CN116004421A - Peanut nodule nitrogen-fixing Huang-Huai-Hai slow-growing rhizobium RY9 and application thereof - Google Patents

Abstract

The invention discloses peanut nodule nitrogen-fixing Huang-Huai-Hai slow-growing rhizobium RY9 and application thereof. The invention provides a strain RY9 of Huang-Huai-Hai slow rooting tumor bacteria (Bradyrhizobium huanghuaihaiense), which is preserved in the China center for type culture collection of Wuhan in the year 2021, month 4 and day 28, and has the preservation number of CCTCC NO: m2021481. The research shows that the RY9 strain can secrete auxin and has the capability of resisting acid and aluminum, can be widely applied to the soil in the south of China, can obviously increase the nodulation quantity of peanuts after being inoculated with the RY9 strain, promote the growth and the fructification of the peanuts, improve the biomass of the peanuts by 41.5 percent and increase the yield of the peanuts by 62.2 percent, thereby promoting the growth of the peanuts and leading the peanuts to reach high yield. The RY9 strain has strong dip dyeing capability, large nodulation quantity, large nodulation volume and strong stress resistance, can lead the peanut to have high yield, and has promotion effect on nodulation and nitrogen fixation of the peanut.

Description

Peanut nodule nitrogen-fixing Huang-Huai-Hai slow-growing rhizobium RY9 and application thereof

Technical Field

The invention belongs to the technical field of agricultural microorganisms. More particularly relates to peanut nodule nitrogen-fixing Huang-Huai-Hai slow-growing rhizobium RY9 and application thereof.

Background

The nitrogen is one of the most important nutrient elements for plant growth, and the nitrogen in the soil mainly consists of three parts of nitrogen decomposed from soil minerals, nitrogen fixed by soil microorganisms and nitrogen applied to chemical fertilizers. The application of nitrogen fertilizer in agricultural production is an important means for ensuring high yield, but the blind application of a large amount of nitrogen fertilizer greatly exceeds the requirements of soil storage capacity and crop absorption, so that the utilization rate of the fertilizer is reduced, and meanwhile, the ecological problems of soil hardening, environmental pollution and the like are caused. Statistics show that the nitrogen fertilizer utilization rate in China is extremely low, and more than 50% of the nitrogen fertilizer is lost through ammonia volatilization, runoff, nitrification-denitrification, leaching and other ways. Biological nitrogen fixation has the characteristics of high efficiency and environmental friendliness, and the value of nitrogen fertilizer required for the growth of food, oil and fiber crops in the world reaches 330 hundred million dollars according to statistical report estimation, wherein the biological nitrogen fixation effect of leguminous crops provides about 50 hundred million dollars of nitrogen sources. It is calculated that agricultural products harvested worldwide each year take about 11000 ten thousand tons of nitrogen from the soil, while the modern synthetic ammonia industry can provide only 4500 ten thousand tons of nitrogen, even though its utilization rate is as high as 60-70%, only 20-30% of the world's agricultural nitrogen needs, and the remaining 70-80%, all from organic fertilizers and biological nitrogen fixation. Inoculating rhizobia is a necessary cultivation for leguminous cropsMeans, the symbiotic energy of leguminous plants and root nodules can make N in the atmosphere ₂ Conversion to NH ⁴⁺ This process is efficient and environmentally friendly. It is estimated that the annual biological nitrogen fixation amount worldwide is up to 1.75 hundred million tons, which is 4.37 times the world's industrial nitrogen fertilizer production, with about 5500 ten thousand tons of nitrogen being fixed by legumes and rhizobia. In the United states, brazil and other countries, the area of the inoculated rhizobia generally accounts for more than 50 percent of the sowing area, thereby improving the soil, protecting the environment and being beneficial to maintaining the nitrogen balance. At present, basic research of symbiotic nitrogen fixation in China reaches the international advanced level, but industrialization and large-area popularization and application of rhizobia agents are very backward, the area of leguminous crop inoculation rhizobia agents only accounts for 1% -3% of the sowing area, and the difference is very large compared with foreign countries.

Peanuts (Arachis hypogaea Linn.) are the major oil crop and can be matured for 1 year 3 under southern appropriate climatic conditions. The peanut inoculated efficient rhizobia has stronger nitrogen fixation capability, and can increase the number of rhizobia and the yield, thereby increasing the benefit. As disclosed in the prior art, the high-efficiency peanut root nodule curing (Rhizobimlegminiosarv) has higher biological nitrogen fixation activity and better salt and alkali resistance and high temperature resistance. However, nitrogen fixation and growth promotion capability after symbiotic rhizobia and leguminous plants are affected by physical and chemical properties of leguminous plants, rhizobia and soil, and the maximum nitrogen fixation efficiency can be exerted only by inoculating the high-efficiency rhizobia strains screened out in a specific environment to the corresponding leguminous plants. At present, peanut azotobacter capable of being applied to different environmental conditions is fewer, and there are few efficient rhizobia strains suitable for the climate and the soil condition in the Hainan, so that the method for screening peanut efficient azotobacter suitable for specific areas from different types of soil for planting peanuts in the Hainan has important practical significance.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings and provide a root nodule azotobacter strain RY9 strain and application thereof.

The invention aims to provide a peanut nodule nitrogen-fixing Huang Huai sea slow rooting tumor strain RY 9.

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 peanut growth and/or nodulation and fixing nitrogen.

It is a further object of the present invention to provide a method of promoting peanut growth and/or nodulation and nitrogen fixation.

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

in the invention, in 2019, 7 months 23, a peanut with a large number of root nodules and luxuriant growth is collected in the Zhenjia city and the town peanut planting field (E109.37N19.74H 3.19 m), a peanut root nodule nitrogen-fixing Huanghuaihai slow rooting tumor strain (Bradyrhizobium huanghuaihaiense) RY9 is obtained by separating and purifying the peanut root nodule nitrogen-fixing Huanghuaihai slow rooting tumor strain, RY9 is a gram-negative bacterium, a bacterial colony is milky and semitransparent punctate in the early growth stage of the strain, and a bacterial colony in the later stage is round, milky, semitransparent, neat in edge and sticky. The colonies were cultured for 4 days of 2-3mm, and the colonies were allowed to grow to the medium by the time of 8 days, and a large amount of mucus was present around the colonies. The optimal growth conditions for RY9 were: temperature 30 ℃, ph=6, rotation speed 180r/min. Can be widely applied to carbon sources and nitrogen sources, can well grow on the comprehensive extract liquid of various plant sources, and can well grow on YMA culture medium than peptone culture medium. The peanut nodule nitrogen-fixing Huang Huai sea slow-growing rhizobium RY9 strain is preserved in the China Center for Type Culture Collection (CCTCC) of Wuhan in 2021, 4 months and 28 days, and the preservation number is CCTCC NO: m2021481, the 16S DNA sequence of which is shown in SEQ ID NO: 1.

The study shows that after the peanut is inoculated with the RY9 strain, the biomass, the yield and the fresh weight of the root nodule of the peanut are obviously improved, the biomass is improved by 41.5 percent after the peanut is inoculated with the root nodule of the RY9 strain, the yield of the peanut is increased by 62.2 percent, and the nodulation quantity of the peanut can be obviously increased, so that the growth of the peanut is promoted. Therefore, rhizobia RY9 is a high-efficiency matched strain of peanuts, and has the advantages of strong dip dyeing capability, large nodulation quantity, large nodulation volume and strong stress resistance after inoculation, can ensure high yield of the peanuts, and has the effect of promoting nodulation and nitrogen fixation of the peanuts.

Therefore, the invention provides the application of the Huang-Huai-Hai slow-growing rhizobium RY9 or bacterial liquid thereof as nodulation nitrogen-fixing bacteria in the aspect of promoting peanut growth or promoting peanut growth under the condition of aluminum acid and preparing peanut growth-promoting and/or nodulation nitrogen-fixing bacteria agent.

The invention provides a peanut growth-promoting and/or nodulation-fixing microbial inoculum which contains Huang-Huai-Hai slow-growing rhizobium RY9 or bacterial liquid thereof.

Preferably, the concentration of the Rhizobium flavum RY9 or bacterial liquid thereof is not less than 1.0X10 ⁹ CFU/mL。

Preferably, the microbial inoculum comprises a mixed microbial adsorbent: turf, vermiculite and perlite.

More preferably, the microbial inoculum is 10mL of microbial inoculum per 50g of turf, 10mL of microbial inoculum per 15g of vermiculite or 10mL of microbial inoculum per 15g of perlite.

Further preferably, the mixing proportion of the microbial inoculum is 40mL of microbial inoculum/100 g of mixed adsorbent, and the microbial inoculum prepared by mixing 50% of turf and 50% of coconut coir has the highest cost performance.

The invention provides a method for promoting peanut growth and/or nodulation and nitrogen fixation, which adopts Huang-Huai-Hai slow-growing rhizobium RY9 or bacterial liquid thereof to carry out seed dressing treatment or directly apply the seed dressing treatment to peanut roots or soil environment.

Preferably, the soil environment is soil under the condition of aluminum acid.

More preferably, the pH value of the soil environment is 5.5-7.0, the NaCl concentration is lower than 0.2mol/L, and Al ³⁺ The concentration is lower than 100 mu mol.L ^-1 Can be widely applied to the soil in the south of China.

The invention has the following beneficial effects:

the invention provides a peanut nodule nitrogen-fixing Huang-Huai-Hai slow rooting tumor strain (Bradyrhizobium huanghuaihaiense) RY9, wherein the RY9 strain can secrete auxin and has the capability of resisting acid aluminum, and can be widely applied to the soil in the south of China, and the peanut nodule quantity can be obviously increased after the RY9 strain is inoculated, so that the peanut growth and the fruiting are promoted, the peanut biomass is increased by 41.5%, and the peanut yield is increased by 62.2%, thereby promoting the peanut growth and ensuring the peanut to reach high yield. The RY9 strain has strong dip dyeing capability, large nodulation quantity, large nodulation volume and strong stress resistance, can lead the peanut to have high yield, and has promotion effect on nodulation and nitrogen fixation of the peanut.

Drawings

FIG. 1 is a diagram of rhizobia RY9 colonies;

FIG. 2 is a gram of rhizobia RY 9;

FIG. 3 is a diagram showing a rhizobia RY9 tieback test;

FIG. 4 is a diagram of Rhizobium RY9 agarose gel electrophoresis;

FIG. 5 is a rhizobia RY9NCBI alignment chart;

FIG. 6 is a graph of a rhizobia RY9 cluster analysis;

FIG. 7 is a graph of the biomass of the ground part of a peanut inoculated with RY 9;

FIG. 8 is a graph showing the variation in yield of inoculated RY9 peanuts;

FIG. 9 is a graph showing the amount of peanut nodulation inoculated with RY 9;

FIG. 10 is a graph showing the results of the secretion of auxin by Rhizobium RY 9;

FIG. 11 is a graph showing growth in a medium of rhizobia RY9 at different pH values;

FIG. 12 is a graph showing growth of Rhizobium RY9 in different NaCl concentration media;

FIG. 13 is a graph showing growth of rhizobia RY9 in various concentrations of aluminum solution;

FIG. 14 is a plot of field inoculation RY9 peanut yield.

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.

Example 1 obtaining and culturing of strains

In 2019, 7 months 23, a peanut with a large number of root nodules and luxuriant growth is collected in the YangZhou city of Hainan province and the peanut planting land (E109.37N19.74H 3.19 meters), the overground part of the peanut is cut off, and the whole root system and the root nodules are put into a fresh-keeping bag and then are stored in an ice bag at low temperature. The root system of the peanut is taken out of the ice bag and washed under tap water for 2-3 times, the root system is washed clean, fresh and complete root nodule with dark red color is cut off by scissors with 2mm root, and the surface is washed clean by 2-grade water in a culture dish, so that the integrity of the root nodule surface is ensured in the process.

Transferring the cleaned root nodule into sterilized culture dish, soaking in 95% (volume concentration) alcohol for 3 min, pouring out alcohol, washing with sterile water for 4-5 times, adding 0.1% (mass volume concentration, 0.1g HgCl) ₂ 100mL of water) HgCl ₂ Sterilizing the aqueous solution for 2-3 minutes, and rapidly adding HgCl ₂ Pouring out and adding sterile water, transferring into ultra clean bench, washing with sterile water for more than 6 times, pouring out sterile water, selecting root nodule, transferring into sterilized culture dish (sterilized by flame), mashing root nodule with sterilized forceps and inoculating loop, dipping milky juice in prepared YMA solid culture medium (mannitol 10g, mgSO 4.7H) ₂ O0.2 g, naCl 0.1g, yeast powder 3g, K ₂ HPO ₄ 0.25g、KH ₂ PO ₄ 0.25g、CaCO ₃ 3g (added during storage), agar 15 g) were streaked. Transferring the scratched plate into a fresh-keeping bag, and culturing in an incubator (28+ -1deg.C in darkness) in an inverted manner. 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.

The determination of whether rhizobia is present during the cultivation is made from the following 3 points:

(1) Colony morphology: the colony water sample in the initial growth stage of rhizobia is semitransparent or white opaque spots, and the colony in the later stage is round, milky white, semitransparent, neat in edge and more or less in viscosity. The bacterial colony diameter reaches 2-4 mm after 2-4 days of culture, and the bacterial colony is 1mm after 5-10 days of culture.

(2) Cell morphology: 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 (0.5-0.9) × (1.2-0.3) 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.

(3) By inoculating isolated rhizobia back onto peanut under aseptic conditions, rhizobia can form nodules.

A strain isolated by the above culture process was initially identified as Rhizobium and designated as Pyricularia No. 9 (RY 9). The bacterial colony at the initial stage of rhizobia growth is a milky and semitransparent dot, and the bacterial colony at the later stage is round, milky, semitransparent, neat in edge and high in viscosity. The colony is cultured for 4 days and 2-3mm, the colony can grow to be full of the culture medium until 8 days, and a large amount of mucus exists around the colony, and the colony morphology is shown in figure 1.

The optimal growth conditions for RY9 were determined by culture: the temperature is 30 ℃ and the rotating speed is 180r/min. Can be widely applied to carbon sources and nitrogen sources, can well grow on the comprehensive extract liquid of various plant sources, and can well grow on YMA culture medium than peptone culture medium.

Gram staining showed that rhizobia RY9 appears to be a purple shape under the microscope as a rod and gram negative as shown in fig. 2.

Tieback verification of rhizobia arachidis hypogaeae: washing the separated and purified root nodule RY9 with sterile water into prepared liquid YMA medium (prepared by removing agar and adjusting pH to 6.0 based on the formula of YMA solid medium), sealing with sealing film, shaking in shaking table at 180r/min for 3 days, and measuring OD of the bacterial solution ₆₀₀ Value, when OD ₆₀₀ When the content of the bacterial strain is more than 0.6 (namely, the bacterial content per mL of bacterial solution is more than 1.0X10) ⁹ And 3) obtaining bacterial liquid which can be used.

Preparing sterile seedlings: selecting peanut seed, soaking in 95% ethanol for 5min, taking out, and adding 0.2% HgCl ₂ After 1 minute, washing 5-10 times with sterile water, then discharging in a sterilized culture dish with germinated paper, soaking the germinated paper with sterilized 0.05mmol/L calcium sulfate solution, and placing in a incubator at 28 ℃ for 3 days in darkness. Transplanting and tieback when the seedlings grow to 5 cm.

Placing the strong sterile seedlings in a culture dish, soaking the seedlings in the prepared bacterial solution for 15 minutes, planting the seedlings in small flowerpots filled with sterile sand by forceps, planting 4 plants in a diamond shape in each pot, and adding the root of each seedling5mL of bacteria liquid is added to ensure that the inoculation quantity of each seedling reaches 1.0x10 ⁹ More than one. After about four weeks, the seedlings were pulled up to see if nodulation, which is rhizobia.

The tieback results are shown in FIG. 3, which shows that the tieback RY9 shows obvious nodulation after tieback test, and can prove that the isolate is pure rhizobia.

Example 2 identification of Rhizobium RY9

To determine the phylogenetic status of rhizobia RY9, the 16S DNA series of the isolated strain was sequenced. First, the DNA of the strain was extracted and PCR-specific amplification was performed using primers, upstream primer 27F:5-AGAGTTTGATCCTGGCTCAG-3; downstream primer 1492R:5-CTACGGCTACC TTGTTACGA-3. The amplification reaction system is as follows: 10 Xreaction Buffer 5.0. Mu. L, dNTPs (10 mM) 1.0. Mu.L, 27F (10. Mu.M) primer 1.5. Mu.L, 1492R (10. Mu.M) primer 1.5. Mu.L, taq DNA polymerase (5U/. Mu.L) 1.0. Mu.L, genomic DNA (20 ng/. Mu.L) 1.0. Mu.L, and ultra-pure water 39. Mu.L. The reaction procedure is: pre-denaturation at 95℃for 5min; denaturation at 95℃for 30s, annealing at 58℃for 30s,35 cycles; extending at 72 ℃ for 90s; finally, the extension is carried out for 7min at 72 ℃.

Taking 3 mu L of amplified product, checking on 1.0% agarose gel electrophoresis, and as shown in figure 4, wherein the amplified band is bright and has NO dragging band, the amplified 16S rDNA is purer 16S rDNA with the fragment size of about 2000bp, and the product is sent to Shenzhen micro-co Union technology group Co., ltd for sequencing, and the sequence of RY9 strain is obtained by sequencing as shown in SEQ ID NO: 1.

The sequence results obtained were aligned in the U.S. National Center for Biological Information (NCBI), and found that rhizobia RY9 was 99.63% similar to the known strain (NR 117945.1) Bradyrhizobium huanghuaihaiense Strain CCBAU23303, the NCBI alignment was as shown in FIG. 5, and then subjected to an adjacent method (Neighbor-cloning) analysis using the Kimura-2 parameter using Mega4.0 software, 1000 replicates, and a phylogenetic tree was generated, as shown in FIG. 6. From the above pair of results and cluster map, it was found that RY9 is a new strain of Bradyrhizobium sp. Chrysotium strain (Bradyrhizobium huanghuaihaiense), which has been deposited at the university of armed university chinese classical culture collection (CCTCC) at 28 th year 2021 under the classification designation Bradyrhizobium huanghuaihaiense RY and has a deposit number of CCTCC NO: m2021481, the preservation address is No. 299 of Wuchang district of Wuhan, hubei province.

EXAMPLE 3 Effect of Rhizobium RY9

To confirm the effect of the RY9 strain on peanuts, this example uses sand culture to tie rhizobium RY9 back to the peanut root, 4 replicates per treatment (see tie-back experiments in example 1 for specific bacterial, sand and seedling treatments), with no tie-back as a Control (CK), and low nitrogen nutrient solution was irrigated throughout. And (5) counting the influence on the physiological index of the peanut after the rhizobium RY9 is tixed.

The results show that: as can be seen from fig. 7, the biomass of the aerial parts of the peanuts is significantly increased after the inoculation of the RY9 strain, which indicates that the growth of the peanuts can be significantly promoted after the inoculation of the RY9 strain; as can be seen in fig. 8, the yield was significantly increased after peanut inoculation with RY9, both with less inoculated strain and with other strains; as can be seen in FIG. 9, the fresh weight of the root nodule after peanut inoculation with RY9 is significantly higher than that of other treatments, indicating that RY9 can increase the nodulation of the peanut, thereby promoting peanut growth. In peanut cultivation, yield is the final indicator for measuring high yield. Compared with the control, the biomass of the peanut inoculated with the rhizobium RY9 is improved by 41.5%, and the peanut yield is increased by 62.2%, so that the rhizobium RY9 is a high-efficiency matched strain of the peanut, and the peanut yield can be remarkably improved after inoculation.

To further determine the growth-promoting ability of peanuts, the ability of RY9 to secrete auxin was measured by a colorimetric method, and rhizobium strains H1 and H2 were used as control groups, and as shown in FIG. 10, it was found that the ability of RY9 to secrete auxin was significantly higher than that of other strains.

In conclusion, the rhizobia RY9 provided by the invention has the characteristics of strong capability of secreting auxin in samples collected in a region where peanuts are planted in the south, high yield of the peanuts, high number of nodulation, large nodulation volume and strong stress resistance after the peanuts are inoculated with the rhizobia, and high yield of the peanuts. 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 abundance of rhizobia in soil, and have the effect of promoting nodulation and nitrogen fixation of peanuts.

Example 4 adaptability of Rhizobium RY9

Rhizobia inoculation is greatly affected by environmental conditions, and factors which have the greatest effect on rhizobia in southern soil are soil pH, salt content and aluminum ion concentration, so that the adaptability of RY9 to the main soil factors needs to be determined before application.

1. Effect of pH on RY9 growth

Through 10 mL.L ^-1 HCl and 10 g.L ^-1 The pH value of the culture medium is regulated by NaOH, solid culture media with pH values of 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0 are respectively prepared, the activated strain is inoculated on the solid culture medium, and the concentration of the liquid culture medium is determined according to the growth condition of the strain on the solid culture medium. YMA liquid media at pH 3.5, 4.5, 5.5, 6.0, 7.0, 8.0 were prepared separately, 50mL per bottle, 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 the temperature of 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 and continuously measured for 7 days.

The growth of strain RY9 in medium of different pH values is shown in FIG. 11, and it can be seen from the graph that strain RY9 is suitable for growth in the pH range of 5.5-7.0, and the growth is best at pH of 6.0.

2. Effect of NaCl content on RY9 growth

Preparation of NaCl concentration of 0, 0.2, 0.4, 0.6mol.L ^-1 4 replicates of YMA broth of (C). 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 the temperature of 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 and continuously measured for 7 days.

As shown in FIG. 12, the growth of the strain RY9 in the medium with different concentrations of NaCl, the sensitivity of the strain RY9 to NaCl and the sensitivity thereof were found, and the strain RY9 could not normally grow in the medium with the NaCl concentration higher than 0.2 mol/L.

3、Al ³⁺ Effect of concentration on RY9 growth

With AlCl ₃ The concentration of the preparation is 0, 25, 100 and 200 mu mol.L ^-1 The pH value of the YMA liquid culture medium is 4.5, 1mL of each strain after activation is added into each treated culture solution respectively for 4 times, the culture is carried out in a shaking table with the temperature of 28 ℃ and the speed of 180r/min, the absorbance of the bacterial liquid at the wavelength of 600nm is measured after 12 hours, and then the bacterial liquid is measured every 12 hours for 7 days continuously.

Strain RY9 at different Al ³⁺ The growth in the medium at the concentration is shown in FIG. 13, from which it can be seen that strain RY9 is subjected to Al ³⁺ The concentration has small influence and exceeds 100 mu mol.L ^-1 It affects its growth.

In summary, strain RY9 is suitable for application to a pH of 5.5-7.0, naCl concentration not higher than 0.2mol/L, al ³⁺ The concentration is not more than 100 mu mol.L ^-1 In the environment.

EXAMPLE 5 rhizobium RY9 microbial inoculum and application

The RY9 strain is taken and subjected to seed dressing treatment after shaking, or is poured into the root of the peanut which is just planted by a liquid microbial inoculum, or is mixed with a mixed microbial adsorbent material, such as turf (10 mL of microbial inoculum/50 g of turf), vermiculite (10 mL of microbial inoculum/15 g of vermiculite), perlite (10 mL of microbial inoculum/15 g of perlite) and other adsorbents to prepare microbial inoculum, and the microbial inoculum is mixed and applied into soil before transplanting. The prepared RY9 turf inoculant was applied to soil and its effect on peanut yield was determined.

According to the tieback test in example 1, sterile seeds and seedlings were prepared, rhizobium was drawn from a solid medium into a triangular flask for cultivation at 28℃for 120 hours, diluted with sterile water and homogenized with a vortex spinner to give an OD (λ=600 nm) of 0.9, ensuring a bacterial content of greater than 10 per seed at field inoculation ⁹ And each. Mixing turf 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, the bacterial liquid is poured into the adsorbent according to the proportion of 40mL/100g, and the bacterial liquid and the adsorbent are uniformly mixed. Seed dressing with a microbial inoculum before sowing. With no inoculation as control, treatment with inoculation RY9, 4 replicates, random block design, 4×2.5=10m per cell area ² Ridging cultivation is carried out, and the sowing density is 20cm multiplied by 10cm (row spacing multiplied by plant spacing). Sowing adopts a hole sowing mode, wherein each hole sowing is used for 2 grains, and the seeds becomeAfter maturation the yield was determined.

As shown in fig. 14, it can be seen that the peanut yield after the application of the microbial inoculum in the soil was significantly higher than that without rhizobia inoculation by 28.97%, showing that the peanut yield after the inoculation of RY9 was significantly improved.

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. Peanut nodule nitrogen-fixing Huang Huai sea slow rooting tumor bacteria (Bradyrhizobium huanghuaihaiense) RY9 strain, which is characterized in that the strain is preserved in China Center for Type Culture Collection (CCTCC) in 2021, 4 months and 28 days, and the preservation number is CCTCC NO: m2021481.

2. The application of the Huang-Huai-Hai slow rooting tumor bacteria RY9 in peanut nodulation and nitrogen fixation.

3. The use of the Huang-Huai-Hai slow rooting tumor bacteria RY9 or bacterial liquid thereof in promoting peanut growth or promoting peanut growth under the condition of aluminum acid.

4. The use of Huang-Huai-Hai slow rooting tumor bacteria RY9 or its bacterial liquid in preparing peanut growth promoting and/or nodulation nitrogen fixing bacteria agent.

5. A peanut growth-promoting and/or nodulation-fixing microbial inoculum is characterized by comprising the Huang-Huai-Hai slow rooting tumor bacterium RY9 or bacterial liquid thereof according to claim 1.

6. The microbial inoculum according to claim 5, wherein the concentration of the Rhizobium flavum RY9 or bacterial liquid thereof is not lower than 1.0X10 ⁹ CFU/mL。

7. The microbial inoculant according to claim 5, wherein the microbial inoculant comprises a mixed cell adsorbent: turf, vermiculite and perlite.

8. A method for promoting peanut growth and/or nodulation and fixing nitrogen is characterized in that the Huang-Huai-Hai slow rooting tumor bacteria RY9 or bacterial liquid thereof in claim 1 is adopted for seed dressing treatment or is directly applied to peanut roots or soil environment.

9. The method of claim 8, wherein the soil environment is a soil under aluminum acid conditions.

10. The method according to claim 9, wherein the soil environment has a pH of 5.5-7.0, naCl concentration of less than 0.2mol/L, al ³⁺ The concentration is lower than 100 mu mol.L ^-1 。

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