Application of bradyrhizobium japonicum

Abstract

The invention provides the application of the bradyrhizobium japonicum, the bradyrhizobium japonicum is used for promoting the growth of soybeans and increasing the yield, and the bradyrhizobium japonicum especially has a remarkable effect on a soybean variety 87 planted in the Xinjiang region. The bradyrhizobium japonicum disclosed by the invention has the characteristics of efficient nodulation, high nodulation quantity significance, strong oxidation resistance and broad-spectrum antibiotic tolerance, solves the problems of poor environmental adaptability, low nodulation rate, unstable inoculation effect and the like of a traditional nodule inoculant, and can adapt to soybean varieties planted in various regions, especially in the Xinjiang region, so that the bradyrhizobium japonicum has a wide application prospect. The bacillus subtilis can be effectively used for promoting the growth and stress resistance of soybean crops, and plays an important role in remarkably improving the field soybean yield.

Description

Technical Field

[0001] The invention belongs to the technical field of soybean rhizobia, and particularly relates to application of slow-growing soybean rhizobium Bradyrhizobium japonicum. Background Art

[0002] Soybean (Glycine max (L.) Merrill) is an annual herbaceous plant of the genus Glycine in the Leguminosae family, 30-90 cm tall. Soybean is the fifth largest food crop after corn, rice, wheat, and potato. It is not only the main source of protein and edible oil in people's lives, but also a raw material for animal husbandry feed, food additives, medicine and chemical industries. Therefore, promoting the sustainable development of the soybean industry is of great significance to ensuring national economic growth and food and oil security.

[0003] The inoculation of rhizobia is often restricted by many factors. First, from the perspective of rhizobia resources, rhizobia strains need to have broad-spectrum hosts, strong stress resistance, broad-spectrum antibiotic tolerance, etc. as excellent strains to lay the foundation for the research and development of microbial agents. Secondly, rhizobia are easily affected by a variety of environmental stresses after inoculation. Environmental stress affects the growth and survival of rhizobia, thereby affecting the signal transduction, infection, nodulation and symbiotic nitrogen fixation between rhizobia and soybean roots. Some known rhizobia with tolerance to biotic and abiotic stresses can be used as inoculated microbial agents to greatly improve the ability of biological nitrogen fixation (Atieno et al., 2019). For example, environmental factors such as salinity, temperature, pH, drought, and heavy metal ions are important factors that affect the infection process of rhizobia-legume symbiotic nitrogen fixation, the development and function of nodules, and the low yield of crops with low nitrogen fixation (Lebrazi et al., 2014). In addition, current rhizobium agents often have problems such as poor environmental adaptability, low nodule occupancy rate and unstable inoculation effect, which restrict their application and development. The main reason is that rhizobium strain resources do not have the advantage of greater adaptability. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide an application of a slow-growing soybean rhizobium Bradyrhizobium japonicum in view of the deficiencies of the above-mentioned prior art. The strain has the characteristics of efficient nodulation, strong antioxidant ability and broad-spectrum antibiotic tolerance. It makes up for the problems of poor environmental adaptability, low nodule occupancy rate and unstable inoculation effect of traditional rhizobia agents, can be adapted to soybean varieties in various regions, and can be used to promote the growth and yield of soybeans.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is: an application of a slow-growing soybean rhizobium Bradyrhizobium japonicum, wherein the slow-growing soybean rhizobium Bradyrhizobium japonicum is used to promote the growth of soybeans and increase the soybean yield.

[0006] Preferably, the bradyrhizobium japonicum is used to increase the number of soybean pods, pod dry weight, number of nodules, and nodule dry weight; and to increase the number of soybean pods, number of grains per plant, grain weight per plant, and yield.

[0007] Preferably, the bradyrhizobium japonicum is used to improve the stress resistance of soybean.

[0008] Preferably, the stress resistance includes resistance to antibiotics and resistance to oxidative stress.

[0009] Preferably, the antibiotic is gentamicin, ampicillin, chloramphenicol, tetracycline, streptomycin, spectinomycin, neomycin, or rifampicin.

[0010] Preferably, the soybean variety is Heinong 87.

[0011] Compared with the prior art, the present invention has the following advantages:

[0012] 1. The slow-growing soybean rhizobium Bradyrhizobium japonicum of the present invention has the characteristics of efficient nodulation, strong antioxidant ability, and broad-spectrum antibiotic tolerance. It makes up for the problems of poor environmental adaptability, low tumor occupancy rate, and unstable inoculation effect of traditional rhizobium agents. It can be adapted to soybean varieties in various regions and can be used to promote soybean growth and increase yield.

[0013] 2. The slow-growing soybean rhizobium Bradyrhizobium japonicum of the present invention can effectively nodulate the Xinjiang soybean variety Heinong 87. Under the same fertility conditions, the inoculation of rhizobia can effectively promote the physiological growth of soybean flowering and pod stage, dry matter accumulation, and grain development. The growth performance and soybean yield of the soybean variety Heinong 87 inoculated with the slow-growing soybean rhizobium Bradyrhizobium japonicum are better than those inoculated with the local indigenous rhizobia ZLBR and ZLSR. It not only lays the foundation for improving the poor environmental adaptability of rhizobia agents, low nodule rate and unstable inoculation effect, but also has important significance for promoting the high-quality development of soybean industry agriculture and the sustainable development of the ecological environment.

[0014] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a diagram showing the soybean nodulation effect obtained by different field test treatments of the present invention. DETAILED DESCRIPTION

[0016] Example 1

[0017] The application of the slow-growing soybean rhizobium Bradyrhizobium japonicum in this embodiment is used to promote the growth of soybeans. The slow-growing soybean rhizobium Bradyrhizobium japonicum is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, the address is No. 3, No. 1 Beichen Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, the deposit number is: CGMCC No. 16747; the deposit time is: November 15, 2018, and the relevant information on the isolation and identification of the strain is disclosed in Chinese patent CN109370956B, also known as strain 5038.

[0018] The slow-growing soybean rhizobium Bradyrhizobium japonicum is used to promote the growth of soybeans (increasing the number of soybean pods, pod dry weight, nodule number, nodule dry weight) and improve soybean yield (increasing the number of soybean pods, the number of single-plant grains, single-plant grain weight, and yield).

[0019] The bradyrhizobium japonicum is used to improve the stress resistance of soybeans. The stress resistance includes resistance to antibiotics and resistance to oxidative stress; the antibiotics are gentamicin, ampicillin, chloramphenicol, tetracycline, streptomycin, spectinomycin, neomycin and rifampicin.

[0020] 1. Host nodulation test of Bradyrhizobium japonicum (field test)

[0021] 1. Overview of the test site

[0022] The experiment was carried out from May to October 2024 at the Sanping Experimental Base in Urumqi, Xinjiang (E87.35893372, N43.93536151). The basic fertilizer of the experimental field is 4kg / mu of ammonium phosphate; 3kg / mu of potassium fertilizer; 3kg / mu of urea, evenly spread and plowed into the soil. The experimental site belongs to the mid-temperate continental semi-arid climate zone, alluvial flood plain, cold winter and hot summer, and the soil is planted with crops for a long time. The background value of the experimental field before sowing: soil organic matter 14.595g / kg, total nitrogen 0.447g / kg, pH7.28, conductivity 157.9us / cm, available potassium 237.115mg / kg, available phosphorus 13.842mg / kg, alkaline nitrogen 0.214g / kg.

[0023] 2. Test materials

[0024] The experimental soybean variety is Heinong 87, which has excellent quality and was jointly bred by the Soybean Research Institute of Heilongjiang Academy of Agricultural Sciences and the Institute of Tillage and Cultivation of Heilongjiang Academy of Agricultural Sciences.

[0025] There were three strains in total, one of which was the slow-growing soybean rhizobium Bradyrhizobium japonicum 5038 of this example, isolated from the nodules of soybean crops grown in Shandong; the other two were native strains isolated from cultivated soybeans in Xinjiang: Bradyrhizobium sp. ZLBR and Sinorhizobium sp. ZLSR.

[0026] 3. Test methods

[0027] Preparation of fermentation broth: Pick a single colony and inoculate it into a 250mL conical flask containing 100mL liquid YMB medium. Shake and culture at 180r / min, 30°C for 4 days until the late logarithmic growth phase. Centrifuge at 8000r / min for 10min, pour off the supernatant to collect the bacteria, and adjust the OD with sterile water. 600 = about 0.9, bacterial concentration is 10 9 CFU·mL -1 stand-by.

[0028] Seed treatment: The inoculation method is to mix the seeds with fermentation liquid, and the effective live bacteria count of the fermentation liquid is 0.5×10 8 cfu / mL, the number of viable bacteria on the surface of each seed is 10 5 -10 6 , mix 3mL of fermentation liquid for every kilogram of seeds.

[0029] 4. Experimental design

[0030] The experiment set up 4 treatments (CK, R5038, ZLBR and ZLSR), each with three replicates, and a total of 12 plots, arranged in random blocks. Each plot was 8m long, 5m wide and 40m in area. 2 , each treatment was separated by 1m protection row; the sowing pattern was one film and two rows, plant spacing was 30cm, film spacing was 55cm, hole spacing was 12cm, and 1-2 seeds were planted in each hole. No basal fertilizer was applied to each treatment, and the management measures were the same as general field management methods. The specific experimental treatments are shown in Table 1:

[0031] Table 1 Field plot design

[0032]

[0033]

[0034] 5. Plant measurement content, methods and statistical analysis

[0035] Collection and processing of plant samples: 6-8 representative plants were randomly selected in each treatment plot at the pod-setting stage (50 days), grain-filling stage (80 days) and maturity stage (115 days) of soybeans, and the plant height, stem thickness, root length, number of pods and soybean nodulation were measured. In addition to the above indicators, the number of grains per plant and the weight of grains per plant were measured at maturity. When measuring the dry weight of the plants, the plants were placed in an oven at 105°C for 30 minutes, then dried at 75°C to constant weight, and their dry weight was measured. The data results were statistically analyzed using Excel 2016 software, and one-way analysis of variance (ANOVA) was performed using SPASS27.0. The significance of the differences between different treatments was tested using LSD and Duncan's multiple test method (P < 0.05).

[0036] 6. Field test results of 5038 host nodulation experiment

[0037] From the test results in Table 2, Table 3, and Table 4, it can be seen that the number of pods, dry weight of pods, number of grains per plant, and weight of grains per plant of Heinong 87 after inoculation with rhizobia are significantly better than those of CK, ZLBR, and ZLSR inoculation treatments, and the inoculation effect is obvious. Compared with the inoculation effects of the two indigenous rhizobia, the number of nodules and dry weight of nodules of Bradyrhizobium japonicum 5038 are significantly better than those of the indigenous rhizobia inoculation effects.

[0038] At the pod-setting stage, the pod number and pod dry weight of the group inoculated with Bradyrhizobium japonicum 5038 (R5038) were significantly increased compared with the CK group, with the growth rates being 46.2% and 46.5%, respectively. In addition, compared with the treatment group inoculated with the indigenous bradyrhizobium ZLBR, the number of pods in the R5038 treatment group was significantly different, and the growth rates of the number of pods and the dry weight of pods were approximately 35.0% and 35.5%, respectively; the number of nodules and the dry weight of nodules both showed a significant increase, with growth rates of approximately 43.3% and 39.4%; compared with the indigenous fast-growing rhizobium ZLSR, the number of pods and the number of nodules of R5038 also showed a significant increasing trend (P < 0.05), the number of pods and the dry weight of pods increased significantly by approximately 10.3% and 25.4%, and the number of nodules and the dry weight of nodules increased significantly by approximately 4.8% and 25.0% (see Table 2 for details).

[0039] At the grain filling stage, the number of pods and dry weight of pods of Bradyrhizobium japonicum 5038 increased significantly compared with CK (P < 0.05), with an increase rate of about 34.7% and 15.9%; compared with the inoculation effect of ZLBR, the number of pods and dry weight of pods increased by about 16.3% and 4.9%; compared with the inoculation effect of ZLSR, the number of pods increased significantly by 6.1%; the number of nodules in all results showed significant differences, and the number of nodules of R5038 was extremely significantly different from that of ZLBR and ZLSR, with an increase rate of 89.3% and 81.3% respectively (see Table 3 for details). The dry weight of nodules of R5038 was significantly different from that of ZLBR and ZLSR, with an increase rate of 78.9% and 65.8% respectively (see Table 3 for details). In summary, whether in the pod-setting stage or the grain-filling stage, the nodulation effect of R5038 was significantly better than that of the two indigenous rhizobia, and the growth-promoting effect was obvious. Figure 1 shown.

[0040] The results of yield data analysis at maturity showed that compared with CK, the number of pods per plant, the number of grains per plant, the grain weight per plant and the yield data of R5038 were significantly different. The growth rates of the number of pods per plant and the number of grains per plant were both 37.5%, and the growth rates of the grain weight per plant and the yield were both about 46.1%. The growth-promoting effect was obvious and the yield was significantly increased. Compared with the indigenous slow-growing rhizobium ZLBR, all indicators of the R5038 treatment group increased significantly, the number of pods increased by about 17.7%, and the growth rates of the number of grains per plant, the grain weight per plant and the yield were about 12.1%, 12.2% and 12.2%. Compared with the indigenous fast-growing rhizobium ZLSR, the number of pods increased by about 5.2%, and the growth rates of the number of grains per plant, the grain weight per plant and the yield were about 3.9%, 9.4% and 9.4%. The analysis results of various indicators at maturity showed that the treatment groups that promoted the yield increase were R5038>ZLSR>ZLBR (see Table 4 for details)

[0041] Table 2 Related indexes of plants in different treatment groups in the experimental area during pod setting period

[0042] Processing area Number of pods per plant Plant pod dry weight (g) Number of plant nodules Plant tumor dry weight (g) CK <![CDATA[63±2 d ]]> <![CDATA[8.73±2.57 b ]]> - - R5038 <![CDATA[117±3 a ]]> <![CDATA[16.33±3.23 a ]]> <![CDATA[83±2 a ]]> <![CDATA[3.20±0.59 a ]]> ZL <![CDATA[76±3 c ]]> <![CDATA[10.54±2.97 ab ]]> <![CDATA[47±2 c ]]> <![CDATA[1.94±0.54 b ]]> ZLSR <![CDATA[105±3 b ]]> <![CDATA[12.19±4.48 ab ]]> <![CDATA[79±2 b ]]> <![CDATA[2.40±0.16 ab ]]>

[0043] Note: Lowercase letters indicate significant differences at the p<0.05 level, and “-” indicates blank treatment without inoculation and no nodulation.

[0044] Table 3 Related indices of plants in different treatment groups in the experimental area during the grain filling period

[0045]

[0046]

[0047] Note: Lowercase letters indicate significant differences at the p<0.05 level, and “-” indicates the blank control treatment without inoculation, with no nodulation.

[0048] Table 4 Related indexes of plants in the experimental area under different treatment groups at maturity

[0049] Processing area Number of pods Number of seeds per plant Single plant grain weight (g) Yield (kg / mu) CK <![CDATA[60±4 c ]]> <![CDATA[160±8 c ]]> <![CDATA[28.67±2.94 c ]]> <![CDATA[248.59±25.5 c ]]> R5038 <![CDATA[96±8 a ]]> <![CDATA[256±9 a ]]> <![CDATA[53.22±3.2 a ]]> <![CDATA[461.47±27.7 a ]]> ZL <![CDATA[79±8 b ]]> <![CDATA[225±10 b ]]> <![CDATA[46.72±3.66 b ]]> <![CDATA[405.11±31.7 b ]]> ZLSR <![CDATA[91±8 ab ]]> <![CDATA[246±10 a ]]> <![CDATA[48.24±2.96 ab ]]> <![CDATA[418.29±25.7 ab ]]>

[0050] Note: Lowercase letters indicate significant differences at the p<0.05 level.

[0051] 2. Experiment on the broad-spectrum antibiotic properties of the slow-growing soybean rhizobium Bradyrhizobium japonicum 5038.

[0052] Preparation of antibiotics: Antibiotics used and storage concentration (mg / mL): Kanamycin (Km / 50), Tetracycline (Tc / 10), Ampicillin (Amp / 100), Streptomycin (Sm / 50), Chloramphenicol (Cm / 30), Spectinomycin (Spe / 10), Gentamycin (Gm / 50), Clindamycin (Cm / 50), Neomycin (Neo / 100). Sterilize by suction through a 0.22 μm microporous filter and store at -20°C.

[0053] YMA solid medium with 9 antibiotics (Km, Gm, Amp, Cm, Tc, Sm, Spe, Neo, Rif) was prepared respectively, and the concentration gradient was set to: 5, 10, 25, 50, 100, 150 μg / mL. B. japonicum 5038 cultured to the logarithmic phase was spread on the solid medium and cultured at 30°C for 5-7 days. The growth of the colonies was observed to determine their antibiotic resistance level. The medium without antibiotics was used as the positive control, and 3 replicates were set for each treatment. The experimental results of the broad-spectrum antibiotic characteristics of Bradyrhizobium japonicum 5038 can be seen from Table 4 that strain R 5038 has a wide range of antibiotic tolerance, is resistant to Gm, Amp, Cm, Tc, Sm, Spe, Neo and Rif, and is sensitive to Km (>50 μg / mL).

[0054] Table 4 Antibiotic tolerance levels of Bradyrhizobium japonicum 5038

[0055]

[0056] Note: “+” means the strain can tolerate it, and the greater the number, the stronger the tolerance; “-” means it is intolerant.

[0057] The slow-growing soybean rhizobium Bradyrhizobium japonicum in this embodiment is used to improve the stress resistance of soybeans, and has broad-spectrum tolerance to antibiotics, including gentamicin, ampicillin, chloramphenicol, tetracycline, streptomycin, spectinomycin, neomycin, and rifampicin.

[0058] Screening of rhizobia with broad-spectrum antibiotic tolerance indicates that they have strong antibiotic resistance, which helps rhizobia increase the resistance of plants to soil pathogens. They survive and reproduce in the environment, so they can be used for environmental restoration and improve the stability of field inoculation effects, and have broad application prospects.

[0059] 3. Experiment on the antioxidant capacity characteristics of slow-growing soybean rhizobium Bradyrhizobium japonicum5038.

[0060] Test method: Centrifuge the bacteria at 10000r / min, 4℃ for 10min, discard the supernatant and wash the bacteria twice with PBS. Use sterile water to prepare the concentration of bacterial suspension to OD 600 =1.0, take 100 μL and spread on YMA plate. Then place a 5 mm diameter sterile filter in the middle of the plate, and spot 5 μL of 3% (v / v) H2O2 on the filter. Incubate at 30°C for 7 days and observe and compare the size of the inhibition zone. For detailed methods, refer to the method described by Noh et al.

[0061] In addition to Bradyrhizobium japonicum 5038, the other strains were used for control experiments in this embodiment. The other strains were collected in the laboratory and isolated from the root nodules of different soybean varieties planted in other regions, specifically as follows: Meng 9793-1 (strain 4253) of soybean varieties planted in Anhui Province in East China, Heilongjiang Province in Northeast China, Heilongjiang Province in Northeast China, Hefeng 55 (strain 5818) of soybean varieties planted in Heilongjiang Province in Northeast China, Huaxia 3 (strain 5136) of soybean varieties in Guangdong Province in South China, and B. diazoefficients USDA110 was donated by Professor Woo-Suk Chang of the Department of Biology, University of Texas, Arlington, Texas, USA. By comparing the size of the hydrogen peroxide inhibition zone, the sensitivity characteristics of different strains to hydrogen peroxide were compared. Strains 4253, 5841, 5818, 5136 and B. diazoefficients USDA110 were more sensitive than strain 5038. These results indicate that strain 5038 is more tolerant to H2O2, as shown in Table 5.

[0062] Table 5 Comparison of responses of different strains to oxidative stress

[0063] Strain number Diameter of inhibition zone (cm) 5038 3.1±0.3a 4253 3.9±0.4d 5841 3.7±0.4c 5818 3.9±0.2d 5136 3.8±0.3bc USDA110 3.6±0.4b

[0064] Note: Lowercase letters indicate significant differences at the p<0.01 level.

[0065] Bradyrhizobium japonicum, a slow-growing soybean rhizobium, is used to improve the resistance of soybeans to oxidative stress. In the process of symbiotic nodulation with host plants, rhizobia can reduce nitrogen in the air into a nitrogen source that can be used by plants, but this process needs to face the challenge of oxidative stress. Therefore, rhizobia with strong oxidizing power can better overcome oxygen repression and ensure the symbiotic nitrogen fixation process. In addition, strong antioxidant capacity is one of the important factors for strains to grow in adverse conditions (drought resistance, saline-alkali environment). The strong antioxidant capacity of growth-promoting bacteria indicates the production of a large number of antioxidant metabolites. These substances alleviate the oxidative damage caused by ROS produced in the plant's stress resistance process, thereby improving antioxidant capacity.

[0066] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. An application of Bradyrhizobium japonicum, characterized in that: The slow-growing soybean rhizobium Bradyrhizobium japonicum is used for promoting the growth of soybeans and increasing the soybean yield.

2. The use of Bradyrhizobium japonicum according to claim 1, characterized in that: The slow-growing soybean rhizobium Bradyrhizobium japonicum is used to increase the number of soybean pods, pod dry weight, nodule number, and nodule dry weight; and to increase the number of soybean pods, the number of grains per plant, the weight of grains per plant, and the yield.

3. The use of Bradyrhizobium japonicum according to claim 1, characterized in that: The bradyrhizobium japonicum is used for improving the stress resistance of soybean.

4. The use of Bradyrhizobium japonicum according to claim 3, characterized in that: The stress resistance includes resistance to antibiotics and resistance to oxidative stress.

5. The use of Bradyrhizobium japonicum according to claim 4, characterized in that: The antibiotics are gentamicin, ampicillin, chloramphenicol, tetracycline, streptomycin, spectinomycin, neomycin and rifampicin.

6. The use of Bradyrhizobium japonicum according to claim 1, characterized in that: The soybean variety is Heinong 87.

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