CN115838655B - A strain of Bradyrhizobium elkanii and its application - Google Patents

Abstract

The present invention discloses a Bradyrhizobium elkanii and its application. The RY8 strain of Bradyrhizobium elkanii has been deposited in the China Center for Type Culture Collection on April 28, 2021, with the deposit number of CCTCC NO: M 2021480. The research of the present invention shows that after the rhizobium RY8 is back-grafted into soybeans, the biomass and yield of the plants can be significantly increased; at the same time, it also has the ability to secrete auxin, and the amount of IAA secreted is significantly better than other growth-promoting rhizobia, and has a significant growth-promoting effect on soybeans. The RY8 strain has a strong ability to infect nodules, and can significantly promote plant growth after soybean inoculation. It also has a strong acid resistance and is suitable for inoculation in acidic soils. It promotes the nodulation and nitrogen fixation of soybeans, and is harmless to humans, animals and plants, and will not pollute the environment.

Description

Engelhardy slow rhizobia and application thereof

Technical Field

The invention belongs to the technical field of agricultural microorganisms. More particularly, it relates to an elkanni slow rhizobia strain and its use.

Background

Nitrogen is an indispensable element constituting proteins, nucleic acids, chlorophyll, enzymes, etc. in plants, and is a key factor in crop yield formation. The sources of nitrogen in the soil are mainly applied to nitrogen fertilizer and biological nitrogen fixation of soil microorganisms. The chemical nitrogenous fertilizer has high energy consumption in production and brings serious economic and environmental pressure after long-term use. Excessive use of the fertilizer also causes acidification and hardening of farmland soil, reduces the water absorption and soil moisture conservation capacity of the soil, and aggravates the atmospheric pollution caused by nitrogen sedimentation.

Biological nitrogen fixation is the conversion of atmospheric N ₂ to NH ⁴⁺ by nitrogen-fixing microorganisms in the soil, which is efficient and environmentally friendly. According to the relationship between the nitrogen-fixing microorganism and the higher plant, biological nitrogen fixation can be divided into three systems of autogenous nitrogen fixation, symbiotic nitrogen fixation and combined nitrogen fixation, wherein symbiotic nitrogen fixation is the most important one. The most common method is that rhizobium and leguminous plants are used for symbiotic nitrogen fixation, and the nitrogen fixation is influenced by leguminous plants, rhizobium and soil physical and chemical properties, and the maximum nitrogen fixation efficiency can be exerted only by inoculating the high-efficiency rhizobium strains screened under specific environments to the corresponding leguminous plants. However, because of different regions or soil properties, the planting of main leguminous crops in some regions cannot be inoculated with rhizobium, or the inoculated rhizobium is not suitable for specific soil conditions, and the high-efficiency nitrogen fixation effect cannot be realized.

Soybean (Glycine max (linn.) merr.) is the major food, oil and feed crop in our country. The whole Hainan island is suitable for planting soybeans, the fresh soybeans are mainly planted in the Hainan island, the annual planting area of the Hainan island is up to 2481 hectares according to statistics, the yield is 7303 tons, and the planting area in recent years is increased year by year. The nitrogen fixation amount of the rhizobium sojae under natural conditions can be up to 95% of the total nitrogen amount required by soybeans, the rhizobium sojae can be inoculated to improve the nodulation rate of the soybeans, the number and the yield of the rhizobium sojae can be increased, and the contents of soil organic matters and quick-acting nitrogen can be improved. According to the previous investigation, the cultivation technology of planting soybean in Hainan is realized by providing nitrogen source through urea application and no rhizobium inoculation. In northern soybean planting areas of China, microbial inoculum for nitrogen fixation inoculation of soybeans is produced and sold, but after Hainan inoculation, the effect is not obvious, mainly caused by the lack of strains suitable for Hainan soil and climatic conditions. The slow rhizobium strain is specially suitable for the slow rhizobium DBPB in northeast China, but is lack of nitrogen-fixing rhizobium strain suitable for different soils and climatic properties, especially suitable for soybean rhizobium strain under the condition of the soil in the Hainan climate, so that the screening of the high-efficiency nitrogen-fixing rhizobium suitable for specific areas from indigenous bacteria in different types of soils for planting soybeans in the Hainan is of great practical significance.

Disclosure of Invention

The invention aims to overcome the defects and the shortcomings and provide the elkanni slow rhizobia and the application thereof.

The invention aims to provide an Eelkanni slow rhizobia strain.

It is a further object of the invention to provide the use of said strain.

The invention further aims to provide a microbial inoculum for promoting growth and/or nodulation and fixing nitrogen.

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

The above object of the present invention is achieved by the following technical scheme:

the invention separates a slow rooting tumor from soybean plants, and the colony is water-stained semitransparent punctate in the initial stage of culture, and the colony in the later stage is round, punctate, semitransparent, neat in edge and less in mucilage. The bacterial colony is cultured for about 1mm for 4 days, and the bacterial colony can grow to be full of a culture dish for about 8 days, and single bacterial colony is arranged at the edge, and the rod is a platy body and belongs to gram-negative bacteria. The most suitable growth condition is that the temperature is 28 ℃, the pH=5.5 and the rotating speed is 180r/min. The soybean is inoculated with obvious nodulation, purified and identified to be the Ehrcut slow rhizobia (Bradyrhizobium elkanii), named as Ehrcut slow rhizobia RY8 strain, and preserved in China Center for Type Culture Collection (CCTCC) No. M2021480 in 2021 and with the 16S DNA sequence shown as SEQ ID No. 1.

The research of the invention shows that the rhizobia RY8 has stronger acid resistance, is sensitive to salt and is not suitable for being inoculated in saline-alkali soil environment, the pH adaptation range is 4.5-7.0, and the growth is best when the pH value is 5.5. The RY8 rhizobia has stronger capability of secreting auxin, has a remarkably higher effect than other soybean rhizobia control strains, can secrete auxin after soybean is inoculated with the rhizobia RY8, so that the soybean yield is increased, the nitrogen content of plants is increased, and the growth of the plants can be remarkably promoted.

Thus, the present invention provides the use of elkanni slow rhizobia as a nodulation nitrogen fixing bacteria in nodulation nitrogen fixation, in promoting soybean growth or in acid soil or in the preparation of a growth-promoting and/or nodulation nitrogen fixing bacteria agent.

The invention provides a microbial inoculum for promoting growth and/or nodulation and fixing nitrogen, which contains elkanni slow-growing rhizobia or bacterial liquid thereof.

Preferably, the Ehrlich Kanny Rhizobium or its bacterial liquid concentration is not less than 1.0X10 ⁹ CFU/mL.

The invention provides a method for promoting soybean growth and/or nodulation and nitrogen fixation in acid soil, which adopts elkanni slow rhizobium or bacterial liquid thereof to carry out seed dressing treatment or directly apply to soybean roots.

Further, the elkanni slow-growing rhizobia or bacterial liquid and the mixed bacterial adsorbing material are prepared into a bacterial agent and then mixed and applied to crop soil.

Preferably, the mixed thallus adsorption material is one or more of turf, coconut husk, vermiculite or perlite.

More preferably, the mixed thallus adsorption material is turf and coconut husk, and the turf and the coconut husk are mixed according to a ratio of 1:1, wherein the mixing ratio is 40mL of bacterial liquid per 100g of adsorption material.

Further, the pH of the bacterial liquid is 4.5-7.0.

Preferably, the pH of the bacterial liquid is 5.5.

The invention has the following beneficial effects:

The invention provides an Eelkanni slow-growing rhizobium (Bradyrhizobium elkanii) RY8 strain, which can increase the fresh weight of the soybean by more than 2.42 times, increase the yield of the soybean by more than 2.83 times, increase the nitrogen content of the soybean by 1.36 times, and obviously increase the biomass and yield of the plant after the RY8 strain is returned to the soybean, wherein the RY8 strain also has the capability of secreting auxin, and the IAA secretion amount is obviously superior to that of other rhizobium-promoting bacteria, reaches 12.78mg.L ^-1, and has obvious growth-promoting effect on the soybean. Meanwhile, the microbial inoculum prepared by sand culture back inoculation microbial inoculum and soil culture application RY8 and a thallus adsorbing material also has the effect of increasing yield. The RY8 strain has strong dip dyeing capability on root nodules, can obviously promote plant growth after soybean inoculation, has strong acid resistance, and is suitable for being inoculated in soil under acidic conditions. When the thalli are released into the natural environment, the thalli are harmless to people, animals and plants, do not pollute the environment, but increase the population of rhizobia in soil, and have the effect of promoting the nodulation and nitrogen fixation of soybeans.

Drawings

FIG. 1 is a diagram of a rhizobia RY8 tieback test;

FIG. 2 is a diagram of rhizobia RY8 colonies;

FIG. 3 is a gram of rhizobia RY 8;

FIG. 4 is a diagram of RY8 agarose gel electrophoresis;

FIG. 5 is a NCBI alignment chart;

FIG. 6 is a graph of RY8 rhizobia cluster analysis;

FIG. 7 is a graph comparing soybean growth after inoculation with RY8 (where the left is the inoculated nodule as Control (CK), the middle plant is treated with the same batch of isolated rhizobia 20190721004-2, and the right is the inoculated RY8 treatment);

FIG. 8 is a graph of fresh weight of the aerial parts of soybeans after inoculating different strains;

FIG. 9 is a graph showing the yield of beans after soybeans are inoculated with different strains;

FIG. 10 is a graph showing the number of nodulation after inoculating soybeans with different strains;

FIG. 11 is a nitrogen content plot of soybean plants after inoculation with RY 8;

FIG. 12 is a graph of RY8 secretion auxin;

FIG. 13 is a graph showing the growth of RY8 in different pH media;

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

FIG. 15 is a graph showing effects after inoculation of RY8 during production;

FIG. 16 is a graph showing the effect on yield after application of RY8 inoculum.

Detailed Description

The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.

Reagents and materials used in the following examples are commercially available unless otherwise specified.

The culture medium adopted by the invention is as follows:

YMA solid culture Medium formulation (L ^-1) mannitol 10g, mgSO 4.7H2O 0.2g, naCl0.1g, yeast powder 3g, K ₂HPO₄ 0.25g、KH₂PO₄ 0.25g、CaCO₃ g (added during storage), agar 15g.

Example 1 isolation culture of strains

1. Sample collection

The invention collects a soybean plant with vigorous growth, more rums and large root nodule from village (E109.96,N 19.77,H 55.9 m) with soil-relieving and brilliant soil in Fushan, chengmai county, hainan province, cuts off the whole root system, removes soil, puts the root system into a fresh-keeping bag, and places the root system into an ice bag for low-temperature preservation. Taking out soybean root system, washing the soybean root system under tap water for 2-3 times to wash the root system, cutting fresh and complete root nodules with large volume and dark red color in a culture dish by using scissors, and washing the surface of the root system by using grade 2 water, wherein the process is to ensure the integrity of the surface of the root nodules.

2. Culture of strains

Transferring the cleaned root nodule into a sterilized culture dish, adding 95% alcohol to soak for 3 min, pouring out the alcohol, flushing with sterile water for 4-5 times, adding 30% hydrogen peroxide to sterilize for 2-3 min, pouring out the hydrogen peroxide quickly, adding the sterile water, transferring into an ultra-clean workbench to operate, flushing with sterile water for more than 6 times, pouring out the sterile water, transferring the selected root nodule into the sterilized culture dish (which can be sterilized on flame), cutting the root nodule into two halves by a sterilized scalpel, mashing the root nodule by a sterilized forceps and an inoculating loop, and drawing lines on the prepared YMA solid culture medium by dipping the milky juice. Placing the scratched plate into a fresh-keeping bag, and then inverting the plate into an incubator (at 28+/-1 ℃ and in darkness) for culturing. The culture medium was checked for colony growth starting on day 3 of culture, and until around day 15, the colonies were marked to record morphology and gloss.

Whether rhizobia is determined from the following three points during the culture:

a. Colony morphology-the semitransparent or white opaque spots of the initial colony water sample of rhizobia later colony is round, milky white, semitransparent, neat in edge and more or less sticky. Culturing for 2-4 days until the colony diameter reaches 2-4 mm, wherein the colony diameter is fast-growing rhizobium, and culturing for 5-10 days until the colony diameter is 1mm, wherein the colony diameter is slow-growing rhizobium.

B. The markers were initially confirmed as rhizobium colonies from which lawn smears were picked, gram stained and observed under a 100-fold microscope. Rhizobia is (0.5 to 0.9) × (1.2 to 0.3) μm of small bacillus. Beta-hydroxybutyric acid is often contained, namely, hollow particles with strong refraction are formed or the thalli are shaped like links, gram negative (G-), no spores exist, and the thalli are single or paired.

C. by inoculating isolated rhizobia back onto soybean under aseptic conditions, rhizobia can form nodules.

4. Morphological characteristics of strains

A slow-growing rhizobium strain is identified through the culture process and named as hot grinding No. 8 (RY 8), water stain-shaped semitransparent punctate is formed on a colony at the initial stage of culture, and the colony at the later stage is round, punctate, semitransparent, neat in edge and less in mucilage. The colony was cultured for about 1mm for 4 days, and the dish was allowed to grow for about 8 days, and single colonies were formed at the edge, and the colony characteristics of RY8 rhizobia were as shown in FIG. 1.

5. Optimal growth conditions for RY8

The optimal growth conditions for RY8 after strain cultivation were determined to be 28℃at pH=5.5 and 180r/min. Can be widely applied to carbon source and nitrogen source, can well grow on the comprehensive extract liquid of various plant sources, and can well grow on YMA culture medium than peptone culture medium.

6. Physiological Properties of RY8

Gram staining of rhizobia RY8 As shown in FIG. 2, rhizobia RY8 was gram-stained purple under a microscope and was in the shape of a rod-like body of gram-negative bacteria. Cells often have uneven staining or a ring shape due to accumulation of non-staining and highly refractive poly-beta-hydroxybutyrate particles.

Colonies other than rhizobia are removed, colonies which are different in time of single colony growth by more than 3 days, and colonies which are different in growth form, size, transparency, refraction under light and color of the colonies on a plate are selected according to the labeled colonies, and the colonies are marked and numbered on a new culture medium. The colony of the new plate is purified once by the method until the growth form, the size and the transparency of the colony on the same plate, and the refraction and the color of the colony under the light are consistent. The purified plates were visualized by stepwise purification and returned to YMA slope for storage.

7. Tieback verification of rhizobia sojae

A. preparation of bacterial liquid

Washing the separated and purified RY8 with sterile water into a prepared liquid YMA culture medium, sealing with a sealing film, measuring the OD ₆₀₀ value of the bacterial liquid after shaking in a shaking table for 5 days at 180r/min, and obtaining the bacterial liquid when the OD ₆₀₀ is more than 0.9 (namely the bacterial content of the bacterial liquid per mL is more than 1.0X10. 10 ⁹), wherein the bacterial liquid can be used for tie-back. The liquid YMA medium is prepared by removing agar in YMA solid medium formula, and adjusting pH to 5.5.

B. Preparation of sterile seedlings

Selecting soybean seeds, soaking the soybean seeds in 95% ethanol for 5 minutes, taking out, treating the soybean seeds in 0.1% HgCl ₂ for 5 minutes, flushing the soybean seeds with sterile water for 5-10 times, discharging the soybean seeds in a sterilized culture dish with germinated paper, soaking the germinated paper in a sterilized 0.05mmol/L calcium sulfate solution, and carrying out dark germination acceleration for 3 days in a 28 ℃ incubator. And when the seedlings grow to 4 cm, transferring the seedlings into a prepared sterilized sand culture box for later use.

C. Tieback

And (3) selecting strong seedlings from a sand culture box, placing the seedlings into a culture dish, soaking the seedlings in the bacterial liquid prepared in the step (7 a) for 15 minutes, planting the seedlings in small flowerpots filled with sterilized sand by using tweezers, planting 2 plants in each pot, adding 5mL of bacterial liquid into the root of each seedling, and ensuring that the bacterial inoculation amount of each bare seedling reaches more than 1.0X10 ⁹. After about four weeks, the seedlings were pulled up to see if nodulation, which is rhizobia.

The tiebacteria RY8 tieback test result is shown in FIG. 3, and the tieback test shows that RY8 has obvious nodulation, and the isolate is pure rhizobia.

Example 2 identification of strains

To determine the phylogenetic status of rhizobia RY8, the 16SDNA series of isolated strains were sequenced. The total DNA was first extracted using the omega company kit, then PCR-specific amplification was performed using primers, the upstream primer 27F:5-AGAGTTTGATCCTGGCTCAG-3 and the downstream primer 142R: 5-CTACGGCTACCTTGTTACGA-3. The amplification Reaction system comprises 10×reaction Buffer 5.0mL, dNTPs (10 mM) 1.0mL, 27F (10 mM) primer 1.5mL, 1492R (10 mM) primer 1.5mL, taq DNA polymerase (5U/mL) 1.0mL, genome DN A (20 ng/mL) 1.0mL, ultrapure water 39mL, and the PCR amplification program comprises 95 ℃ pre-denaturation for 5min, 95 ℃ denaturation for 30s,58 ℃ annealing for 30s 35 cycles, 72 ℃ extension for 90s and 72 ℃ final extension for 7min.

3ML of the amplified product is taken and tested on 1.0% agarose gel electrophoresis, the detection result is shown as an agarose gel electrophoresis diagram of RY8 in figure 4, the amplified product is sent to Shenzhen micro-co Union technology group Co., ltd for sequencing, and the sequence of RY8 is shown as SEQ ID NO. 1.

The sequence results of the obtained RY8 were aligned in the National Center for Biological Information (NCBI) and the rhizobium strain RY8 was found to have 99.78% similarity to the known strain (NR 112927.1) Bradyrhizobium ELKANII STRAIN NBRC14791, the alignment is shown in FIG. 5. Then, by using Mega4.0 software and Kimura-2 parameters, an adjacent method (Neighbor-training) analysis is performed, and the system development tree is generated by repeating 1000 times. A graph of RY8 rhizobia cluster analysis is shown in FIG. 6. From the comparison results and the cluster map, it is known that the RY8 strain belongs to a new strain of Rhizobium (Bradyrhizobium sp.) which is a novel strain of Ehrkania slow rooting tumor (Bradyrhizobium elkanii) and has a classification designation of Bradyrhizobium ELKANII RY8 and is preserved in China Center for Type Culture Collection (CCTCC) of Wuhan university at 4 months 28 of 2021, wherein the preservation number is CCTCC M2021480 and the preservation address is eight 299 of Wuchang district of Wuhan, hubei province.

Example 3 role of rhizobia RY8 in nodulation and Nitrogen fixation

To confirm the effect of RY8 on soybean, root nodule RY8 was tiebased to the root of soybean by sand culture, each treatment was repeated 4 times (treatment of bacteria, sand and seedlings see tiebased experimental procedure in example 1), tiebased with non-inoculated nodule as Control (CK), and the same batch of isolated rhizobium 20190721004-2 and conventional rhizobium strain BDYD1 as reference strain. And irrigating with low nitrogen nutrient solution in the whole process. After rhizobium RY8 inoculation, the growth, the fresh weight of the overground parts, the yield and the nodulation related indexes of the soybeans are evaluated.

As shown in FIG. 7, the growth results of the soybeans after the inoculation of RY8 are shown, and the growth vigor, the strain shape and the pod bearing amount of the soybeans after the inoculation of rhizobia RY8 are significantly better than those of the soybeans after the inoculation of the same batch of the separated 20190721004-2 strain (the intermediate plants in the figure are 20190721004-2 strain treatment). After soybean inoculation with rhizobia RY8, the fresh weight of the overground parts (figure 8) and the fresh weight of the beans (figure 9) and the root nodule number (figure 10) are both significantly higher than those of other treatment groups, and after soybean inoculation with RY8, the fresh weight of the overground parts of the soybeans and the fresh weight of the beans are both over 2.4 times that of the strains which are not inoculated, and the root nodule number is also significantly increased compared with that of other strains. The yield of the beans is a main index for measuring the production performance of the beans, the plants with vigorous growth are the basis of the high yield of the beans, and the strain which can promote the growth of the beans and remarkably increase the yield of the beans after being inoculated with the strain is a high-efficiency strain. Inoculating rhizobia RY8 strain can increase fresh weight of aerial parts and beans of soybean by 142.41% and 182.72% respectively compared with that of reference strain by 36.71% and 49.32% respectively. As can be seen from FIG. 11, the nitrogen content of plants after the soybeans are inoculated with RY8 is increased by 36.17% compared with the plants after the soybeans are inoculated with RY8, so that the RY8 strain is a high-efficiency strain for the soybeans.

In conclusion, the rhizobia RY8 is obviously added to soybeans to increase biomass and yield by more than 2.4 times compared with the control, and has obvious growth promotion effect on the soybeans. RY8 shows strong dip-dyeing ability after soybean is inoculated with rhizobium, can obviously increase the nodulation quantity of soybean roots, and also increases the nitrogen content of plants. Therefore, the RY8 strain can increase the population of rhizobia in soil, does not pollute the environment, and has the effect of promoting nodulation and nitrogen fixation of soybeans.

Example 4 ability of Rhizobium RY8 to secrete auxin

Rhizobia can secrete auxin in addition to nitrogen fixation to promote crop growth, and to determine the auxin secretion capacity of RY8 rhizobia, the auxin secretion capacity of RY8 was determined by the following method.

(1) Liquid culture medium configuration

The culture medium adopts a modified Congo red liquid culture medium with pH of 6.8. The modified Congo red medium formulation was ：MgSO4.7H₂O 0.2g.L^-1、KH₂PO₄.3H₂O 0.5g.L^-1、NaCl 0.1g.L^-1、NH₄NO₃ 1g.L^-1、 tryptophan 0.10g.L ^-1, mannitol 10g.L ^-1, yeast extract g.L ^-1, 0.25% Congo red solution g.L ^-1.

(2) Colorimetric solution arrangement

12G of FeCl ₃ is dissolved in 300mL of distilled water, 429.7mL of concentrated sulfuric acid is slowly added, the volume is fixed to 1L after cooling, and the IAA is measured to be 0.3-20mg.L ^-1.

(3) Standard IAA colorimetric liquid formulation

0.1G IAA was weighed and dissolved in 0.5L of absolute ethanol to prepare a mother solution having a concentration of 200. Mu.g.mL ^-1, and IAA solutions having concentrations of 20, 2, 0.2 and 0. Mu.g.mL ^-1 were diluted with absolute ethanol based on the mother solution for qualitative measurement of auxin, and IAA solutions having concentrations of 0, 2.5, 5, 7.5, 10.0, 12.5, 15 and 17.5. Mu.g.mL ^-1 were diluted with absolute ethanol based on the mother solution for quantitative measurement of auxin.

(4) Strain inoculation and management

The activated standby RY8 strain and the D1, D2, D4, D5 strains (rhizobium strains isolated from Rhizobium japonicum in Hainan. Sojae, which were used as controls with a better inoculation effect) were inoculated in 50mL of medium and cultured in a shaker at 28℃and a rotation speed of 125r/min for 5 days for measurement.

(5) Qualitative measurement of IAA secretion by strains

Qualitative determination 100. Mu.L of the standard IAA solution for qualitative determination was placed on a white porcelain plate, and 100. Mu.L of a colorimetric solution was added as a reference. Also, 100. Mu.L of rhizobia suspension was placed on a white porcelain plate, and after 15 minutes, a color change was observed by adding 100. Mu.L of a colorimetric solution. And determining the capability of the strain to secrete auxin by taking the color of the standard colorimetric solution as a reference, wherein the color shows pink positive, the deeper the pink color shows stronger capability of secreting IAA, and the colorless negative shows that the strain cannot secrete IAA.

The quantitative determination comprises the steps of sucking 2 ml of the bacterial liquid cultured for 5 days, centrifuging for 10 minutes at 10000r/min, taking the bacterial liquid with the same volume, uniformly mixing the bacterial liquid with the colorimetric liquid, standing for 0.5h in the dark, immediately measuring an OD ₅₃₀ value by an ultraviolet spectrophotometer, preparing standard yeast by using the standard colorimetric liquid, and calculating the IAA secretion content of the bacterial liquid according to the standard yeast.

As shown in the following Table 1 and FIG. 12, the RY8 rhizobia has a strong ability to secrete auxin, and the effect is significantly better than that of other Hainan soybean rhizobia, and can reach more than 2 times, and is significantly higher than that of the control strain, so that the growth of the strain can be significantly promoted by secreting auxin after soybean inoculation with RY 8.

TABLE 1 RY8 secretion of auxin

Example 5 adaptation of rhizobia RY8 to soil

Rhizobia, soil environment and plant are an interactive system, and the real high yield and high efficiency can be achieved only by integrating the three. The soil in Hainan is mainly acid red soil and coastal saline soil, and in order to determine the adaptation condition of RY8 to main influencing factors (acid-base and saline-alkali) of the soil, the acid resistance and the salt resistance of RY8 are determined by the following method. Provides a preferable technical support for more scientifically and reasonably using RY8 rhizobium japonicum in the future.

To understand the adaptability of the strain RY8 to acid-base environment, YMA liquid culture media with pH values of 3.5, 4.5, 5.5, 6.0, 7.0 and 8.0 are prepared respectively, 50mL of each bottle is repeated, 1mL of each activated strain is added into each treated culture solution, the culture solution is transferred to a shaking table with the temperature of 28 ℃ and 180r/min for culture, the absorbance of the bacterial solution at the wavelength of 600nm is measured after 12 hours, and then the bacterial solution is measured once every 12 hours for 7 days continuously.

As shown in FIG. 13, the strain RY8 has strong acid resistance, but is not suitable for alkaline environment, the pH application range is 4.5-7.0, and the growth is best when the pH value is 5.5.

The ability to adapt to salts was determined by the growth of RY8 in liquid media of varying concentrations. YMA liquid media were prepared at NaCl concentrations of 0, 0.2, 0.4, 0.6 mol.L ^-1, respectively, in 4 replicates. 1mL of each activated strain is added into each treated culture solution, the culture solution is transferred to a shaking table with the temperature of 28 ℃ and 180r/min for culture, the absorbance of the bacterial solution at the wavelength of 600nm is measured after 12 hours, and then the bacterial solution is measured every 12 hours for 9 days continuously.

As shown in FIG. 14, the growth conditions of the RY8 on the liquid culture mediums with different NaCl concentrations show that the strain RY8 is sensitive to salt and can not grow normally under other salt concentrations except the control can grow normally, so that the strain RY8 is suitable for being inoculated on soybeans planted in acid soil and is not suitable for being inoculated on soybeans planted in saline-alkali soil.

Example 6 growth promoting effect of Rhizobium RY8

The microbial inoculum prepared by the rhizobium RY8 provided by the invention can be subjected to seed dressing treatment after fungus shaking, or can be poured into the root of soybean which is just planted by a liquid microbial inoculum, or can be mixed with a thallus adsorption material (grass carbon and coconut coir are mixed according to a ratio of 1:1) to prepare the microbial inoculum which is mixed and applied into soil before transplanting.

Effect of RY8 inoculant application in soil on soybean yield:

Activating RY8 strain, inoculating to YMA liquid culture medium, culturing until OD ₆₀₀ is more than 0.9, mixing grass carbon powder and coconut husk at a ratio of 1:1, pulverizing, sieving with 2mm sieve, sterilizing at 121deg.C for 40 min, and cooling. Before sowing, pouring different bacterial solutions into turf according to the proportion of 40mL/100g, fully mixing to enable the turf to absorb the bacterial solutions, and dressing seeds with a bacterial agent before sowing. The method is characterized by taking non-inoculation as a control, taking inoculated RY8 as a treatment, setting 4 repeats, randomly setting a block design, enabling the area of each cell to be 4 multiplied by 2.5=10m ², performing ridging planting, and enabling the sowing density to be 20cm multiplied by 10cm (row spacing multiplied by plant spacing). The seeding adopts a hole seeding mode, and each hole is seeded with 2 grains.

The growth of plants after the inoculation of RY8 microbial inoculum in the soybean field is shown in figure 15, and the growth of the soybean is obviously promoted after the inoculation of RY8 rhizobia microbial inoculum in the soybean, the growth vigor of the soybean is obviously better than that of a control group without the inoculation of microbial inoculum, and the yield of the soybean after the inoculation of RY8 is obviously higher than that of the soybean without the inoculation of rhizobia (figure 16), so that the improvement degree is 35.24%.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (10)

1. A Bradyrhizobium elkanii RY8 strain, characterized in that the strain has been deposited in the China Center for Type Culture Collection on May 5, 2021, with the deposit number CCTCC NO: M 2021480. 2. Use of the Bradyrhizobium erkannii RY8 strain according to claim 1 as a nodulation and nitrogen-fixing bacterium in soybean nodulation and nitrogen fixation. 3. Use of the Bradyrhizobium erkannii RY8 strain or its bacterial solution according to claim 1 in promoting soybean growth or promoting soybean growth in acidic soil. 4. Use of the Bradyrhizobium erkannii RY8 strain or its bacterial solution according to claim 1 in the preparation of a growth-promoting and/or nodulation nitrogen-fixing bacterial agent. 5. A bacterial agent for promoting growth and/or nodulation and nitrogen fixation, characterized in that it contains the Bradyrhizobium erkannii RY8 strain or its bacterial solution as described in claim 1. 6 . The bacterial agent according to claim 5 , characterized in that the concentration of the Bradyrhizobium ercanii RY8 strain or its bacterial solution is not less than 1.0×10 ⁹ CFU/mL. 7. A method for promoting soybean growth and/or nodulation and nitrogen fixation in acidic soil, characterized in that the Bradyrhizobium erkannii RY8 strain or its bacterial solution as described in claim 1 is used for seed dressing treatment or directly applied to the soybean roots. 8. The method according to claim 7, characterized in that the Bradyrhizobium ercanii RY8 strain or its bacterial liquid according to claim 1 is mixed with a mixed bacterial adsorption material to prepare a bacterial agent and then mixed and applied to the crop soil. 9. The method according to claim 8, characterized in that the mixed bacterial adsorption material is one or more of peat, coconut bran, vermiculite or perlite. 10. The method according to any one of claims 7 to 9, characterized in that the pH of the bacterial solution is 4.5-7.0.