Peanut bradyrhizobium WYCCWR15459 as well as culture method and application thereof

Abstract

The invention belongs to the field of microorganisms, and relates to rhizobium, in particular to bradyrhizobium anhuoshanense. The classification name of the strain is Bradyhizolium zhenyangense, the strain is preserved in the Guangdong Microbial Culture Collection Center on October 10, 2024, the preservation number is GDMCC No: 1.4969, and the preservation address is the 5th floor of the Experimental Building, No. 100 Court, Middle Xianlie Road, Guangzhou District, Guangdong Province. The bacterial strain can greatly improve the symbiotic efficiency of peanuts and promote the growth of peanut plants in the seedling raising process of aseptic potted peanut seedlings, the number of plant nodules of a peanut culture group treated by the bradyrhizobium is 115 per plant, the chlorophyll content is increased by 100.36%, the total dry weight is increased by 192.63%, the effect is remarkable, and the method is suitable for industrial production. The microbial inoculum can be used as a peanut growth-promoting efficient microbial inoculum.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms, relates to rhizobia, and particularly refers to a strain of Bradyrhizobium arachidis. Background Art

[0002] Peanut ( Arachis hypogaea L.) originated in South America and is an important oilseed cash crop. It ranks third among the world's top five oil crops due to its high oil content. Peanut is the oilseed crop with the highest yield potential per unit area and is planted throughout China, which is divided into 7 peanut production areas. Among them, the Northeast Peanut Production Area, the Yellow River Basin Peanut Production Area, the Yangtze River Basin Peanut Production Area, and the Southern Peanut Production Area are the main peanut production areas. Most of Henan Province is located in the Yellow River Basin Peanut Production Area, and its peanut planting is further divided into four areas, namely the southern Henan production area, the eastern Henan production area, the central Henan production area, and the northern Henan production area. In the past two years, the peanut planting area in Henan Province has ranked first in the country, all above 22 million mu. Peanut is an important oilseed crop. Vigorously developing peanut production is of great significance in China's agriculture. In recent years, due to the abuse of chemical fertilizers and pesticides and unscientific fertilization methods, the yield and quality of leguminous plants have greatly declined, and at the same time, problems such as soil compaction and acid-base imbalance have emerged. Biological nitrogen fixation is the natural process of converting atmospheric nitrogen into ammonia, which can be achieved by free-living, associative, endophytic, and symbiotic rhizosphere bacteria, collectively referred to as rhizobia. They form a symbiotic relationship with leguminous plants, where the plant host provides nutrients to the rhizobia, and the rhizobia provide fixed atmospheric nitrogen to the host in the form of ammonia. Eventually, it can achieve the purposes of promoting crop growth, reducing crop diseases, and improving crop yield and quality.

[0003] A large number of studies have shown that rhizobial inoculants have been applied in the cultivation of leguminous crops such as soybeans, Chinese milk vetch, and alfalfa. Since rhizobia can infect the root hairs of host plants to form root nodules in the roots, and then synthesize nitrogen in the air into ammonium through symbiotic nitrogen fixation and supply the nitrogen demand of the host, significantly improving the yield and other growth indicators of the host, achieving the goal of replacing chemical nitrogen fertilizers, which is a green ecological planting mode. Peanut rhizobia can form symbiotic nodules with the roots of peanut plants, increase the biomass of the plants, and achieve the purpose of increasing production and replacing chemical nitrogen fertilizers. At the same time, research has also shown that there are significant differences in the composition of symbiotic rhizobia of different leguminous plant hosts, that is, there is a certain matching and specificity between rhizobia and hosts for nodulation. Generally, peanut rhizobia do not form root nodules with soybeans, Chinese milk vetch, alfalfa, etc., and the symbiotic rhizobia of soybeans, Chinese milk vetch, alfalfa, etc. will not form root nodules with peanuts either. Therefore, each leguminous plant has its matching rhizobial strain, and it is necessary to classify and screen the strains according to local conditions. At the same time, due to the variability of the planting environment of leguminous plants, in order to select rhizobia suitable for different soil environments, in addition to considering the strong nitrogen fixation ability of rhizobia during seed selection, their traits such as salt tolerance, drought tolerance, and high temperature tolerance should also be taken into account. The application with the publication number CN116731888A discloses a Bradyrhizobium arachidis strain ZB15, which has the ability to produce high yields of IAA, but its performance is greatly reduced under nicotine stress and high temperature conditions. In order to further screen excellent rhizobia, this study has conducted in-depth exploration. Summary of the Invention

[0004] The present invention provides a Bradyrhizobium arachidis WYCCWR15459, its cultivation method and application, to solve the technical problem that the growth promotion effect of existing strains becomes worse under acidic and alkaline, high temperature, salt, and drought conditions.

[0005] The technical solution of the present invention is realized as follows: A Bradyrhizobium arachidis WYCCWR15459, whose taxonomic name is Bradyrhizobium zhengyangense and was deposited at the Guangdong Provincial Culture Collection of Microorganisms on October 10, 2024, with the deposit number GDMCC No: 1.4969 and the deposit address being the 5th floor of the Experimental Building, No. 100 Compound, Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province.

[0006] The cultivation method of the above-mentioned Bradyrhizobium arachidis WYCCWR15459 is to inoculate Bradyrhizobium arachidis WYCCWR15459 on a YMA solid medium and incubate it at a constant temperature of 28 °C for 5 - 7 d. Then pick single colonies into a YMA liquid medium and culture them in a constant temperature shaker at 180 rpm and 28 °C until the logarithmic growth phase, and adjust the OD of the rhizobial liquid 600 = 0.8.

[0007] A bacterial agent containing the above-mentioned Bradyrhizobium arachidis WYCCWR15459.

[0008] The application of the above-mentioned bacterial agent as a peanut symbiotic bacterial agent.

[0009] The application of the above-mentioned bacterial agent in increasing the nodulation number of peanut plants.

[0010] The application of the above-mentioned bacterial agent in promoting the growth of peanut plants.

[0011] The application of the above-mentioned bacterial agent in promoting plant growth under stress conditions.

[0012] The above-mentioned stress is selected from one or more of the following stresses: ① Acid stress with pH = 6; ② Alkaline stress with pH = 11; ③ High salt stress containing 2% NaCl; ④ High temperature stress at 45 °C; ⑤ Drought stress.

[0013] The steps of the above-mentioned application are as follows: Dissolve the bacterial agent in water to prepare a bacterial solution, and then immerse the peanut seeds that have been cleaned and disinfected in the bacterial solution, and conduct dark cultivation until rooting and then plant.

[0014] The concentration of Bradyrhizobium arachidis WYCCWR15459 in the above-mentioned bacterial solution is (8 - 10) × 10 8 CFU / mL; The conditions for dark cultivation are to cultivate at 28 °C for 3 - 4 days.

[0015] The present invention has the following beneficial effects: 1. The present invention discloses a strain of Bradyrhizobium arachidis Bradyrhizobium sp. WYCCWR 15459 = WYCCWR15459. In addition to the unique nitrogen fixation ability of rhizobia, this strain of rhizobia also has the characteristics of acid and alkali resistance (pH 6 - 11), high temperature resistance (45 °C), salt tolerance (2% NaCl), and drought tolerance (10% PEG).

[0016] 2. During the seedling raising process of peanut sterile potted seedlings, the strain of the present application can greatly improve the symbiotic efficiency of peanuts, promote the growth of peanut plants. The number of nodules of peanut plants in the culture group treated with the slow-growing rhizobia of the present application is 115 nodules / plant, the chlorophyll content increases by 100.36%, and the total dry weight increases by 192.63%. The effect is remarkable, and it can be used as an efficient bacterial agent for promoting the growth of peanuts. Description of the Drawings

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0018] Figure 1 It is the phylogenetic tree of the 16S rRNA sequence of Rhizobium WYCCWR15459.

[0019] Figure 2 It is the plate map of Rhizobium WYCCWR15459.

[0020] Figure 3 It is the microscope picture of Rhizobium WYCCWR15459.

[0021] Figure 4 It is the plate growth situation of WYCCWR15459 under acid-base stress conditions.

[0022] Figure 5 It is the plate growth situation of WYCCWR15459 under salt stress conditions.

[0023] Figure 6 It is the plate growth situation of WYCCWR15459 under drought conditions. Specific embodiments

[0024] The following will clearly and completely describe the technical solutions of the present invention in combination with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0025] Unless otherwise specified, the test methods used in the following experimental examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0026] The present application provides a Bradyrhizobium arachidis WYCCWR15459, whose taxonomic name is Bradyrhizobium zhengyangense , which was deposited in the Guangdong Provincial Microbial Culture Collection Center on October 10, 2024, with the deposit number GDMCC No: 1.4969, and the deposit address is on the 5th floor of the Experimental Building, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

[0027] The above-mentioned cultivation method of Bradyrhizobium arachidis WYCCWR15459 is to inoculate Bradyrhizobium arachidis WYCCWR15459 on YMA solid medium and cultivate it at a constant temperature of 28 °C for 5 - 7 d. Then pick single colonies into YMA liquid medium and cultivate them in a constant temperature shaker at 180 rpm and 28 °C until the logarithmic growth phase, and adjust the OD 600 of the rhizobium bacterium solution

[0028] to 0.8.

[0029] A bacterial agent containing the above-mentioned Bradyrhizobium arachidis WYCCWR15459.

[0030] Application of the above-mentioned bacterial agent as a peanut symbiotic bacterial agent.

[0031] Application of the above-mentioned bacterial agent in increasing the nodulation number of peanut plants.

[0032] Application of the above-mentioned bacterial agent in promoting the growth of peanut plants under stress conditions.

[0033] The above-mentioned stress conditions are selected from one or more of the following stresses: ① Acidic stress with pH = 6; ② Alkaline stress with pH = 11; ③ High-salt stress containing 2% NaCl; ④ High-temperature stress at 45 °C; ⑤ Drought stress.

[0034] The above-mentioned application steps are as follows: Dissolve the bacterial agent in water to prepare a bacterial solution, and then immerse the peanut seeds that have been cleaned and disinfected in the bacterial solution and conduct dark cultivation until roots grow, and then plant them.

[0035] The concentration of Bradyrhizobium arachidis WYCCWR15459 in the above-mentioned bacterial solution is (8 - 10) × 10 8 CFU / mL; the conditions for dark cultivation are to cultivate at 28 °C for 3 - 4 days.

[0036] The specific experiments are as follows: Example 1: Collection of soil information and climate information in Xingyang, Henan Method for collecting soil samples and climate information: (1) Collect soil samples in the peanut planting areas of Henan Province, select the same county and district locations at each sampling point as replicates, collect soil at a depth of 10 - 20 cm at each sampling point, crush the collected soil samples to a uniform state, transport them back to the laboratory under low-temperature conditions, and record information such as the longitude, latitude and altitude of the sampling points.

[0037] (2)After air-drying the collected soil samples, they were crushed and sieved. Approximately 300 grams of each soil sample were collected, and then the samples were sent to Henan Bainxin Technology Testing Company to detect the organic matter content (OM), available nitrogen (AN), available phosphorus (AP), available potassium (AK), and soil pH value. Meanwhile, according to the longitude and latitude information of the sampling points, DIVA-GIS software was used to collect the climate information data of the sampling points, including the altitude, annual rainfall, and annual maximum and minimum temperatures of the sampling points. The results of the soil sample and climate information collection are shown in Table 1.

[0038] Table 1 Soil Samples and Climate Information in Xingyang, Henan As can be seen from Table 1, the soil in Xingyang, Henan is alkaline.

[0039] Example 2: Peanut Rhizobia Bradyrhizobium sp. Isolation and Screening of WYCCWR15459 Peanut seeds were treated with 95% ethanol by volume for 30 s under sterile conditions, disinfected with 0.2% mercuric chloride by mass for 5 min, and washed 7 times with sterile water. They were kept in a dark culture environment and cultivated at 28 °C until the seeds germinated. Vermiculite was intermittently sterilized twice with a plant low-nitrogen nutrient solution and then used for planting, adopting a planting mode of vermiculite + soil sample + vermiculite.

[0040] Peanuts were planted using a double-layer pot device. Sterile vermiculite was filled in the upper pot body and germinated seeds were transplanted. Water was stored in the lower pot body, and they were connected by a gauze strip in the middle to provide water for the plant growth. After planting, it was placed in an artificial climate incubator. After growing for 45 days, the peanut roots were rinsed clean, and the root nodules were removed with sterile forceps and disinfected with 0.2% mercuric chloride by mass for 30 s. The root nodules were mashed, and the liquid was aspirated and streaked in a "Z" shape on a YMA medium. It was incubated in the dark at a constant temperature of 28 °C for 5 - 7 days, and was purified by streaking 3 times until there were no miscellaneous colonies on the plate. The plate diagram is shown in Figure 2 and the microscope diagram is shown in Figure 3 A total of 30 strains of bacteria were obtained.

[0041] The DNA of the WYCCWR15459 strain was extracted using the GUTC method, and the extracted DNA was subjected to 16S rRNA PCR amplification. The results are as Figure 1 indicating that it belongs to Bradyrhizobium.

[0042] Example 3: Method for Applying the Above Peanut Bradyrhizobium WYCCWR15459 to Detect the Symbiotic Effect of Peanuts (1) Inoculate the strain of Bradyrhizobium arachidis in 5 mL of YMA liquid medium and culture it in a constant temperature shaker at 28 °C with a rotation speed of 180 rpm (OD600 = 0.8 - 1.0). Before planting peanut seedlings, adjust the OD600 of the bacterial solution to 0.8.

[0043] (2) Select peanut seeds (Yuanza 9102) with intact and uniform surfaces, add an appropriate amount of 95% ethanol and stir for 30 s. Add an appropriate amount of 0.2% mercuric chloride solution for disinfection treatment for 5 min. Finally, wash with sterile water 7 times. Place the seeds evenly on the surface of the water agar medium and incubate in the dark at 28 °C for 3 - 4 days until the root length of the seeds reaches 2 - 3 cm.

[0044] (3) Use a double-layer pot device for planting peanuts (after sterilizing the upper pot body, fill it with sterile vermiculite, store water in the lower pot body, and connect them with a gauze strip in the middle to provide necessary moisture for plant growth). Use sterile forceps to plant a normally germinated peanut seed at the position of the soil layer in the upper pot body with the root facing down. After planting, place it in an artificial climate incubator and set the culture parameters: 16 h of light at 25 °C and 8 h of darkness at 20 °C. When the seedling grows until the bud tip touches the sealing film, make a "cross"-shaped cut on the sealing film, and supplement water irregularly during this period.

[0045] (4) After 45 days of growth, carefully take out each plant and carefully wash off the vermiculite attached to the roots with clean water. Pay attention to avoiding damaging the root nodules and the integrity of the plant during the operation. Then measure the root length, use a portable chlorophyll meter to detect the chlorophyll content of the plant leaves, and finally dry the plant to a constant weight and measure its dry weight.

[0046] Use the conventional cultivation method without adding Bradyrhizobium bacterial solution as a control.

[0047] After treatment with Bradyrhizobium Bradyrhizobium sp. The statistical results of the symbiotic indexes of peanut plants treated with WYCCWR 15459 and the peanut plants in the control group after 45 days of cultivation are shown in Table 2: Table 2: Statistical data of the roots of Bradyrhizobium arachidis WYCCWR15459 and the control group As can be seen from Table 2, after using the Bradyrhizobium arachidis WYCCWR15459 of the present application, the number of nodules per plant is 115, the chlorophyll content increases by 100.36%, and the total dry weight increases by 192.63%, with significant effects.

[0048] Example 4: Stress resistance experiment of Bradyrhizobium arachidis WYCCWR15459 strain (1) Detection of acid and alkali tolerance of peanut rhizobia: Prepare YMA solid media with different pH values (5, 6, 7, 8, 9, 10, 11) and sterilize them. Use pH 7 as the control group. Inoculate the tested strains cultured on YMA plates with different pH values. Set 3 replicates for each treatment. After making marks, incubate them upside down in a constant temperature incubator at 28 °C for 5 - 7 d and then record the results. "+" indicates growth is possible, and "-" indicates no growth. (The judgment criterion is that colonies grow on more than two of the three plates). The results show that WYCCWR15459 can grow on the acidic stress plate with pH 6 and the alkaline stress plate with pH 11. The results are shown in Figure 4 .

[0049] (2) Salt tolerance detection of peanut rhizobia: Inoculate the tested strains on YMA plates containing different concentrations of NaCl. The concentration gradients of NaCl are set as 0.01%, 1%, 2%, 3%, and 4% in sequence. Use the YMA plate with 0.01% NaCl concentration as the control. Adopt the spot inoculation method and conduct 3 repeated experiments for each strain. Then place them in a constant temperature incubator at 28 °C for 5 - 7 d and record the results. "+" indicates growth is possible, and "-" indicates no growth. The results show that WYCCWR15459 can grow on the salt stress plate of NaCl. The results are shown in Figure 5 .

[0050] (3) Study on temperature tolerance of peanut rhizobia: Inoculate each rhizobia strain into YMA medium and YMA liquid medium and incubate them at a constant temperature at different temperatures. The temperatures are set at 4 gradients: 28 °C, 37 °C, 45 °C, and 60 °C. For 45 °C and 60 °C, use liquid YMA medium. After inoculating the tested strains into the liquid YMA medium, first heat shock them in a water bath at 45 °C and 60 °C for 10 min respectively, and then place them in a constant temperature incubator at 28 °C for cultivation. For the other temperature gradients, use solid YMA plates. For the 28 °C and 37 °C treatments, directly place the inoculated plates upside down in a constant temperature incubator at the corresponding temperature for cultivation. Use the YMA plate treated at 28 °C as the positive control. Set 3 replicates for each treatment. Then place them in an incubator at 28 °C for 5 - 7 d and record the results. When recording the results for the liquid test tubes treated at 45 °C and 60 °C, observe their turbidity and measure the OD600 value. Use the YMA liquid test tube without inoculation as the blank control to identify whether it can grow at this temperature. "+" indicates growth is possible, and "-" indicates the strain does not grow, that is, 3 strains do not grow among the 3 replicates. The results show that WYCCWR15459 can grow after heat shock at 45 °C. See Table 3.

[0051] Table 3 Detection of high temperature tolerance of Bradyrhizobium sp. WYCCWR15459 at 45 °C (4)Research on drought tolerance of peanut rhizobia: Artificial drought conditions were simulated using polyethylene glycol 6000 (PEG 6000) to determine the drought tolerance of rhizobia. Different amounts of PEG were added to the YMA medium to make the final concentration of PEG in the culture solution (W / V was 0%, 3%, 5%, 7%, 10% and 15% in turn). The test strains were inoculated on YMA plates with different gradients, and then placed in a constant temperature incubator at 28 °C for 5-7 d, and the experimental results were recorded. "+" indicates that it can grow, and "-" indicates that it cannot grow. The results showed that the strain WYCCWR15459 could grow on the drought-resistant plate with 10% PEG6000 content. The results are shown in Figure 6 .

[0052] In summary, the peanut rhizobia WYCCWR15459 has characteristics in acid stress at pH = 6; alkaline stress at pH = 11; high salt stress containing 2% NaCl; high temperature stress at 45 °C; drought stress; the stress resistance detection results of peanut rhizobia are shown in Table 4.

[0053] Table 4: Statistical data of stress resistance results of Bradyrhizobium sp. WYCCWR15459 As can be seen from Table 4, the Bradyrhizobium sp. WYCCWR15459 of the present application, in addition to having the unique nitrogen fixation ability of rhizobia, also has acid-base tolerance (pH 6-11), high temperature tolerance (45 °C), salt tolerance (2% NaCl) and drought tolerance (10% PEG).

[0054] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Bradyrhizobium sp. WYCCWR15459, with the taxonomic name of Bradyrhizobium zhengyangense , was deposited at the Guangdong Microbial Culture Collection Center on October 10, 2024, with the deposit number GDMCC No: 1.4969 and the deposit address at the 5th floor of the Experimental Building, No. 100 Compound, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

2. The cultivation method of Bradyrhizobium sp. WYCCWR15459 as claimed in claim 1, characterized in that: Inoculate Bradyrhizobium arachidis WYCCWR15459 on YMA solid medium and culture it at a constant temperature of 28 °C for 5 - 7 d. Pick single colonies into YMA liquid medium and culture them in a constant temperature shaker at 180 rpm and 28 °C until the logarithmic growth phase, and adjust the OD of the rhizobium bacterium solution 600 = 0.

8.

3. A bacterial agent containing Bradyrhizobium arachidis WYCCWR15459 as described in claim 1.

4. Use of the bacterial agent as described in claim 3 as a symbiotic bacterial agent for peanuts.

5. Use of the bacterial agent as described in claim 3 in increasing the nodulation number of peanut plants.

6. Use of the bacterial agent as described in claim 3 in promoting the growth of peanut plants.

7. Use of the bacterial agent as described in claim 3 in promoting plant growth under stress conditions.

8. The application according to claim 7, characterized in that, The stress conditions are selected from one or more of the following stresses: ① Acid stress with pH = 6; ② Alkaline stress with pH = 11; ③ High salt stress containing 2% NaCl; ④ High temperature stress at 45°C; ⑤ Drought stress.

9. The application according to any one of claims 3-8, characterized in that: Dissolve the bacterial agent in water to prepare a bacterial solution, and then immerse the peanut seeds that have been cleaned and disinfected in the bacterial solution, and conduct dark cultivation until roots grow and then plant them.

10. The application according to claim 9, wherein: The concentration of Bradyrhizobium arachidis WYCCWR15459 in the bacterial liquid is (8 - 10) × 10 8 CFU / mL; the conditions for dark cultivation are to cultivate at 28 °C for 3 - 4 days.

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