn and numbered. Each time the colony on the new plate is purified by the above method until the colony growth form, colony size, colony transparency, refraction under light, and colony color on the same plate are consistent. After step-by-step purification, the purified plate was subjected to microscopic examination and back-ligation, and then placed in a YMA slant for storage.

7. Back inoculation verification of soybean rhizobia

a. Preparation of bacterial solution

Wash the isolated and purified RY8 into the prepared liquid YMA medium with sterile water, seal it with a parafilm, shake the bacteria in a shaker at 180r/min for 5 days, then measure the OD ₆₀₀ value of the bacterial solution, when the OD ₆₀₀ is greater than 0.9 (that is, the amount of bacteria per mL of bacterial liquid is more than 1.0×10 ⁹ ) to obtain the bacterial liquid, which can be used for backgrafting. The liquid YMA medium is that the agar is removed in the formula of the YMA solid medium medium, and the pH value is adjusted to 5.5.

b. Preparation of sterile seedlings

Soak a few selected soybean seeds in 95% ethanol for 5 minutes, take them out and treat them in 0.1% HgCl ₂ for 5 minutes, rinse them with sterile water for 5-10 times, and then put them on the sterilized and germinated paper Wet the germination paper with sterile 0.05mmol/L calcium sulfate solution in a petri dish, and place it in an incubator at 28°C for 3 days in the dark to accelerate germination. When the seedlings grow to 4 cm, move the seedlings into the prepared sterilized sand culture box for use.

c. Tieback

Select strong seedlings from the sand culture box and put them in a petri dish, soak them in the bacterial solution prepared in step 7a for 15 minutes, plant the seedlings in small flowerpots filled with sterilized sand with tweezers, and plant 2 plants in each pot. Add 5 mL of bacterial solution to the root of each seedling to ensure that the inoculation amount of each naked seedling reaches more than 1.0×10 ⁹ . After about four weeks, pull up the seedlings to see if there are nodules, which are rhizobia.

The results of the back grafting test of rhizobia RY8 are shown in Figure 3. After the back grafting test, RY8 had obvious nodules, which proved that the isolate was a pure rhizobia.

The identification of embodiment 2 bacterial strains

In order to determine the phylogenetic status of Rhizobium RY8, the 16SDNA series of the isolated strains were sequenced. Firstly, the total DNA was extracted using the kit from Omega, and then PCR-specific amplification was performed using primers. The upstream primer 27F used: 5-AGAGTTTGATCCTGGCTCAG-3; the downstream primer 1492R: 5-CTACGGCTACCTTGTTACGA-3. The amplification reaction system is: 10×Reaction Buffer 5.0mL, dNTPs (10mM) 1.0mL, 27F (10mM) primer 1.5mL, 1492R (10mM) primer 1.5mL, Taq DNA polymerase (5U/mL) 1.0mL, genomic DN A (20ng/mL) 1.0mL, ultrapure water 39mL; PCR amplification program: pre-denaturation at 95°C for 5 minutes; 35 cycles of denaturation at 95°C for 30s, annealing at 58°C for 30s; extension at 72°C for 90s; final extension at 72°C for 7 minutes.

Take 3mL of the amplified product and test it on 1.0% agarose gel electrophoresis. The obtained sequence of RY8 is shown in SEQ ID NO:1.

The sequence results of the obtained RY8 were compared at the National Center for Biological Information (NCBI), and it was found that the Rhizobium strain RY8 was 99.78% similar to the known strain (NR112927.1) Bradyrhizobium elkanii Strain NBRC14791. The comparison results are shown in Figure 5 Show. Then use Mega4.0 software, adopt Kimura-2 parameter, carry out Neighbor-Joining method (Neighbor-Joining) analysis, repeat 1000 times, generate phylogenetic tree. The cluster analysis diagram of RY8 rhizobia is shown in Figure 6. From the comparison results and the clustering diagram, it can be seen that the RY8 strain belongs to the new strain Bradyrhizobium elkanii (Bradyrhizobium elkanii) of the Bradyrhizobium sp. It was deposited in the Chinese Center for Type Culture Collection (CCTCC) of Wuhan University on March 28, with the preservation number CCTCC M 2021480, and the preservation address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

The role in the nodulation and nitrogen fixation of embodiment 3 rhizobia RY8

In order to confirm the effect of RY8 on soybean, the root nodule RY8 was reattached to the soybean root with the method of sand culture, and each treatment was repeated 4 times (for the processing of bacteria, sand and seedlings, refer to the reintroduction experimental steps in Example 1), so as not to The root nodules were inoculated as the control (CK), and the same batch of isolated rhizobia 20190721004-2 and the conventional rhizobia strain BDYD1 were used as reference strains for backgraft comparison. The whole process is irrigated with low-nitrogen nutrient solution. After inoculation with Rhizobium RY8, the soybean growth, aboveground fresh weight, yield and nodulation related indexes were evaluated.

The growth results of soybeans inoculated with RY8 are shown in Figure 7. It can be seen that the growth, plant shape and pod production of soybeans inoculated with Rhizobium RY8 are significantly better than those of the control and inoculated with the 20190721004-2 strain isolated from the same batch (the middle plant in the figure For 20190721004-2 strain treatment). After soybeans were inoculated with Rhizobium RY8, the fresh weight of aboveground parts (Fig. 8), the fresh weight of beans (Fig. 9) and the number of nodules (Fig. 10) were all significantly higher than those of other treatment groups. The fresh weight was more than 2.4 times that of the non-inoculated strains, and the number of nodules was also significantly increased compared with those inoculated with other strains. The yield of carob is the main index to measure the production performance of soybean, and the vigorously growing plants are the basis of high yield of carob, and the strains that can promote the growth of soybean and significantly increase the yield of carob after inoculation are high-efficiency strains. Inoculation of Rhizobium RY8 strain can increase the fresh weight of soybean shoots and beans by 142.41% and 182.72% compared with non-inoculation, respectively, and increase by 36.71% and 49.32% compared with the reference strain. Inoculation of RY8 can not only increase the yield of soybean but also significantly increase the nitrogen content of the plant. It can be seen from Figure 11 that the nitrogen content of the soybean plant after inoculation with RY8 is 36.17% higher than that without inoculation, so the RY8 strain is an efficient strain for soybean.

To sum up, it can be seen that the rhizobia RY8 significantly increased the biomass and yield after being reintroduced into soybean, both of which were 2.4 times higher than that of the control, and had a significant growth-promoting effect on soybean. After inoculating soybean with rhizobia, RY8 showed a strong dipping ability, which can significantly increase the number of nodules on soybean roots, and also increase the nitrogen content of plants. Therefore, the use of the RY8 strain can increase the population of rhizobia in the soil without polluting the environment, and can promote the nodulation and nitrogen fixation of soybeans.

The ability of embodiment 4 rhizobium RY8 to secrete auxin

In addition to nitrogen fixation, rhizobia can also secrete auxin to promote crop growth. In order to determine the auxin secretion ability of RY8 rhizobia, the auxin secretion ability of RY8 was determined by the following method.

(1) Liquid medium configuration

The medium was modified Congo red liquid medium with a pH of 6.8. The formula of the improved Congo red medium is: MgSO4.7H ₂ O 0.2gL ^-1 , KH ₂ PO ₄ .3H ₂ O 0.5gL ^-1 , NaCl 0.1gL ^-1 , NH ₄ NO ₃ 1g.L ^-1 , tryptophan Acid 0.10gL ^-1 , mannitol 10g.L ^-1 , yeast extract gL ^-1 , 0.25% Congo red solution gL ^-1 .

(2) Colorimetric solution configuration

Dissolve 12g of FeCl ₃ in 300mL of distilled water, slowly add 429.7mL of concentrated sulfuric acid, after cooling down to 1L, the measured IAA range is 0.3-20mg·L ^-1 .

(3) Standard IAA colorimetric solution configuration

Weigh 0.1g of IAA and dissolve it in 0.5L of absolute ethanol to prepare a mother liquor with a concentration of 200μg·mL ^-1 , and dilute with absolute ethanol based on the mother liquor to obtain IAA solutions with a concentration of 20, 2, 0.2, and 0μg·mL ^-1 , for qualitative measurement of auxin; IAA solutions diluted with absolute ethanol to concentrations of 0, 2.5, 5, 7.5, 10.0, 12.5, 15, and 17.5 μg·mL ^-1 based on the mother liquor were used for quantitative measurement of auxin.

(4) Strain inoculation and management

Inoculate the RY8 bacterial strain and the D1, D2, D4, D5 bacterial strains (from the Rhizobium strain isolated from Hainan Soybean Rhizobium, whose inoculation effect is better, set as the control) in 50mL culture medium, at temperature The temperature was 28°C and the rotation speed was 125r/min in a shaker for 5 days for measurement.

(5) Qualitative measurement of strain secretion IAA

Qualitative determination: Take 100 μL of the above-mentioned standard IAA solution for qualitative use, place it on a white porcelain plate, and add 100 μL of colorimetric solution as a reference. Also take 100 μL of rhizobia suspension and place it on a white porcelain plate, add 100 μL of colorimetric solution, and observe the color change after 15 minutes. Use the color of the standard colorimetric solution as a reference to determine the ability of the strain to secrete auxin. If the color is pink, it means that the strain can secrete IAA. The darker the pink color, the stronger the ability to secrete IAA; Secretes IAA.

Quantitative determination: absorb 2 ml of the above-mentioned bacterial liquid cultured for 5 days, centrifuge at 10,000 r/min for 10 minutes, take the same volume of bacterial liquid and colorimetric solution, mix them evenly, and measure them with an ultraviolet spectrophotometer after standing in the dark for 0.5 h OD ₅₃₀ value; use the above-mentioned standard colorimetric solution, prepare standard music with the same method, and calculate the IAA content secreted by the bacterial liquid according to the standard music.

The auxin secretion of RY8 rhizobia is shown in Table 1 and Figure 12 below. It can be seen that the strain RY8 has a strong ability to secrete auxin, and the effect is significantly better than that of other Hainan soybean rhizobia, which can reach more than 2 times, which is significantly higher than that of the control strain. Soybean inoculated with RY8 can secrete auxin to significantly promote its growth.

Table 1 Auxin secreted by RY8

Adaptation to soil of embodiment 5 rhizobia RY8

Rhizobium, soil environment and plants are an interactive system, and only when the three are integrated and considered can real high yield and high efficiency be achieved. The soil in Hainan is mainly acidic red soil and coastal saline soil. In order to determine the adaptability of RY8 to the main influencing factors of soil (acid-base and salinity-alkaline), the acid and salt resistance of RY8 were determined by the following methods. Provide optimal technical support for the more scientific and rational use of RY8 soybean rhizobia in the future.

In order to understand the adaptability of the strain RY8 to the acid-base environment, YMA liquid medium with pH values of 3.5, 4.5, 5.5, 6.0, 7.0, and 8.0 were prepared respectively, each bottle was 50 mL, and 4 replicates were taken. 1ml was added to the culture solution of each treatment, and transferred to a shaker at a temperature of 28°C and 180r/min. After 12 hours, the absorbance of the bacterial solution at a wavelength of 600nm was measured, and then measured every 12 hours for 7 consecutive days.

The growth of RY8 on liquid media with different pH values is shown in Figure 13. It can be seen that the strain RY8 has strong acid resistance, but is not suitable for alkaline environments. The pH adaptation range is: 4.5-7.0, and it grows at a pH value of 5.5 most.

The adaptability of RY8 to salt was determined by the growth of RY8 in different concentrations of liquid medium. Prepare YMA liquid medium with NaCl concentration of 0, 0.2, 0.4, 0.6 mol·L ^-1 respectively, with 4 replicates. Take 1 mL of each activated bacterial strain and add it to the culture solution of each treatment, transfer to a shaker with a temperature of 28 °C and 180 r/min for cultivation, and start to measure the absorbance of the bacterial solution at a wavelength of 600 nm after 12 hours, and then every 12 hours Measure once and measure continuously for 9 days.

The growth of RY8 on liquid media with different NaCl concentrations is shown in Figure 14. It can be seen that the strain RY8 is sensitive to salt and cannot grow normally under other salt concentrations except the control. Therefore, the strain RY8 is suitable for inoculation in acidic Soybeans grown in soil are not suitable for inoculation on soybeans grown in saline-alkali soil.

The growth-promoting effect of embodiment 6 rhizobia RY8

Adopt the inoculum prepared by Rhizobium RY8 provided by the invention, this inoculum can carry out seed dressing treatment after shaking the bacteria, or pour on the soybean root just planted with liquid inoculum, also can mix thalline adsorption material (peat, peat, Coconut peat (mixed at a ratio of 1:1) was made into a fungal agent and mixed with the soil before transplanting.

Effect of RY8 bacterial agent applied in soil on soybean yield:

Activate the RY8 strain, inoculate it into YMA liquid medium and cultivate it until OD ₆₀₀ >0.9, mix the peat powder and coconut peat at a ratio of 1:1, grind it, pass it through a 2mm sieve, sterilize it at 121°C for 40 minutes, and cool it down for later use. Before sowing, pour different bacterial solutions into the peat at a ratio of 40mL/100g, and mix well to allow the peat to absorb the bacterial solution, and then use the bacterial agent to dress the seeds before sowing. Taking no inoculation as the control and inoculation with RY8 as the treatment, set 4 replicates, randomized block design, each plot area is 4×2.5=10m ² , planted in ridges, and the sowing density is 20cm×10cm (row spacing×plant spacing). Sowing is done by hole-sowing, with 2 seeds per hole.

The growth of soybeans after inoculation of RY8 bacterial agent in the soybean field is shown in Figure 15. It can be seen that after soybeans were inoculated with RY8 rhizobium bacterial agent, the growth of soybeans was significantly promoted, and the growth of soybeans was significantly better than that of the control group that was not inoculated with bacterial agents; The yield of soybean after RY8 was significantly higher than that without inoculation of rhizobia ( FIG. 16 ), and the degree of increase was 35.24%.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (10)

1. An Elkinsonia slow rhizobium (Bradyrhizobium elkanii) RY8 strain, which is preserved in China Center for Type Culture Collection (CCTCC) at 28 months 4 and 2021, and has a preservation number of CCTCC NO: m2021480.

2. The use of the RY8 strain of bradyrhizobium ehrliani as claimed in claim 1 as a nodulation nitrogen fixing bacterium in nodulation nitrogen fixation in soybean.

3. The use of the slow rhizobium erlangonii RY8 strain of claim 1 or a bacterial solution thereof for promoting soybean growth or for promoting soybean growth in acid soil.

4. The use of the slow rhizobium eryngii RY8 strain of claim 1 or a bacterial solution thereof in the preparation of a growth-promoting and/or nodulation nitrogen-fixing bacterial agent.

5. A microbial inoculum for promoting growth and/or nodulation and fixing nitrogen, which is characterized by comprising the RY8 strain of the slow-growing rhizobium aeneriani or a bacterial solution thereof in claim 1.

6. The microbial preparation according to claim 5, wherein the concentration of the slow rhizobium erlangsiense RY8 strain or bacterial liquid thereof is not less than 1.0X 10 ⁹ CFU/mL。

7. A method for promoting the growth and/or nodulation and nitrogen fixation of soybeans in acid soil, which is characterized in that the slow rhizobium erlangonii RY8 strain or the bacterial solution thereof disclosed by claim 1 is used for seed dressing treatment or is directly applied to the roots of the soybeans.

8. The method according to claim 7, wherein the slow rhizobium erlangonii RY8 strain according to claim 1 or a bacterial solution thereof is mixed with a mixed bacterial adsorption material to prepare a bacterial preparation, and then the bacterial preparation is applied to the soil of the crops.

9. The method as claimed in claim 7, wherein the mixed thallus adsorbing material is one or more of turf, coconut coir, vermiculite or perlite.

10. The method according to any one of claims 7 to 9, wherein the pH of the bacterial liquid is: 4.5-7.0.

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