A strain of Priesteria gigantea with carbon fixation function and its fermentation liquid and application

Abstract

The invention discloses a strain of Priesteria gigantea with carbon fixation function, a fermentation liquid and an application thereof, and belongs to the technical field of microbial application. The invention screens out a strain of Priesteria gigantea ( Priestia megaterium )C17, its biological preservation number is CCTCC NO: M 20241724; strain C17 has the carbon fixation gene cbbL gene, can produce carbon fixation enzymes, has good carbon fixation ability, can be used in the field of microbial carbon fixation, promote the conversion of inorganic carbon in the soil into organic carbon, and at the same time can improve the soil and increase the number of carbon fixation bacteria in the soil.

Description

Technical Field

[0001] The invention relates to the technical field of microbial application, and in particular to a strain of Priesteria gigantea with carbon fixation function, a fermentation liquid thereof and an application thereof. Background Art

[0002] Affected by the rapid development of global industry and human activities, global changes caused by greenhouse gases have become a key research area in the academic community's study of sustainable development. The main gas causing the greenhouse effect is CO 2 At the same time, CO 2 It is also the most abundant carbon resource on earth, and it can reasonably fix CO 2 It can also convert inorganic carbon into a huge renewable carbon resource. 2 concentration changes, researchers have explored many methods for fixing CO 2 The methods mainly include chemical carbon fixation, physical carbon fixation and biological carbon fixation.

[0003] Currently, nature fixes CO through biological processes. 2 It has attracted the attention of researchers from all over the world for its direct, economical, green and pollution-free characteristics. 2 The main organisms that fix carbon are plants and microorganisms. Carbon-fixing microorganisms are widely distributed in nature, and even exist in some extremely harsh environments, such as deep-sea sediments and acidic volcanic lakes. Guo Jun et al. used a carbon-free inorganic culture medium to separate and screen CO2-fixing microorganisms from activated sludge, biogas slurry and facility soil. 2 strains as carbon sources; Hu Jiajun and others screened carbon-fixing microorganisms from seawater and sediments that do not require hydrogen or light. Carbon-fixing microorganisms convert atmospheric carbon dioxide or carbonates into organic matter through photosynthesis or chemosynthesis, which is an important part of the global carbon cycle. This process helps to mitigate the greenhouse effect and combat global warming. At the same time, they also play an important role in soil remediation. They fix CO 2 Increase soil organic matter content, improve soil fertility, reduce the use of chemical fertilizers, and promote the diversity of soil microbial communities.

[0004] Priesteria gigantea has many functions and good effects in agricultural applications. For example, patent CN116970517A discloses a strain of Priesteria gigantea and its application in alleviating the impact of cadmium on rice. The Priesteria gigantea releases organic acids to complex cadmium ions in the environment that are toxic to plants, thereby alleviating the adverse effects of cadmium toxicity on rice growth. Patent CN117025469A discloses a Priesteria gigantea and its application. The Priesteria gigantea can not only significantly inhibit the growth of tobacco phytophthora hyphae, but also has a broad-spectrum antibacterial property, and has a certain inhibitory effect on plant pathogenic fungi such as Fujikura Fusarium, Fusarium oxysporum or rice blast fungus, and can be used to prevent and control plant diseases caused by a variety of plant pathogenic fungi. Patent CN117701470A discloses a strain of Priesteria gigantea for preventing and controlling plant powdery mildew and its microbial agent, which provides an efficient microorganism for preventing and controlling powdery mildew of boxwood, and opens up an effective prevention and control approach. However, there are no reports on the role of Priesteria gigantea in carbon fixation, increasing soil organic carbon content, and promoting crop organic carbon accumulation. Summary of the invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide a strain of Priesteria gigantea with carbon fixation function and its fermentation liquid and application.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] In a first aspect of the present invention, a strain of Priesteria gigantea having carbon fixation function is provided. Priestia megaterium )C17; the strain was deposited in the China Center for Type Culture Collection (CCTCC for short, address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province) on August 2, 2024, and its biological deposit number is CCTCCNO: M 20241724.

[0008] The second aspect of the present invention provides the fermentation broth of the above-mentioned Priesteria gigantea, which is prepared by the following method:

[0009] (1) inoculating the activated Priesteria gigantea into NB culture medium and culturing on a shaking table for 12-20 hours to obtain a Priesteria gigantea seed solution;

[0010] (2) inoculating the Priesteria gigantea seed liquid into a fermentation medium, and fermenting and culturing for 15-20 hours to obtain a Priesteria gigantea fermentation liquid;

[0011] The composition of the NB culture medium is: 10 g / L peptone, 3 g / L beef extract powder, and 5 g / L sodium chloride;

[0012] The fermentation medium is composed of, by mass percentage, 1.25% corn steep liquor dry powder, 1.25% peptone, 3% corn starch, 0.5% glucose, 0.1% dipotassium hydrogen phosphate, 0.1% potassium dihydrogen phosphate, 0.3% calcium chloride, 0.3% sodium chloride, 0.04% protease, 0.04% amylase, and the remainder is water.

[0013] Preferably, in step (2), the inoculation amount of Priesteria gigantea seed solution is 2-3% of the volume of the fermentation medium.

[0014] The third aspect of the present invention provides the use of the above-mentioned Priesteria gigantea or the fermentation broth of Priesteria gigantea in the following (1) or (2):

[0015] (1) Carbon fixation;

[0016] (2) Produce carbon-fixing enzyme.

[0017] Preferably, the carbon fixation enzyme is RubisCO enzyme.

[0018] The fourth aspect of the present invention provides the use of the above-mentioned Priesteria gigantea or the fermentation broth of Priesteria gigantea in the following (1) or (2):

[0019] (1) Increase the organic carbon content in crops;

[0020] (2) Prepare products that increase the organic carbon content in crops.

[0021] The fifth aspect of the present invention provides the use of the above-mentioned Priesteria gigantea or the fermentation broth of Priesteria gigantea in the following (1) or (2):

[0022] (1) Increase the organic carbon content in the soil;

[0023] (2) Prepare products that increase the organic carbon content in soil.

[0024] The sixth aspect of the present invention provides the use of the above-mentioned Priesteria gigantea or the fermentation broth of Priesteria gigantea in the following (1) or (2):

[0025] (1) Increase the number of carbon-fixing bacteria in the soil;

[0026] (2) Prepare products that increase the number of carbon-fixing bacteria in the soil.

[0027] The seventh aspect of the present invention provides the use of the above-mentioned Priesteria gigantea or the fermentation liquid of Priesteria gigantea in promoting the growth of crops.

[0028] Beneficial effects of the present invention:

[0029] 1. The present invention screened out a strain of Priesteria gigantea with carbon fixation function ( Priestia megaterium )C17, which has the carbon fixation gene cbbL gene, can produce carbon fixation enzymes, has good carbon fixation ability, can be used in the field of microbial carbon fixation, promote the conversion of inorganic carbon in the soil into organic carbon, and at the same time can improve the soil and increase the number of carbon fixation bacteria in the soil.

[0030] 2. The Priesteria gigantea C17 of the present invention exhibits drug resistance to 13 pesticides, indicating that the bacterium can be used in combination with a variety of fungicides, herbicides and insecticides at the same time and has practical production application value.

[0031] 3. The Priesteria gigantea C17 of the present invention can promote the growth of a variety of crops. When applied to rapeseed, it can increase the organic carbon (total carbon) content of rapeseed, promote plant growth, increase the SPAD value, and increase dry matter accumulation; when applied to wheat, it significantly increases the plant height, fresh weight and dry weight of wheat, and the plant height, fresh weight and dry weight increase by 16.24%, 45.59% and 66.43% respectively, promotes wheat growth, and increases plant dry matter accumulation; when applied to soybeans, it can promote soybean growth, increase soybean plant height, and ultimately achieve increased yield, and its yield is increased by 9.51% compared with the control group, indicating that Priesteria gigantea C17 has a good growth-promoting and yield-increasing effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 :Chart of carbon fixation gene detection of strains;

[0033] Figure 2 : Colony morphology of strain C17;

[0034] Figure 3 :Bacterial morphology of strain C17;

[0035] Figure 4 :Hemolytic test diagram of strains;

[0036] Figure 5 :Microscopic examination pictures of strain C17 after fermentation;

[0037] Figure 6 :Drug resistance test diagram of strain C17;

[0038] Figure 7 : The index determination diagram of the wheat hydroponic experiment; wherein (a) is the test result of wheat plant height of different treatment groups; (b) is the test result of wheat fresh weight of different treatment groups; (c) is the test result of wheat dry weight of different treatment groups;

[0039] Figure 8 :Pictures of wheat hydroponic experiment;

[0040] Fig. 9: The index determination diagram of rapeseed hydroponic experiment; wherein (a) is the test result of rapeseed leaf length of different treatment groups; (b) is the test result of rapeseed leaf width of different treatment groups; (c) is the test result of rapeseed fresh weight of different treatment groups; (d) is the test result of rapeseed dry weight of different treatment groups;

[0041] Fig.10 :The total carbon content of rapeseed plants in the hydroponic experiment;

[0042] Fig.11 : Pictures of rapeseed hydroponic experiment;

[0043] Fig.12 : Determination of organic carbon content in wheat soil in pot experiments;

[0044] Fig.13 : Color diagram of rapeseed plants in different treatment groups after drying and grinding;

[0045] Fig.14 :Yield determination diagram of soybean in the field of different treatment groups;

[0046] Fig.15 :The effect of applying C17 bacterial powder on soybeans in the field. DETAILED DESCRIPTION

[0047] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0048] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0049] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0050] The culture medium used was as follows:

[0051] Carbon-free inorganic liquid medium: MnSO 4 0.02 g, Na 2 HPO 4 1 g, KH 2 PO 4 1.8 g MgSO 4 0.4 g, CaCl 2 0.05 g, NaCl 1 g, NH 4 Cl 0.5 g, FeCl 3 0.02 g, Na 2 S2 O 3 10 g, add distilled water to 1000 mL, and sterilize at 121℃ for 20 min. Carbon-free inorganic solid medium is prepared by adding 2% agar powder to carbon-free inorganic liquid medium.

[0052] NB medium: 10 g peptone, 3 g beef extract powder, 5 g sodium chloride, add distilled water to 1000 mL, sterilize at 121℃ for 20 min. NA medium is NB medium with 2% agar powder added. 1.5% NA medium is NB medium with 1.5% agar powder added.

[0053] Example 1: Isolation and identification of strains

[0054] 1. Isolation and initial screening of strains

[0055] Farmland soil, sludge and other samples were collected from Taishan District, Shandong Province. 10 g of the sample was added to a conical flask containing 90 mL of physiological saline and mixed thoroughly. The sample was placed on a shaker at 30°C and 180 r / min for 30 min to prepare a suspension for gradient dilution. -3 , 10 -4 , 10 -5 Dilute the concentration, pipette 0.1 mL of the sample suspension into a sterile culture dish containing a carbon-free inorganic solid culture medium, spread it evenly, culture it upside down at 30°C for 5 days, pick a single colony, mark it, separate and purify it by streaking for multiple times, and store it at 4°C after transferring to a slant.

[0056] The collected samples (sludge, farmland soil, etc.) were initially screened for bacterial strains, and a total of 65 carbon-fixing microorganisms were screened.

[0057] 2. Rescreening of strains

[0058] DNA was extracted from the 65 strains obtained in the preliminary screening, and the carbon fixation gene cbbL gene was detected using the primers in Table 1, the reaction system in Table 2, and the reaction procedure in Table 3.

[0059] (1) Amplification primer name and sequence:

[0060] Table 1 Amplification primer names and sequences

[0061]

[0062] (2) PCR reaction system:

[0063] Table 2 PCR reactants and reaction volumes

[0064]

[0065] (3) Reaction procedure:

[0066] Table 3 Reaction procedure

[0067]

[0068] The carbon fixation gene cbbL was detected in the initially screened carbon fixation bacteria. The results showed that more than 10 strains including strain C17 had the target band near 500 bp, that is, they had the carbon fixation gene cbbL gene ( Figure 1 ).

[0069] 3. RubisCO enzyme activity assay

[0070] The RubisCO enzyme activity of the bacterial liquid of the strain with the carbon fixation gene cbbL was determined according to the instruction manual of the 1,5-bisphosphate ribulose carboxylase / oxygenase (RubisCO) enzyme-linked immunosorbent assay kit provided by Hengyuan Biotechnology.

[0071] Table 4 RubisCO enzyme activity of strains

[0072]

[0073] The results showed that the RubisCO activity of the strains with the carbon fixation gene cbbL was between 25.65 and 193.26 U / L, and the strain C17 had the highest RubisCO activity, reaching 193.26 U / L. Therefore, based on the results of carbon fixation gene detection and RubisCO activity determination, strain C17 was finally selected as a carbon fixation functional strain.

[0074] 4. Identification of strains

[0075] 4.1 Bacterial and colony morphology of strains

[0076] Colony morphology: A single colony of strain C17 was streaked onto a NA solid plate medium, and the plate was inverted in a constant temperature incubator and cultured at 30°C for 24 h. The colony was nearly round, milky white, with a moist and smooth surface and a dot-like protrusion in the center ( Figure 2 ).

[0077] Bacterial morphology: Gram staining was used to observe that the strain C17 was rod-shaped, thick, arranged in chains, with spores in the spores, and Gram staining was positive ( Figure 3 ).

[0078] 4.2 Physiological and biochemical identification

[0079] The physiological and biochemical characteristics of strain C17 were identified using Haibo Biotech HBI Bacillus biochemical identification strips. The results are shown in Table 5.

[0080] Table 5 Physiological and biochemical reactions of strain C17

[0081]

[0082] Note: “+” represents positive, and “-” represents negative.

[0083] Strain C17 was able to utilize a variety of sugars (D-xylose, L-arabinose, starch), was unable to utilize citrate and propionate, was able to reduce nitrate, and was unable to grow at pH 5.7 and 7% NaCl.

[0084] 4.3 Safety evaluation of bacterial strains - hemolytic test

[0085] The strain C17 was streaked in a "Z" shape on Columbia blood agar (Qingdao Haibo) and cultured for 24 hours to observe whether there was a hemolytic ring. The results showed that no hemolytic ring appeared after the strain was cultured, proving that the strain C17 was non-hemolytic and had high safety during production and use ( Figure 4 ).

[0086] 4.4 Molecular Biological Identification

[0087] The 16S rDNA gene sequence analysis method was used to identify the genus and species of the isolated strain C17. The 16S rDNA gene sequence is shown in SEQ ID NO.1. The identification result showed that the strain C17 belonged to Priesteria gigantea ( Priestia megaterium ).

[0088] Based on the results of morphological, physiological and biochemical, and molecular biological identification, strain C17 was identified as Priesteria gigantea and named Priesteria gigantea ( Priestia megaterium ) C17, and deposited the strain biologically. The deposit information is as follows:

[0089] Bacteria species: Priesteria gigantea C17

[0090] Latin name: Priestia megaterium

[0091] Collection institution: China Center for Type Culture Collection

[0092] Abbreviation of depository institution: CCTCC

[0093] Address: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province

[0094] Date of deposit: August 2, 2024

[0095] Deposit number: CCTCC NO: M 20241724.

[0096] Example 2: Preparation of Priesteria megaterium C17 fermentation broth

[0097] Fermentation process optimization: First, optimize the optimal culture medium in a 20 L fermenter, and then perform 10 T fermentation verification after determining the optimal culture medium. The specific optimization is as follows:

[0098] 1. 20 L fermentation tank:

[0099] (1) Activation of strains: Activate Priesteria gigantea C17 from a glycerol tube, inoculate it on NA medium, and culture it at 37°C for 18 h to obtain activated Priesteria gigantea;

[0100] (2) Preparation of seed solution: Take one loopful of activated Priesteria gigantea and inoculate it into NB medium. Incubate it on a shaking table for 16 h and then inoculate it into a 20 L fermenter at a 2.5% inoculation rate for fermentation.

[0101] (3) Fermentation conditions: temperature 37°C, initial pH 7, initial stirring rate 400 r / min, changed to 700 r / min after bacterial growth, ventilation volume 1.5 m 3 / h, tank pressure 0.05 MPa.

[0102] (4) Screening of fermentation medium: There are 7 groups of fermentation medium for 20L fermenter, and the composition of the medium is as follows:

[0103] ① Calculated by mass percentage: corn starch 4%; soybean meal 4%; glucose: 0.5%; dipotassium hydrogen phosphate 0.1%; potassium dihydrogen phosphate 0.1%; magnesium sulfate 0.02%; manganese sulfate 0.05%; protease 0.04%; amylase 0.04%; calcium carbonate 0.1%; sodium chloride 0.3% and the rest is water.

[0104] ② Calculated by mass percentage: corn starch 5%; soybean meal 4%; glucose: 0.5%; dipotassium hydrogen phosphate 0.1%; potassium dihydrogen phosphate 0.1%; magnesium sulfate 0.02%; manganese sulfate 0.05%; protease 0.04%; amylase 0.04%; calcium carbonate 0.1%; sodium chloride 0.3% and the rest is water.

[0105] ③ Calculated by mass percentage: corn starch 5%; soybean meal 4%; glucose: 0.5%; potassium dihydrogen phosphate 0.1%; magnesium sulfate 0.02%; manganese sulfate 0.1%; protease 0.04%; amylase 0.04%; calcium chloride 0.3%; sodium chloride 0.3%, and the rest is water.

[0106] ④ By mass percentage, corn starch 5%; soybean meal 4%; glucose: 0.5%; potassium dihydrogen phosphate 0.1%; magnesium sulfate 0.02%; manganese sulfate 0.1%; amylase 0.04%; calcium chloride 0.3%; sodium chloride 0.3%, and the rest is water.

[0107] ⑤ By mass percentage, corn starch 5%; corn steep liquor powder 1.25%; peptone 1.25%; glucose: 0.5%; potassium dihydrogen phosphate 0.1%; magnesium sulfate 0.02%; manganese sulfate 0.1%; amylase 0.04%; protease 0.04%; calcium chloride 0.3%; sodium chloride 0.3%, and the rest is water.

[0108] ⑥ Calculated by mass percentage: corn starch 3%; soybean meal 4%; glucose: 0.5%; dipotassium hydrogen phosphate 0.1%; potassium dihydrogen phosphate 0.1%; magnesium sulfate 0.1%; manganese sulfate 0.02%; protease 0.04%; amylase 0.04%, and the rest is water.

[0109] ⑦ By mass percentage, corn steep liquor powder 1.25%, peptone 1.25%, corn starch 3%, glucose 0.5%, dipotassium hydrogen phosphate 0.1%, potassium dihydrogen phosphate 0.1%, calcium chloride 0.3%, sodium chloride 0.3%, protease 0.04%, amylase 0.04%, and the rest is water.

[0110] (5) Detection indicators and their measurement methods

[0111] Determination of total viable count: After the fermentation is completed, the fermentation liquid is aseptically taken from the fermentation tank for viable count. The method is as follows: 1 mL of the evenly mixed fermentation liquid is added to 99 mL of sterile water, shaken on a shaker for 30 min, then diluted step by step, and counted on a shaker plate using 1.5% NA culture medium.

[0112] Determination of spore count: Take the sterile fermentation liquid and bathe it in a water bath at 80℃ for 30 min. Then take 1 mL of the evenly mixed fermentation liquid and add it to 99 mL of sterile water. Oscillate on a shaker for 30 min. Then dilute it step by step and count the spores on a shaking plate using 1.5% NA culture medium.

[0113] Spore rate = number of spores / total number of viable bacteria × 100%.

[0114] Table 6 Statistics of fermentation results of Priesteria gigantea C17 in 20 L fermenter

[0115]

[0116] The fermentation medium of Priesteria gigantea C17 was explored in a 20L fermenter. After 7 batches of fermentation, the optimal medium was determined to be ⑦ (by mass percentage, corn steep liquor powder 1.25%, peptone 1.25%, corn starch 3%, glucose 0.5%, dipotassium hydrogen phosphate 0.1%, potassium dihydrogen phosphate 0.1%, calcium chloride 0.3%, sodium chloride 0.3%, protease 0.04%, amylase 0.04%, and the rest was water). The spore rate reached 96% after 30 hours of fermentation, and the number of viable bacteria was 6.5 billion cfu / mL. The microscopic examination pictures at the end of fermentation are as follows Figure 5 .

[0117] 2. 10T fermentation tank:

[0118] (1) Activation of bacterial strains: After activating Priesteria gigantea C17 from a glycerol tube, inoculate it on NA medium and culture it at 37°C for 1 day to reactivate it to obtain secondary activated Priesteria gigantea.

[0119] (2) Preparation of seed solution: Take the activated Priesteria gigantea, inoculate one loopful into NB medium, and culture on a shaking table for 16 h.

[0120] (3) Fermentation process

[0121] Fermentation medium: by mass percentage, corn steep liquor powder 1.25%, peptone 1.25%, corn starch 3%, glucose 0.5%, dipotassium hydrogen phosphate 0.1%, potassium dihydrogen phosphate 0.1%, calcium chloride 0.3%, sodium chloride 0.3%, protease 0.04%, amylase 0.04%, and the rest is water.

[0122] Primary scale-up: Inoculate the culture medium at 2.5% in the presence of 500 L of inoculum in a 700 L fermenter (liquid volume: 500 L) for 5 h.

[0123] The fermentation conditions were as follows: temperature 37°C, initial pH 7, stirring rate 125 r / min initially, changed to 150 r / min after bacterial growth, ventilation 15 m 3 / h, tank pressure 0.05 MPa.

[0124] Secondary amplification: The primary amplification fermentation liquid was inoculated into a 10T fermentation tank (liquid volume was 6T) for fermentation. The fermentation conditions were temperature 37°C, initial pH 7, stirring rate 125 r / min, ventilation volume 180 m 3 / h, tank pressure 0.05 MPa.

[0125] Post-fermentation treatment: After the fermentation in the 10T fermenter is completed, the fermentation liquid is aseptically taken from the fermenter for live bacteria count. The rest is spray dried and other treatments are carried out. After the bacterial powder is evenly mixed, part of the bacterial powder is taken for live bacteria count.

[0126] The method is as follows: take 1 mL of the evenly mixed fermentation liquid (or 1 g of bacterial powder) into 99 mL of sterile water, shake on a shaker for 30 minutes, then dilute step by step, and use 1.5% NA culture medium for shaking plate counting.

[0127] Table 7 Statistics of fermentation results of Priesteria gigantea C17 in 10T fermenter

[0128]

[0129] As can be seen from Table 7, the optimal culture medium ⑦ was selected for fermentation in a 10T fermenter. At the same time, the pH was controlled during the fermentation process to increase the viable bacterial count and spore rate of Priesteria gigantea. The viable bacterial count in the fermentation broth obtained after 17 h of fermentation reached 10.4 billion cfu / mL. After crystal violet staining and oil immersion microscopic observation, the spore rate reached more than 95%. The viable bacterial count of the spray-dried bacterial powder reached 160 billion cfu / g, which can significantly improve the fermentation spore count and product quality.

[0130] Example 3: Drug resistance test of Priesteria gigantea C17

[0131] 1. Test materials

[0132] Test strain: Priesteria gigantea C17 screened in Example 1 of the present invention.

[0133] Test pesticides: 15 kinds in total, see Table 8, dilute and use according to the recommended ratio.

[0134] Table 8 Order and name of pesticides

[0135]

[0136] 2. Test methods

[0137] The fermentation broth of Priesteria gigantea C17 prepared in Example 2 (bacterial concentration diluted to 10 6 cfu / mL) was added to the NA plate for coating, and then a sterilized filter paper was placed. Then 5 μL of the diluted pesticides (15 pesticides in Table 8) were respectively pipetted and dropped onto the filter paper, and then placed in a 30℃ constant temperature incubator for 24 h to observe the results.

[0138] 3. Test results

[0139] Priesteria gigantea C17 was intolerant to 5 (Zhongshengmycin) and 6 (Chlorothalonil), but showed resistance to the remaining 13 pesticides, indicating that the bacterium can be used in combination with multiple fungicides, herbicides, and insecticides at the same time ( Figure 6 ).

[0140] Example 4: Wheat hydroponic test

[0141] 1. Test materials

[0142] Test strains: Priesteria megaterium C17 and strain BE59 (Pantoea agglomerans), strain BE60 (Pantoea spp.), strain BC3 (Priesteria megaterium), and strain B19007 (Ochrobacterium spp.) screened in Example 1 of the present invention.

[0143] Test crop: wheat.

[0144] Preparation of nutrient solution: Weigh 1.26 g of Hoagland nutrient solution (without calcium nitrate, purchased from Haibo Biotechnology) and 0.945 g of calcium nitrate, dissolve them in 1000 mL of distilled water by heating, divide them into portions, sterilize them by high pressure at 115°C for 20 min, and set aside.

[0145] Preparation of bacterial solution: The test strains were activated by NA medium slant transfer and then inoculated into NB medium respectively, pH 7.0, 30℃, 180 r / min shaking flask culture for 24 h to obtain fermentation broth for later use.

[0146] 2. Test treatment and index determination

[0147] (1) Experimental design

[0148] Wheat with uniform growth was selected and placed in hydroponic bottles containing 400 mL of nutrient solution. Three control groups and five treatment groups were set up, with six replicates in each group. The experimental treatments are shown in Table 9.

[0149] Dilute the bacterial solution to 10 7 cfu / mL, and sprayed on the wheat leaves of the corresponding groups respectively. The control group was sprayed with the same volume of clean water, NB medium and brown sugar according to the experimental design until the leaves were covered with water droplets. The spraying was carried out once every 7 days, for a total of 3 times.

[0150] Table 9 Hydroponic test treatments

[0151]

[0152] (2) Index determination

[0153] Plant height: Use a ruler to measure the length from the base to the tip of the leaves at the top of the plant.

[0154] Fresh weight: Fresh samples are weighed directly after absorbing water.

[0155] Dry weight: After the fresh samples are collected, they should be promptly sterilized. Place the samples in an oven at 105°C for 30 minutes, then lower the temperature to 60-80°C, continue to bake until constant weight, and then weigh them.

[0156] 3. Test results

[0157] Depend on Figure 7 , Figure 8 It can be seen that compared with the CK2 (NB) group, the plant height, fresh weight and dry weight of the T4 (Priesteria gigantea C17) treatment group increased significantly by 16.24%, 45.59% and 66.43%, respectively. Comprehensive analysis showed that spraying Priesteria gigantea C17 could promote wheat growth and increase plant dry matter accumulation.

[0158] Example 5: Rapeseed hydroponic experiment

[0159] 1. Test materials

[0160] Test strains: Priesteria megaterium C17 and strain BE59 (Pantoea agglomerans), strain BE60 (Pantoea spp.), strain BC3 (Priesteria megaterium), and strain B19007 (Ochrobacterium spp.) screened in Example 1 of the present invention.

[0161] Test crop: rapeseed.

[0162] Preparation of nutrient solution: Weigh 1.26 g of Hoagland nutrient solution (without calcium nitrate, purchased from Haibo Biotechnology) and 0.945 g of calcium nitrate, dissolve them in 1000 mL of distilled water by heating, divide them into portions, sterilize them by high pressure at 115°C for 20 min, and set aside.

[0163] Preparation of bacterial solution: The above strains were activated by NA medium slant transfer and then inoculated into NB medium respectively, pH 7.0, 30℃, 180 r / min shaking flask culture for 24 h to obtain bacterial solution for later use.

[0164] 2. Test treatment and index determination

[0165] (1) Experimental design

[0166] Select rapeseed seedlings with the same growth potential and place them in hydroponic bottles containing 400 mL of nutrient solution. Set up 3 control groups and 5 treatment groups, with 6 replicates in each group. The experimental treatments are shown in Table 10. Dilute the bacterial solution to 10 7 cfu / mL, respectively, were sprayed on the rapeseed leaves of the corresponding groups. The control group was sprayed with the same volume of clean water, NB medium, and brown sugar according to the experimental design until the leaves were covered with water droplets. The spraying was carried out every 7 days, for a total of 3 times.

[0167] Table 10 Hydroponic test treatments

[0168]

[0169] (2) Index determination

[0170] Maximum leaf length and width: Length is measured from the leaf attachment point to the top of the leaf; width is measured at the widest part of the leaf.

[0171] Fresh weight and dry weight of a single plant: including the above-ground and underground parts. Fresh weight is the weight of the fresh sample after it absorbs water. The weighed sample is placed in a 105℃ oven for 30 minutes, then lowered to 60-80℃ and dried to a constant weight. The dry weight is the weight.

[0172] Plant organic carbon content determination: according to K 2 Cr 2 O 7 ―H 2 SO 4 The volumetric method was used for determination. After digestion, 3-4 drops of o-phenanthroline indicator were added and 0.2 mol / L standard ferrous sulfate (FeSO 4 ) solution is titrated. The end point is when the solution changes from yellow to green, gray-blue and finally to brown-red. Note the amount of ferrous sulfate used.

[0173] Calculation formula: ;

[0174] Where: V 0 For blank calibration, the FeSO 4 Standard solution volume (mL);

[0175] V is the FeSO used to titrate the test solution 4 Standard solution volume (mL);

[0176] C is FeSO 4 Concentration of the standard solution;

[0177] 0.003 is the molar mass of 1 / 4C (kg / mol);

[0178] W is the weight of the dried sample (mg).

[0179] 3. Test results

[0180] Depend on Fig. 9 It can be seen that compared with the CK2 (NB) group, the leaf length, leaf width, fresh weight and dry weight of the T4 (Priesteria gigantea C17) treatment group increased significantly by 20.95%, 30.42%, 55.75% and 60.16%, respectively, and the leaf length, leaf width, fresh weight and dry weight of the T5 (strain B19007) treatment group increased significantly by 18.18%, 33.33%, 33.32% and 50.95%, respectively. Fig.10 It can be seen that after the hydroponic rapeseed was sprayed with Priesteria gigantea C17 solution, the organic carbon (total carbon) content of the plants increased significantly, increasing by 11.75% compared with the CK2 group.

[0181] Comprehensive analysis showed that spraying Priesteria gigantea C17 solution could significantly promote the growth of rapeseed leaves and increase the organic carbon content of plants ( Fig.11 ).

[0182] Example 6: Wheat pot test

[0183] 1. Test materials

[0184] Test strains: Priesteria megaterium C17 and strain BE59 (Pantoea agglomerans), strain BE60 (Pantoea spp.), strain BC3 (Priesteria megaterium), and strain B19007 (Ochrobacterium spp.) screened in Example 1 of the present invention.

[0185] Test crop: wheat.

[0186] Preparation of bacterial solution: The above test strains were activated by NA medium slant transfer and then inoculated into NB medium respectively, pH 7.0, 30℃, 180 r / min shaking flask culture for 24 h to obtain fermentation broth for later use.

[0187] 2. Test treatment and index determination

[0188] (1) Experimental design

[0189] A total of 2 control groups and 5 treatment groups were set up, with 5 replicates in each group. The experimental design is shown in Table 11. Sandy loam was selected for potted plant experiments. After the debris in the soil was sieved, it was fully mixed and packaged. The weight of each pot was kept consistent. Then wheat was sown. Wheat seeds of basically the same size and without seed coat damage were selected. Ten seeds were sown in each pot. After sowing, the bacterial solution was irrigated and diluted to 10 6 cfu / mL, each pot was irrigated with 50 mL, and the control group was irrigated with the same volume of clean water and diluted NB medium according to the experimental design, once every 14 days, for a total of 2 times.

[0190] Table 11 Pot experiment design

[0191]

[0192] (2) Index determination

[0193] Determination of soil organic carbon content: according to K 2 Cr 2 O 7 ―H 2 SO 4 Determined by volumetric method.

[0194] (3) Test results

[0195] Depend on Fig.12It can be seen that the organic carbon content of the soil samples before the experiment was generally low (the organic carbon content was: 0.34%). After the experiment, the organic carbon content of the water group and the NB group decreased, which may be related to the consumption of organic matter in the soil by plant growth and development. The organic carbon content of each bacterial solution group increased to varying degrees, increasing by 20.59%-55.88% compared with the soil samples before the experiment. This may be because the bacterial solution can promote the conversion of inorganic carbon in the soil into organic carbon. Especially Priesteria gigantea C17, its organic carbon content was 0.53%, an increase of 55.88% compared with the soil samples before the experiment, and it has a good carbon fixation ability.

[0196] Example 7: Rapeseed pot test

[0197] 1. Test materials

[0198] Test strains: Priesteria megaterium C17 and strain BE59 (Pantoea agglomerans), strain BE60 (Pantoea spp.), strain BC3 (Priesteria megaterium), and strain B19007 (Ochrobacterium spp.) screened in Example 1 of the present invention.

[0199] Test crop: rapeseed.

[0200] Preparation of bacterial solution: The above test strains were activated by NA medium slant transfer and then inoculated into NB medium respectively, pH 7.0, 30℃, 180 r / min shaking flask culture for 24 h to obtain fermentation broth for later use.

[0201] 2. Test treatment and index determination

[0202] (1) Experimental design

[0203] Two control groups and five treatment groups were set up, with five replicates in each group. The experimental treatments are shown in Table 12. Sandy loam was selected for potted plant experiments. After the debris in the soil was sieved, it was fully mixed and packaged. The weight of each pot was kept consistent. Rapeseed seeds with basically the same size and no damage to the seed coat were selected, and 10 seeds were sown in each pot. The bacterial solution was diluted to 10 6 cfu / mL. After sowing, the treatment group was irrigated with bacterial solution, 50 mL per pot, and the control group was irrigated with the same volume of water and diluted NB medium, once every 14 days, for a total of 2 times.

[0204] Table 12 Pot experiment design

[0205]

[0206] (2) Index determination

[0207] Maximum leaf length and width: Length is measured from the leaf attachment point to the top of the leaf; width is measured at the widest part of the leaf.

[0208] Dry weight: Place the sample in a 105°C oven for 30 min, then lower the temperature to 60-80°C and dry to constant weight.

[0209] SPAD value determination: SPAD 502 chlorophyll content meter was used for determination.

[0210] Soil carbon-fixing bacteria count: Weigh 10 g of the collected soil sample, add it to a conical flask containing 90 mL of physiological saline and mix thoroughly. Place it on a shaker at 30°C and 180 r / min for 30 min to make a suspension, and perform gradient dilution. -3 , 10 -4 , 10 -5 Dilute the concentration, pipette 0.1 mL of the sample suspension into a sterile culture dish containing inorganic solid culture medium without a carbon source, spread it evenly, and culture it upside down at 30°C for 5 days.

[0211] (3) Test results

[0212] Table 13 Determination of potted rapeseed indexes

[0213]

[0214] Note: Different lowercase letters in the same column indicate significant differences ( P <0.05, the same letters in the same column indicate no significant difference ( P >0.05).

[0215] As shown in Table 13, the leaf length, leaf width, SPAD value, and dry weight of the rapeseed in the Priesteria gigantea C17 treatment group increased by 24.95%, 24.84%, 13.02%, and 36.11% respectively compared with the clear water group, and increased by 1.71%, 8.36%, 13.32%, and 8.89% respectively compared with the NB group. At the same time, the number of live carbon-fixing bacteria was the highest, increasing by 243.37% and 182.18% compared with the clear water group and the NB group, respectively. This shows that the application of Priesteria gigantea C17 bacterial solution can promote plant growth, increase SPAD value, increase dry matter accumulation, and improve soil and increase the number of carbon-fixing bacteria in the soil. When the color of the plant dry sample is greener than the control after grinding, it means that the sample may contain a higher concentration of chlorophyll or other green pigments. Generally, the higher the SPAD value, the greener the leaf color will usually be. Fig.13 It was proved that the SPAD value of plants was increased after applying Priesteria gigantea C17 bacterial solution.

[0216] Example 8: Application test of Priesteria gigantea C17 on soybean in the field

[0217] A control group and a treatment group were set up at the four-node stage (V4 stage) of soybean vegetative growth and the initial pod stage (R3 stage) of reproductive growth, respectively. On the basis of conventional fertilization, the treatment group was sprayed with Priesteria gigantea C17 bacterial solution (the bacterial powder prepared in Example 2 was diluted to 10 6 cfu / mL), the control group was sprayed with the same volume of water, and the indicators were measured at the harvest stage.

[0218] Yield and yield components: After the soybean seeds matured, 10 soybean plants were selected from each group and placed in mesh bags. After natural air drying outdoors, they were tested to investigate yield components such as the number of valid pods per plant, the number of valid grains per plant, and 100-grain weight.

[0219] Test results:

[0220] Depend on Fig.14 , 15 It can be seen that the use of Priesteria gigantea C17 can promote soybean growth, increase soybean plant height, and ultimately increase yield. Its yield increased by 9.51% compared with the control group, indicating that Priesteria gigantea C17 has a good effect of promoting growth and increasing yield.

[0221] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A strain of Priesteria gigantea with carbon fixation function ( Priestia megaterium ), characterized in that, Its biological deposit number is CCTCC NO: M 20241724.

2. A fermentation broth of Priesteria gigantea according to claim 1, characterized in that: Prepared by the following method: (1) inoculating the activated Priesteria gigantea described in claim 1 into NB culture medium and culturing on a shaking table for 12-20 hours to obtain a Priesteria gigantea seed solution; (2) inoculating the Priesteria gigantea seed liquid into a fermentation medium, and fermenting and culturing for 15-20 hours to obtain a Priesteria gigantea fermentation liquid; The fermentation medium is composed of, by mass percentage, 1.25% corn steep liquor dry powder, 1.25% peptone, 3% corn starch, 0.5% glucose, 0.1% dipotassium hydrogen phosphate, 0.1% potassium dihydrogen phosphate, 0.3% calcium chloride, 0.3% sodium chloride, 0.04% protease, 0.04% amylase, and the remainder is water.

3. The fermentation broth according to claim 2, characterized in that In step (2), the inoculation amount of Priesteria gigantea seed solution is 2-3% of the volume of the fermentation medium.

4. Use of the Priesteria gigantea according to claim 1 or the fermentation broth of the Priesteria gigantea according to claim 2 in the following (1) or (2): (1) Carbon fixation; (2) Produce carbon-fixing enzyme.

5. The use according to claim 4, characterized in that: The carbon fixation enzyme is RubisCO enzyme.

6. Use of the Priesteria gigantea according to claim 1 or the fermentation broth of the Priesteria gigantea according to claim 2 in the following (1) or (2): (1) Increase the organic carbon content in crops; (2) Prepare products that increase the organic carbon content in crops.

7. Use of the Priesteria gigantea according to claim 1 or the fermentation broth of the Priesteria gigantea according to claim 2 in the following (1) or (2): (1) Increase the organic carbon content in the soil; (2) Prepare products that increase the organic carbon content in soil.

8. Use of the Priesteria gigantea according to claim 1 or the fermentation broth of the Priesteria gigantea according to claim 2 in the following (1) or (2): (1) Increase the number of carbon-fixing bacteria in the soil; (2) Prepare products that increase the number of carbon-fixing bacteria in the soil.

9. Use of the Priesteria gigantea according to claim 1 or the fermentation liquid of the Priesteria gigantea according to claim 2 in promoting the growth of crops.

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