Application of Bradyrhizobium G94 in Promoting Corn Growth

Abstract

The invention discloses the application of Bradyrhizobium G94 in promoting corn growth, and belongs to the field of microbial technology. Bradyrhizobium G94 of the invention is classified and named Bradyrhizobium centrosematis G94, and its deposit number is GDMCC.No: 63598. Compared with CK, after inoculation with 1%-94 and 2%-94, the dry weight of corn root system increased by 52.67% and 104.46% respectively; the dry weight of aboveground part increased by 15.2% and 19.28% respectively; the height of corn plant increased by 31.08% and 36.52% respectively; the number of root tips of corn root system increased by 117.39% and 195.85% respectively; the number of branches of corn root system increased by 157.53% and 223.81% respectively; the total root length of corn root system increased by 115.04% and 164.09% respectively; the volume of corn root system increased by 39.7% and 116.33% respectively.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and more specifically, relates to the application of Bradyrhizobium G94 in promoting corn growth. Background Art

[0002] Acidic soils are widely distributed throughout the world and have a tendency to deteriorate. Acidic soils in my country are mainly distributed in the vast tropical and subtropical areas south of the Yangtze River and in Yunnan, Guizhou and Sichuan. The pH value of most soils is less than 5.5, and a large part of them is less than 5.0 or even 4.5. On the one hand, due to the serious loss of cations, acidic soils can also enhance the fixation of phosphate and molybdate in the soil, reduce the effectiveness of soil phosphorus and molybdenum, and cause low soil nutrient content, which is not conducive to the growth and development of crops. On the other hand, soil acidification will produce insoluble aluminum silicates converted into exchangeable aluminum, causing aluminum toxicity stress to plants. Aluminum toxicity is the main abiotic factor restricting crop growth and production, and is considered to be the second largest abiotic adversity after drought. Plants absorb excessive aluminum, which will greatly affect the growth of plant roots and even directly cause plant poisoning and death. At present, the improvement of acidic soils is achieved through breeding to obtain plants with strong adaptability; on the other hand, alkaline substances such as lime and mineral powder are applied.

[0003] Plant growth has different response mechanisms to different rhizosphere microorganisms and different environments. Rhizosphere microorganisms can improve the rhizosphere environment by relying on their own metabolites, such as secreting organic acids, dissolving insoluble nutrients, producing growth hormones, secreting various enzymes, alkaline substances, and increasing soil buffering properties to promote plant growth. In recent years, through more in-depth research on rhizosphere soil microorganisms, rhizosphere microorganisms are regarded as the "second genome" of plants and have an important influence on plant phenotype shaping. Therefore, it is urgent to obtain a strain that has a growth-promoting effect on plants in acidic soil. Summary of the invention

[0004] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide an application of Bradyrhizobium G94 in promoting corn growth for screening excellent growth-promoting strains.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] The application of Bradyrhizobium G94 in promoting corn growth, the Bradyrhizobium G94 is classified and named Bradyrhizobium centrosematis G94, which is preserved in: Guangdong Provincial Microbial Culture Collection Center, the preservation date is: August 14, 2023, the preservation number is GDMCC.No: 63598, and the preservation address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] Application of Bradyrhizobium G94 in improving soil pH.

[0008] Application of Bradyrhizobium G94 in promoting the increase of dry weight of corn.

[0009] Application of Bradyrhizobium G94 in promoting the increase of corn plant height.

[0010] Application of Bradyrhizobium G94 in promoting the increase of root tips in corn root system.

[0011] Application of Bradyrhizobium G94 in promoting the increase of root branches in corn.

[0012] Application of Bradyrhizobium G94 in promoting the increase of total root length of corn root system.

[0013] Application of Bradyrhizobium G94 in promoting the increase of corn root volume.

[0014] Application of Bradyrhizobium G94 bacterial solution in improving soil pH and promoting corn growth. The preparation method of the bacterial solution of Bradyrhizobium G94 is as follows: taking out the preserved strain from the cryopreservation tube, rejuvenating it by streaking on a YMA medium plate, culturing it at 28°C for 5-7 days, picking a robust strain and culturing it in a YMA liquid medium at 28°C and 200rrpm until OD 600 The cells were obtained by centrifugation and diluted with sterile water to OD 600 When the concentration is 0.5, bacterial solution is obtained.

[0015] The use of the bacterial solution of Bradyrhizobium G94 in improving soil pH and promoting corn growth is characterized in that the promotion of corn growth is to promote the increase of corn dry weight, promote the increase of corn plant height, promote the increase of the number of root tips of the corn root system, promote the increase of the number of root branches of the corn root system, promote the increase of the total root length of the corn root system, and promote the increase of the volume of the corn root system.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1) The Bradyrhizobium G94 of the present invention can effectively increase soil pH. Compared with CK, the soil pH increased from 4.453 to 4.677 and 4.673 after inoculation of 1%-94 and 2%-94.

[0018] 2) The Bradyrhizobium G94 of the present invention can effectively promote the increase of corn dry weight. Compared with CK, after inoculation with 1%-94 and 2%-94, the dry weight of the corn root system increased by 52.67% and 104.46%, respectively; the dry weight of the aboveground part increased by 15.2% and 19.28%, respectively.

[0019] 3) The Bradyrhizobium G94 of the present invention can effectively promote the increase of corn plant height. Compared with CK, after inoculation with 1%-94 and 2%-94, the corn plant height increased by 31.08% and 36.52%, respectively.

[0020] 4) The Bradyrhizobium G94 of the present invention can effectively promote the increase in the number of root tips of corn root system. Compared with CK, after inoculation with 1%-94 and 2%-94, the number of root tips of corn root system increased by 117.39% and 195.85%, respectively.

[0021] 5) The Bradyrhizobium G94 of the present invention can effectively promote the increase in the number of maize root branches. Compared with CK, after inoculation with 1%-94 and 2%-94, the number of maize root branches increased by 157.53% and 223.81%, respectively.

[0022] 6) The Bradyrhizobium G94 of the present invention can effectively promote the increase of the total root length of corn root system. Compared with CK, after inoculation with 1%-94 and 2%-94, the total root length of corn root system increased by 115.04% and 164.09%, respectively.

[0023] 7) The Bradyrhizobium G94 of the present invention can effectively promote the increase of corn root volume. Compared with CK, after inoculation with 1%-94 and 2%-94, the corn root volume increased by 39.7% and 116.33%, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a diagram for identifying the autogenous nitrogen fixation function of Bradyrhizobium G94;

[0025] Figure 2 This is a diagram for identifying the IAA production ability of Bradyrhizobium G94;

[0026] Figure 3 This is a diagram for identifying the ability of Bradyrhizobium G94 to utilize ACC nitrogen sources;

[0027] Figure 4 This is a diagram for identifying the phosphate solubilization ability of Bradyrhizobium G94;

[0028] Figure 5 The growth diagram of Bradyrhizobium G94 under different pH conditions;

[0029] Figure 6 This is the effect of different inoculation amounts on soil pH;

[0030] Figure 7 This is the effect of different inoculation amounts on the dry weight of corn potted seedlings;

[0031] Figure 8 This is the effect of different inoculation amounts on the height of corn potted seedlings;

[0032] Fig. 9This is the effect of different inoculation amounts on the number of root tips of the maize potted seedlings;

[0033] Fig.10 This is the effect of different inoculation amounts on the number of root branches of corn potted seedlings;

[0034] Fig.11 This is the effect of different inoculation amounts on the total root length of corn potted seedlings;

[0035] Fig.12 This is a graph showing the effect of different inoculation amounts on the root volume of corn potted seedlings. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. In the following embodiments, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0037] Strongly acidic (pH about 4.5) sugarcane rhizosphere soil was collected from a sugarcane field in Natong Town, Long'an County, Nanning City, Guangxi. Rhizosphere soil samples were randomly collected from each plot using a five-point sampling method and brought back to the laboratory and stored in a 4°C refrigerator.

[0038] YMA medium: MgSO4.7H2O 0.2g, K2HPO4 0.8g, KH2PO4 0.2g, CaSO4.2H2O0.1g, FeCl30.01g, Na2aMoO4.2H2O 0.002g, mannitol 20g, yeast extract 0.5g, pH7.2.

[0039] Axubei nitrogen-free medium (1L): KH2PO4 0.2g, MgSO 2. 7H2O 0.2g, NaCl 0.2g, CaCO3 5g, mannitol 10g, CaSO 4. 2H2O 0.1g, make up to volume with ddH2O, adjust pH=6.5.

[0040] DFa medium (1L): Na2HPO4 6g, KH2PO4 4g, MgSO4·7H2O 0.2g, glucose 2g, gluconic acid 2g, citric acid 2g, (NH4)2SO4 2g, FeSO4·7H2O 1mg, H3BO3 10μg, MnSO4·H2O 11.19μg, ZnSO4·7H2O 124.6μg, CuSO4·5H2O 72.22μg, MnO3

[0041] 10μg, ACC solution 3.0mmol / L, agar 15-20g, pH7.2.

[0042] Tricalcium phosphate medium (1L): glucose 10g, sodium chloride 0.3g, magnesium sulfate 0.3g, manganese sulfate 0.03g, ammonium sulfate 0.5g, potassium chloride 0.3g, iron sulfate 0.03g, calcium phosphate 10g, agar 15g, pH 7.2-7.4.

[0043] Color developing solution: Take 5mL 95% ethanol, 1mL 1% phosphoric acid solution, and 0.2mL 10% pure sulfuric acid, mix well, and then dilute to 50mL volumetric flask with sterile water to obtain Salkowski color developing solution.

[0044] Example 1

[0045] 1. Isolation and purification of bacteria

[0046] Weigh 10g of acidic sugarcane rhizosphere soil and add it to a triangular flask containing 20-30 sterile glass beads and 90mL sterile water, place it in a shaking incubator, culture it at 28℃, 200rpm / min for 2h, and place it at room temperature in a stable environment for 20min to obtain soil bacterial suspension; draw 1mL of soil bacterial suspension and add it to a test tube containing 9mL sterile water, and take another test tube to dilute and obtain 10 -1 , 10 -2 , 10 -3 and 10 -4 20 μL of soil bacterial suspension of each concentration gradient was evenly spread on YMA medium, and repeated five times until a purified single colony was obtained. After 7 days of cultivation at 28°C, observe that when regular, moist, milky white colonies appear on the surface of the plate, purify the colonies on a new plate with a bacterial loop in time, and inoculate the colonies on YMA liquid medium for about 7 days at 28°C to obtain a bacterial solution with OD = 0.5. After molecular biological identification, 50% glycerol and OD = 0.5 bacterial solution were stored in a -80°C refrigerator at a ratio of 1:1.

[0047] 2. Identification of the growth-promoting function of bacteria

[0048] 1) Autogenous nitrogen fixation: 10u of bacterial solution with LOD = 0.5 was spotted on the Axubei nitrogen-free culture medium using the spotting method and cultured at 28°C for 5 days.

[0049] The results are as follows Figure 1 As shown, complete colonies grew, indicating that the strain has the ability to fix nitrogen spontaneously.

[0050] 2) IAA production: inoculate the bacterial liquid with OD=0.5 into YMA medium containing L-Try (1 mg / mL) at a ratio of 1%, and culture at 28°C, 180 rpm for 5 days; centrifuge the culture medium for 10 minutes, take 200uL of the supernatant and mix it with 200uL of color developing solution in equal volumes, and mix 200uL of the supernatant and 200uL of sterile water in equal volumes for the control, and place it in the dark for 0.5h. The IAA production capacity is qualitatively judged based on whether color is developed.

[0051] The results are as follows Figure 2 As shown, the pink color after adding the color developing solution indicates that the strain has the ability to produce IAA, while the control does not show color.

[0052] 3) Production of ACC deaminase: 10 uL of bacterial solution with OD = 0.5 was spotted in DFa medium using the spotting method and cultured at 28°C for 5 days.

[0053] The results are as follows Figure 3 As shown, bacterial rings appeared after 5 days of cultivation, indicating that the strain has the ability to utilize ACC nitrogen source.

[0054] 4) Phosphate solubilization ability: 10 uL of bacterial solution with OD=0.5 was spotted in tricalcium phosphate medium by spotting method and cultured at 28°C for 5 days.

[0055] The results are as follows Figure 4 As shown, complete colonies appeared after 5 days of culture, indicating that the strain has the function of solubilizing phosphate.

[0056] Table 1 Functional identification of Bradyrhizobium G94

[0057] Function Autogenous nitrogen fixation Produces IAA hormone ACC deaminase Phosphate solubilization Dominant (+, -) + + + +

[0058] After screening, Bradyrhizobium G94 was obtained, and its classification name was (Bradyrhizobium centrosematis) G94, which was preserved in Guangdong Microbial Culture Collection Center (GDMCC), with a preservation date of August 14, 2023, a preservation number of GDMCC.No: 63598, and a preservation address of No. 100, Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences.

[0059] Example 2

[0060] The pH of the YMA liquid culture medium was adjusted to 4, 4.5, 5, 5.5, and 6.5 with 1% hydrochloric acid and 1% sodium hydroxide, and sterilized at 121° C. for 15 minutes for use.

[0061] Take out the preserved strains from the cryopreservation tube, rejuvenate by streaking on the YMA medium plate, culture at 28℃ for 5-7 days, pick the robust strains and culture the bacteria in YMA liquid medium at 28℃ and 200rrpm / min until the OD600 The bacterial solution was inoculated into the culture medium with different pH gradients at 1% and cultured at 28°C and 200rrpm / min. The OD was measured at 12:00 noon every day. 600 The absorbance value was used to draw a growth curve.

[0062] The results are as follows Figure 5 As shown, although the growth activity of G94 increased with the increase of medium pH, G94 could grow normally at pH 4.5, and the OD value reached 0.723 on the 11th day. The results showed that G94 had strong acid resistance.

[0063] Example 3

[0064] The corn Zhengdan 958 variety was used as the test object. The corn seeds were first germinated. They were first soaked in 75% alcohol for 30s + 1% sodium hypochlorite solution for 10min to sterilize the corn seeds. After being cleaned with sterile water, they were placed on gauze in a sterilized glass dish. Sterile water was sprayed to keep the gauze moist. Then the glass dish was placed in a clean bench to allow the corn to germinate. Seedlings with the same germination degree were selected and transferred to a PET bottle with a soil pH of 4.5, about 600g of soil per bottle, and a hole in the bottom of the bottle. The experimental design was 1% inoculation amount, 2% inoculation amount (i.e., 1mL and 2mL of bacterial liquid per 100g of air-dried soil) and control without inoculation. Each treatment was set up with 5 groups of replicates; one plant per pot; the moisture content of the experimental soil was controlled at 70%±5% of the field water holding capacity. The walk-in incubator simulated the normal growth environment of corn, and the light, temperature and humidity were suitable. All treatments were not fertilized separately, and the management measures of each treatment were the same. Destructive sampling was performed after 20 days to measure indicators such as plant height, biomass, and root morphology.

[0065] The results are as follows Figure 6 As shown, compared with CK, the soil pH increased from 4.453 to 4.677 and 4.673 after inoculation of 1%-94 and 2%-94, and the G94 strain can effectively and significantly increase the soil pH.

[0066] The results are as follows Figure 7 As shown, compared with CK, after inoculation with 1%-94 and 2%-94, the dry weight of corn roots increased with the increase of inoculum dosage, which increased by 52.67% and 104.46% respectively, and both were significantly different; the dry weight of the aboveground part also increased with the increase of inoculum dosage, which were 15.2% and 19.28% respectively, but did not reach a significant difference.

[0067] The results are as follows Figure 8 As shown, compared with CK, after inoculation with 1%-94 and 2%-94, as the inoculation amount increased, the height of corn plants increased by 31.08% and 36.52% respectively, and both were significantly different.

[0068] The results are as follows Fig. 9 As shown, compared with CK, after inoculation with 1%-94 and 2%-94, the number of root tips increased with the increase of the inoculum amount, which increased by 117.39% and 195.85% respectively. There were significant differences between inoculation and non-inoculation, and there were significant differences between the inoculation amounts.

[0069] The results are as follows Fig.10 As shown, compared with CK, after inoculation with 1%-94 and 2%-94, the number of root branches increased with the increase of inoculation amount, increasing by 157.53% and 223.81% respectively, and there was a significant difference between inoculation and non-inoculation.

[0070] The results are as follows Fig.11 As shown, compared with CK, after inoculation with 1%-94 and 2%-94, the total root length increased with the increase of inoculum dosage, increasing by 115.04% and 164.09% respectively, and there was a significant difference between inoculation and non-inoculation.

[0071] The results are as follows Fig.12 As shown, compared with CK, after inoculation with 1%-94 and 2%-94, the root volume increased with the increase of inoculum dosage, increasing by 39.7% and 116.33% respectively, and there was a significant difference when the inoculum dosage was 2%.

Claims

1. Application of Bradyrhizobium G94 in promoting corn growth. The Bradyrhizobium G94 is classified and named Bradyrhizobium centrosematis G94, which is deposited in: Guangdong Provincial Microbial Culture Collection Center, with a deposit date of August 14, 2023, a deposit number of GDMCC.No: 63598, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. Use of the Bradyrhizobium G94 according to claim 1 in improving soil pH.

3. Use of the Bradyrhizobium G94 according to claim 1 in promoting the increase of dry weight of corn.

4. Use of the Bradyrhizobium G94 according to claim 1 in promoting the increase of corn plant height.

5. Use of the Bradyrhizobium G94 according to claim 1 in promoting the increase in the number of root tips of corn root system.

6. Use of the Bradyrhizobium G94 according to claim 1 in promoting the increase in the number of root branches of corn.

7. Use of the Bradyrhizobium G94 according to claim 1 in promoting the increase of the total root length of corn root system.

8. Use of the Bradyrhizobium G94 according to claim 1 in promoting the increase of corn root volume.

9. The use of the bacterial solution of Bradyrhizobium G94 according to claim 1 in improving soil pH and promoting corn growth, characterized in that: The preparation method of the bacterial solution of Bradyrhizobium G94 is as follows: taking out the preserved bacterial strain from the cryopreservation tube, rejuvenating it by streaking on a YMA medium plate, culturing it at 28°C for 5-7 days, picking a robust strain and culturing it in a YMA liquid medium at 28°C and 200rpm until the OD 600 The cells were obtained by centrifugation and diluted with sterile water to OD 600 When the concentration is 0.5, bacterial solution is obtained.

10. The use of the bacterial solution of Bradyrhizobium G94 according to claim 9 for improving soil pH and promoting corn growth, characterized in that: The corn growth promotion is to promote the increase of corn dry weight, promote the increase of corn plant height, promote the increase of the number of root tips of the corn root system, promote the increase of the number of branches of the corn root system, promote the increase of the total root length of the corn root system, and promote the increase of the volume of the corn root system.

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