Acidovorax vorenii and application thereof in growth promotion and yield increase of rice

Abstract

The invention relates to acidovorax vorenii and application thereof in growth promotion and yield increase of rice, and belongs to the technical field of microorganisms. A strain of acidovorax wonderi D2 is separated from rice roots through a dilution plate coating method, the acidovorax wonderi D2 is identified as acidovorax wonderi through 16S rRNA gene sequence analysis, the two strains (D2 and SM13) are both preserved in the China Center for Type Culture Collection (CCTCC), the preservation numbers are respectively CCTCC M 2025811 and CCTCC M 2025812, and the preservation date is April 17, 2025. The acidovorax warneri D2 can cooperate with bacillus subtilis SM13 to enhance growth promotion (IAA production, phosphorus solubilization and nitrogen fixation) on rice and tolerate soda salt (Na2CO3: NaHCO3 = 1: 9) stress (200 mmol / L), and the rice yield is increased by 12.48%. The ratio of the two strains is 1: 1, the composite bacterial liquid can be used as a microbial agent of a microbial fertilizer for the rice in the cold region, the tillering number, the plant height, the overground dry matter weight and the rice yield in the tillering stage of the rice are remarkably increased, data support is provided for application of synthetic microbial communities in the rice cultivation process, and the application prospect is wide.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms, and in particular relates to an acidovorax walii and an application thereof in promoting the growth and increasing the yield of rice. Background Art

[0002] In recent years, the use of microbial agents to promote plant growth and increase crop yields has become a hot topic in agricultural research. Currently, Bacillus subtilis has been widely studied and applied, promoting crop growth by secreting plant hormones, antagonizing pathogens, and inducing systemic resistance. However, its effectiveness when used alone is limited by environmental conditions (such as soil pH and nutrient competition), resulting in unstable results in field applications. Therefore, further exploration and optimization of synergistic microorganisms is expected to significantly enhance its effectiveness in promoting crop growth and increasing yield.

[0003] Acidovorax wautersii has been shown to promote plant growth and alleviate saline-alkali stress in agricultural applications. Related research has shown that the bacterium can also biotransform nitrates, which is important for reducing the use of phosphate fertilizers and protecting the ecological environment. However, there have been no reports of synergistic effects between the bacterium and Bacillus subtilis. Summary of the Invention

[0004] The present invention aims to provide a strain of Acidovorax wollastonii and its application in promoting rice growth and yield. By screening a strain of Acidovorax wollastonii (named D2) from rice roots, the present invention can enhance the growth-promoting effect of Bacillus subtilis, breaking through the application bottleneck of a single strain.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] Acidovorax wautersii is classified as Acidovorax wautersii D2, deposited in the China Center for Type Culture Collection with a deposit number of CCTCC M 2025811 and a deposit date of April 17, 2025, and its 16S rRNA gene sequence is shown in SEQ ID NO: 1.

[0007] Furthermore, the A. walii has the ability to secrete IAA, dissolve inorganic phosphorus and organic phosphorus, fix nitrogen, and tolerate soda salts at a concentration of 0-150 mmol / L, and significantly improves the yield-increasing ability of Bacillus subtilis on crops.

[0008] An application of the above-mentioned Acidovorax wollastonii in promoting the growth and increasing the yield of rice is specifically: compounding Acidovorax wollastonii with Bacillus subtilis to form a composite bacterial liquid.

[0009] Furthermore, the Bacillus subtilis is classified as Bacillus subtilis SM13, deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC M 2025812 and a deposit date of April 17, 2025.

[0010] Furthermore, the ratio of the number of viable bacteria of the compound of Acidovorax walii and Bacillus subtilis is 1:1.

[0011] Furthermore, the composite bacterial solution has the ability to have no antagonism between strains, produce IAA, dissolve inorganic phosphorus and organic phosphorus, fix nitrogen, and tolerate soda salts at a concentration of 0-200 mmol / L.

[0012] Furthermore, the application is specifically as follows: during the rice greening period, 22.8L / mu of composite bacterial solution (D2 strain and SM13 strain in a ratio of 1:1) is applied with irrigation water, and the bacterial concentration reaches 10 9 CFU / mL.

[0013] Furthermore, the composite bacterial solution can increase the number of rice tillers, panicles, panicle weight and panicle length, and the rice yield increase is 12.48%.

[0014] Compared with the prior art, the present invention has the following characteristics:

[0015] 1. The present invention isolates a strain of Acidovorax walii D2 from the root system of rice and has the functions of promoting plant growth such as soda salt and alkali resistance, nitrogen fixation, dissolution of inorganic phosphorus, dissolution of organic phosphorus, and production of IAA, and has the ability to synergize with Bacillus subtilis.

[0016] 2. The composite bacterial liquid provided by the present invention uses Bacillus subtilis SM13 and Acidovorax wautersii D2. Both strains are deposited in the China Center for Type Culture Collection with the deposit numbers CCTCC M 2025812 and CCTCC M 2025811, and the deposit date is April 17, 2025; the deposit address is Wuhan University, Wuhan City; the composite bacterial liquid has the characteristics of no antagonism between strains, IAA production, solubility of inorganic phosphorus and organic phosphorus, and soda salt resistance.

[0017] 3. The composite bacterial liquid provided by the present invention can significantly increase the number of tillers, plant height, above-ground dry matter weight and rice yield during the tillering period of rice, providing data support for the application of synthetic microbial communities in the rice cultivation process and providing microbial agents for increasing rice yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1This is a morphological diagram of a single colony isolated from a plate of Paracidovorax wautersii D2 in Example 1 of the present invention.

[0019] Figure 2 This is a transmission electron micrograph of Paracidovorax wautersii D2 in Example 1 of the present invention.

[0020] Figure 3 Schematic diagram of the phylogenetic tree of 16S rRNA of Paracidovorax wautersii D2 in Example 1 of the present invention.

[0021] Figure 4 This is a colony morphology diagram of the growth of Paracidovorax wautersii D2 in Example 1 of the present invention under different soda salt (Na2CO3:NaHCO3=1:9) concentrations (mmol / L) stress.

[0022] Figure 5 This is a colony morphology diagram of the growth of Paracidovorax wautersii D2 in Example 1 of the present invention on CAS solid culture medium.

[0023] Figure 6 This is a diagram showing the compatibility of Paracidovorax wautersii D2 and Bacillus subtilis SM13 on a NA solid plate in Example 2 of the present invention.

[0024] Figure 7 This is a colony morphology diagram of the growth of the composite bacterial solution in Example 2 of the present invention on a CAS solid plate.

[0025] Figure 8 This is a graph showing the colony morphology ability of the composite bacterial solution in Example 2 of the present invention under stress of different soda salt (Na2CO3: NaHCO3=1:9) concentrations (mmol / L).

[0026] Figure 9 This is a diagram showing the dynamic changes in the number of tillers and plant height of potted rice from the greening stage to the end of tillering in Example 3 of the present invention.

[0027] Figure 10 This is a graph showing the dynamic changes in the activities of five soil enzymes at various growth stages of rice potted plants in Example 3 of the present invention. DETAILED DESCRIPTION

[0028] The technical solutions of the present application are further described below in combination with the drawings and examples, but are not limited thereto, and any modification or equivalent replacement to the technical solutions of the present application without departing from the spirit and scope of the present application shall be covered in the protection scope of the present application.

[0029] The screening and function determination method of Paracidovorax wautersii D2 of the present application are as follows:

[0030] 1. Collection of rice root samples: The rice root samples were collected in Duerbet Mongolian Autonomous County, Daqing City, Heilongjiang Province (N46°6', E124°18'), the collected root samples were placed in sterile sealed bags, and the information such as collection site, date, etc. was marked and taken back to the laboratory on the same day for processing and stored in a 4°C refrigerator.

[0031] 2. Isolation and purification of strains: The endophytic bacteria in rice roots were isolated by dilution plate coating method, and the strains were purified by plate streaking method after a period of culture. The above steps were repeated for 3 times until the purified D2 strain was obtained.

[0032] 3. Strain identification: The purified D2 strain was subjected to morphological observation, transmission electron microscope observation and 16S rRNA gene sequence analysis.

[0033] 4. Determination of growth-promoting function of strains: The growth-promoting function of D2 strain was verified, and the determination indexes included IAA production ability, inorganic phosphorus solubilization ability, organic phosphorus solubilization ability, siderophore production ability, nitrogenase activity and soda salt tolerance.

[0034] Further, the application method of Paracidovorax wautersii D2 comprises the following steps:

[0035] 1. Strain compounding: The compatibility of Bacillus subtilis SM13 and Paracidovorax wautersii D2 was detected by plate cross streaking method.

[0036] 2. Mixing with Bacillus subtilis SM13: The Paracidovorax wautersii D2 bacterial liquid and the Bacillus subtilis SM13 bacterial liquid were mixed according to the ratio of 1:1 of viable bacteria, to obtain a compound bacterial liquid.

[0037] 3. Function determination of compound bacterial liquid: The IAA production ability, inorganic phosphorus solubilization ability, organic phosphorus solubilization ability, siderophore production ability, nitrogenase activity and soda salt tolerance of the compound bacterial liquid were determined.

[0038] Further, the application method of the compound bacterial liquid on rice crops comprises the following steps: 22.8L / acre of mixed liquid (D2 strain and SM13 strain according to the ratio of 1:1) is applied with irrigation water at the rice green-up stage, and the bacterial concentration reaches 10 9CFU / mL. During the growth period of rice planting, the plant height and tillering dynamics of rice at tillering stage, the activity of potted soil enzymes (phosphatase, urease, catalase, dehydrogenase, invertase), the dry matter weight of potted aboveground and underground parts, the root-shoot ratio, the yield and yield component factors of rice were determined to verify the growth promotion and yield increasing effect of the compound bacterial liquid on rice through the above indexes.

[0039] Example 1: Isolation, purification, identification and function determination of Acidovorax valsis D2

[0040] I. Isolation and purification of Acidovorax valsis D2 strain

[0041] The preparation of NA solid medium (g / L): 10.0 g of proteose peptone, 5.0 g of sodium chloride, 3.0 g of beef extract, 15.0 g of agar powder, distilled water to 1000 mL, pH 7.2, wet heat sterilization 121℃, 25 min.

[0042] Rice root samples were collected from Duerbet Mongolian Autonomous County of Daqing City in Heilongjiang Province (N46°6', E124°18'), and the samples were put into sterile bags and taken back for processing on the same day, and stored in a 4℃ refrigerator. The surface of the rice roots was washed thoroughly with sterile water, the excess water on the root surface was absorbed with sterilized filter paper, the roots were soaked in 75% ethanol for 5 min, after pouring out the ethanol, 0.1% HgCl2 was added for 10 min, and finally the roots were washed 6-7 times with sterile water, and the excess water on the root surface was absorbed with sterilized filter paper. 100 μL of the water used to wash the root surface the last time was spread on the NA solid medium plate to detect the sterilization effect of the rice root surface, and no cultivable bacteria were found, indicating that the root surface was completely sterilized. 1 g of the surface-sterilized rice root sample was cut into 1-2 cm pieces with sterile scissors, and was ground thoroughly in a sterilized mortar containing sterile quartz sand and 9 mL of sterile water. The root sample was ground thoroughly, and then diluted with sterile water to obtain bacterial suspensions with dilution degrees of 10 -1 , 10 -2 and 10 -3 , respectively. 100 μL of the bacterial suspensions with different dilution degrees were spread on the NA solid medium, and each dilution degree was repeated 3 times, and cultured at 30℃ for 2 d. After 2 d, colonies with different colony morphology, color and size were selected for isolation, and the isolated strains were streaked on the NA solid medium for purification for more than 3 times, and the purification results are shown in Figure 1 .

[0043] II. Morphological and molecular identification of Acidovorax valsis D2 strain

[0044] The strain was streaked and purified on the NA solid medium, and the colony morphology was observed after 24 h of culture as follows: the colony was round, small, light yellow, the surface was moist, the edge was smooth and neat, and the center was convex, and the results are shown in Table 1. The colony morphology results are shown in Figure 1 Transmission electron microscopy results are shown in FIG. 2. The size of the strain was measured to be 0.54 μm x 1.80 μm. Figure 2 Transmission electron microscopy results are shown in FIG. 2. The size of the strain was measured to be 0.54 μm x 1.80 μm.

[0045] Table 1 Colony morphological characteristics

[0046]

[0047] DNA extracted according to the method of Tian Gen Biochemical Technology (Beijing) Co., Ltd. bacterial genomic DNA kit was used as a template, and bacterial universal primers 27F and 1492R were used for PCR amplification. The PCR system was 50 μL: 2 x phantaMax Mix 25 μL, primer 27F (50 pmol) 0.5 μL, primer 1492R (50 pmol) 0.5 μL, DNA template 1 μL, and deionized water 23 μL. The PCR reaction conditions were: 94°C pre-denaturation for 5 min; 94°C denaturation for 1 min, 55°C annealing for 1 min, 72°C extension for 1 min, a total of 30 cycles; 72°C final extension for 10 min. The amplified PCR product was tested by agarose gel electrophoresis, and then the PCR product was sent to Shenzhen Huada Gene Technology Service Co., Ltd. for sequencing, and a sequence of 1391 bp in length was obtained. The sequencing results were subjected to BLAST comparison in the GenBank (NCBI) database, and the gene sequence of a model strain with high homology was used as a reference object, and a phylogenetic tree was constructed by MEGA 11.0 software, and the results are shown in FIG. 3. Figure 3 The results show that the homology of the D2 strain and Paracidovorax wautersii V4640 (directly submitted sequence, not published) is as high as 100.00%.

[0048] III. Determination of the growth-promoting function of the Paracidovorax wautersii D2 strain

[0049] 1. Determination of the IAA-producing ability of the Paracidovorax wautersii D2 strain

[0050] Preparation of Salkowski colorimetric reagent: (1) 0.5 M FeCl3 solution: 0.811 g of FeCl3 was dissolved in 10 mL of distilled water; (2) 35% HCIO4 solution: 25 mL of 70% HCIO4 solution was diluted to 50 mL with distilled water. (3) Mixing: 1 mL of (1) and 49 mL of (2) were mixed.

[0051] First, the activated Paracidovorax wautersii D2 strain was inoculated in NA liquid medium, and when the bacterial concentration OD 600When the OD value reaches 0.8, the strain was used and inoculated into 10 mL of NA liquid medium (tryptophan content: 500 mg / L) at a 1% inoculum volume. Repeat three times; 0.1 mL of sterile water was used as a blank control. Culture was shaken at 30°C and 180 rpm for 7 days. The bacterial suspension was collected and centrifuged at 10,000 rpm for 10 minutes. 100 μL of the supernatant was added to an ELISA plate, followed by 200 μL of Salkowski colorimetric solution. The reaction was allowed to stand in the dark for 35 minutes. The OD value of each concentration of the standard solution was measured using a spectrophotometer. 530 The results were analyzed by measuring the absorbance of standard concentration A530. The regression equation y = 0.0242x + 0.0187 (R 2 =0.9982), and the results are shown in Table 2. The IAA concentration of strain D2 calculated from the regression equation was 14.52 ± 2.94 μg / mL, indicating that strain D2 can use tryptophan (Trp) as a precursor to synthesize IAA through a series of enzymatic reactions, thereby promoting plant growth and development. The results are shown in Table 3.

[0052] Table 2 IAA standard curve determination

[0053]

[0054] Table 3 Quantitative determination of IAA secretion by D2 strain

[0055]

[0056] 2. Determination of the Inorganic Phosphate Solubility of Acidovorax walii D2

[0057] Preparation of inorganic phosphorus liquid culture medium (g / L): 10.0 g glucose, 0.5 g ammonium sulfate, 0.5 g yeast extract powder, 0.3 g sodium chloride, 0.3 g potassium chloride, 0.3 g magnesium sulfate, 0.03 g ferrous sulfate, 0.03 g manganese sulfate, 5.0 g calcium phosphate, dilute to 1000 mL with distilled water, pH 7.2, and sterilize at 121°C for 25 min.

[0058] First, activate the Acidovorax walii D2 in NA liquid medium, and centrifuge the activated D2 strain at 10,000 r / min for 5 minutes to obtain a bacterial suspension. Determine the OD value of the bacterial suspension. 600After the inoculum is 0.8, it is inoculated into 10 mL of liquid culture medium in inorganic phosphorus culture medium at a rate of 1%, and repeated 3 times; an inorganic phosphorus culture medium inoculated with 1% sterile water is used as a control. Culture at 30°C and 180 r / min for 5 days, collect the bacterial suspension and centrifuge at 10,000 r / min for 10 minutes, take 1 mL of the supernatant and put it into a 50 mL volumetric flask, add distilled water to about 30 mL, add 2 drops of dinitrophenol indicator, adjust the solution with sodium hydroxide solution or sulfuric acid solution until it is just slightly yellow, then add 5.0 mL of molybdenum antimony anti-color agent, dilute to volume with distilled water, and repeat 3 times. The results are analyzed using the measured absorbance of the standard concentration A530. 700 The absorbance is taken as the ordinate and the inorganic phosphorus concentration is taken as the abscissa, and the regression equation y=0.0572x+0.0534(R 2 =0.999), the results are shown in Table 4. The dissolved inorganic phosphorus concentration of strain D2 converted from the regression equation was 20.44±1.17 mg / L, indicating that strain D2 has the ability to dissolve insoluble phosphate in the environment, thereby promoting the solubility and utilization of unavailable phosphorus in the soil by plants, as shown in Table 5.

[0059] Table 4 Standard curve of inorganic phosphorus

[0060]

[0061] Table 5 Quantitative determination of inorganic phosphorus released by strain D2

[0062]

[0063] 3. Determination of the ability of Acidovorax walii D2 to degrade organic phosphorus

[0064] Preparation of organophosphorus liquid culture medium (g / L): 10.0 g glucose, 0.5 g ammonium sulfate, 0.3 g sodium chloride, 0.3 g magnesium sulfate, 0.03 g manganese sulfate, 0.3 g potassium sulfate, 0.03 g ferrous sulfate, 5.0 g calcium phosphate, 0.2 g lecithin, dilute to 1000 mL with distilled water, pH 7.2, and sterilize at 121°C for 25 min.

[0065] The determination method is the same as the above-mentioned inorganic phosphorus decomposition capacity. 700 With absorbance as the ordinate and organophosphorus concentration as the abscissa, the regression equation y=0.0542x+0.0794(R 2 =0.9994). The dissolved organic phosphorus concentration of strain D2 was calculated from the regression equation to be 2.65 ± 0.22 mg / L, as shown in Table 7. This indicates that strain D2 is able to absorb and utilize organic phosphorus in the soil, maintaining a dynamic balance between inorganic and organic phosphorus in the plant-soil system, reducing the amount of chemical phosphorus fertilizer applied, and promoting phosphorus activation.

[0066] Table 6 Standard curve of organophosphorus

[0067]

[0068] Table 7 Quantitative determination of organic phosphorus degraded by strain D2

[0069]

[0070] 4. Determination of Nitrogenase Activity of Acidovorax walii D2

[0071] After 18 hours of nitrogen-free incubation, 5 mL of bacterial culture was transferred to a 25 mL test tube. The tube was tightly sealed with a rubber stopper to prevent air leakage. 1 mL of air was withdrawn from the tube and 1 mL of acetylene gas was injected into the tube. After the air in the tube was mixed, 1 mL of gas was drawn into the gas chromatograph using a 1 mL syringe and injected into the gas chromatograph to measure the peak areas of ethylene and acetylene. After 3 hours of incubation in a constant temperature shaker, 1 mL of gas was again drawn into the gas chromatograph using a 1 mL syringe and injected into the gas chromatograph to measure the peak areas of ethylene and acetylene. Gas chromatograph parameters included: column oven temperature 60°C, injector 120°C, flame ionization detector (FID) at 250°C, and flow rates of N2, H2, and dry air at 30, 30, and 300 mL / min, respectively.

[0072] Calculate the acetylene reduction (ARA value) (μmol / mL·h): Λ=(Ae-Aa) / V / t

[0073] Ae: initial acetylene concentration; Aa: acetylene concentration after incubation; t: reaction time (h); V: container volume.

[0074] The formula shows that the acetylene reduction capacity of the D2 strain reaches 103.01±20.53 pmol / ml / h. Nitrogenase enables plants to utilize nitrogen in the environment more efficiently, reduce dependence on chemical nitrogen fertilizers, and provide a direct source of nitrogen for plant growth.

[0075] 5. Determination of the soda salt tolerance of A. walii D2 strain

[0076] Preparation of soda salt medium: Based on sterilized NA medium, NaCO3 and NaHCO3 were filtered into NA solid medium at a molar ratio of 1:9 using a 0.22 μm filter membrane.

[0077] The activated D2 strain was centrifuged at 10000 r / min for 5 min to obtain a bacterial suspension. The OD of the bacterial suspension was measured. 600The pH value was 0.8, and 2 μL of bacterial suspension was inoculated into NA solid medium containing 0 mmol / L, 50 mmol / L, 100 mmol / L, 150 mmol / L, 200 mmol / L, and 250 mmol / L soda salt concentrations. The medium was inverted and cultured at 28°C for 5 days, and the growth of the colonies was recorded. The D2 strain could tolerate soda salt in the range of 0-150 mmol / L. The results are shown in Table 8 and Figure 4 shown.

[0078] Table 8 Growth of D2 strain at different soda salt concentrations

[0079]

[0080] Note: +: strain grew, -: strain did not grow.

[0081] 6. Determination of Siderophore Production by Acidovorax walii D2

[0082] Preparation of CAS solid medium (mg / L): 60.50 mg chrome azurol S, 72.90 mg hexadecyltrimethylammonium bromide (HDTMA), 2.65 mg ferric chloride hexahydrate, 296.25 mg sodium dihydrogen phosphate dihydrate, 1213.50 mg disodium hydrogen phosphate dodecahydrate, 125.00 mg ammonium chloride, 37.50 mg potassium dihydrogen phosphate, 62.50 mg sodium chloride, 9000.00 mg agar powder, dilute to 1000 mL with distilled water, pH 6.8, and sterilize at 121°C for 25 min.

[0083] The activated D2 strain was centrifuged at 10000 r / min for 5 min to prepare the OD 600 0.8 bacterial suspension, aspirate 2 μL of bacterial suspension and inoculate into CAS solid medium, invert and culture at 28℃ for 2 days, record the growth of colonies, the results are as follows Figure 5 As shown in the figure, the D2 strain can grow on CAS solid medium, but it does not form a transparent zone and does not have the ability to produce siderophores.

[0084] Example 2: Synergistic application of Acidovorax walii D2 and Bacillus subtilis SM13

[0085] 1. Compatibility test of Acidovorax walii D2 strain and Bacillus subtilis SM13

[0086] The D2 and SM13 strains were inoculated into NA liquid medium for activation. A small amount of D2 and SM13 strains were picked up with an inoculation loop and streaked on NA solid medium. The culture was carried out at 28°C for 2 days, and the colony formation was recorded. The results were as follows: Figure 6 As shown in the figure, there is no antagonistic effect between D2 and SM13, and it can be used for the construction of composite bacteria in the future.

[0087] 2. Preparation of the composite bacterial solution of Acidovorax walii D2 strain and Bacillus subtilis SM13

[0088] The D2 strain and SM13 strain were inoculated into NA liquid medium for activation and the OD 600 The bacterial cell number ratio of D2 and SM13 was 1:1 and they were mixed for subsequent growth-promoting function determination and rice growth-promoting and yield-increasing effect determination.

[0089] 3. Determination of the growth-promoting function of the complex bacteria of Acidovorax walii D2 and Bacillus subtilis SM13

[0090] The growth-promoting properties of the composite bacteria were determined using the following indicators: IAA production, inorganic and organic phosphorus solubilization, nitrogenase activity, soda salt tolerance, and siderophore production. The assay method was the same as for the single strain A. wollastonii D2 described above. The results of the composite bacteria were compared with those of the individual strains to verify whether the D2 and SM13 composite solution had a synergistic effect on SM13. Table 9 shows the results of IAA secretion, inorganic and organic phosphorus solubilization, and nitrogenase activity of the composite solution and SM13. This table indicates that the composite solution showed significant differences compared to the individual strains D2 and SM13, indicating that the combination of the two strains achieved a superior growth-promoting effect.

[0091] Table 9 Summary of the growth-promoting functions of the composite bacterial solution and SM13

[0092]

[0093] The tolerance of soda salt is shown in Table 10 and Figure 8 As shown in the figure, the soda salt tolerance of the composite bacterial solution reached 200 mmol / L, which was more tolerant to soda salt than the single strain D2 (150 mmol / L). Figure 7 As shown in the figure, it can be seen that the composite bacteria do not have the ability to produce siderophores. In summary, the composite bacterial solution of D2 and SM13 can synergize with SM13 and improve the ability of SM13 to secrete IAA, dissolve inorganic phosphorus, organic phosphorus and tolerate soda salt.

[0094] Table 10 Growth of composite bacterial solution at different soda salt concentrations

[0095]

[0096] Note: +: strain grew, -: strain did not grow.

[0097] Example 3: Effect of promoting rice growth and increasing yield

[0098] Based on the composite bacterial solution and SM13 of the above examples, the rice variety Kenjing No. 8 was selected for testing at the rice potting field of Heilongjiang Bayi Agricultural University in Daqing City, Heilongjiang Province (125.155°E, 46.581°N).

[0099] The pot experiment adopted a single-factor experimental design with three treatments and 10 replicates for each treatment. The treatment settings are shown in Table 11. The potting bucket was 32.0 cm high, 30.0 cm in diameter at the top, and 27.0 cm in diameter at the bottom. Each pot had four holes. Seven days after the seedlings were transplanted, the bacterial solution of each treatment was applied at a concentration of 1×10 9 CFU / mL, the amount of bacteria applied is 22.8L / mu, and the application of chemical fertilizers refers to Song Weimin et al. (2022).

[0100] Table 11 Potted plant treatment settings

[0101]

[0102] The tillering and plant height of rice were investigated during the greening period and the tillering peak period of rice growth. 10 fixed rice holes were selected as the survey objects for each treatment. The plant height and tiller number of rice were measured. The results are as follows: Figure 9 As shown in the results, the application of the composite bacterial solution at the end of tillering (34-37 days after application) had a significant promoting effect on rice plant height and tiller number.

[0103] During the rice growth period, fresh rice rhizosphere soil (soil depth 0-15 cm) was collected at the tillering stage, jointing stage, heading stage, grain filling stage, and maturity stage to measure soil enzyme activity. The measured indicators included soil sucrase, catalase, dehydrogenase, urease, and phosphatase activities. The soil enzymes were measured using the 3,5-dinitrosalicylic acid colorimetric method, potassium permanganate titration method, TTC reduction method, phenol-sodium hypochlorite colorimetric method, and disodium phenyl phosphate colorimetric method, respectively. The results are as follows: Figure 10 As shown, application of the composite bacterial solution significantly boosted sucrase activity during the tillering stage, increasing the efficiency of carbohydrate conversion in the soil and enhancing the ability of soil microorganisms to decompose organic matter, thereby providing more absorbable nutrients for rice growth and promoting rice tillering. Furthermore, during the heading stage, the composite bacterial solution significantly boosted catalase activity. This increased catalase activity helps maintain soil redox balance, ensuring healthy root growth and nutrient absorption.

[0104] In contrast, in the soil enzyme activity tests at different periods, the effect of the composite bacterial solution on the activities of dehydrogenase, urease and phosphatase did not reach a significant level, but there was a trend of increasing enzyme activity in certain growth stages, such as soil dehydrogenase.

[0105] The samples were taken at the grain filling stage of rice growth, and the representative plants of each treatment were selected according to the average stem number, 4 holes were taken for each treatment, that is, 4 times of repetition for each treatment, the fresh samples were collected, the leaves, stems, sheaths, ears and roots of rice were separated respectively and placed in a constant temperature oven for drying, 105 DEG C killed green, 80 DEG C dried to constant weight and weighed, and the results are shown in Table 12. Compared with the control (CK), the composite bacterial solution can significantly increase the stem, sheath, ear, total dry weight and root dry weight of rice at the grain filling stage, and compared with SM13, the composite bacterial solution can significantly increase the stem, total dry weight and root dry weight of rice at the grain filling stage.

[0106] Table 12 Dry weight of aboveground and underground parts of rice under different treatments

[0107]

[0108] Theoretical and actual yield of rice was determined after the rice entered the mature stage. 10 hole samples of each rice potting treatment were taken back to the laboratory for investigation of ear traits and theoretical yield, and the determination indexes included: ear number, filled grain number, empty grain number, primary branch (branch number, filled grain number, empty grain number, filled grain weight) and secondary branch (branch number, filled grain number, empty grain number, filled grain weight) traits. The ear trait index, yield and its constituent factors were calculated by determining the index, and the results are shown in Tables 13-15. Compared with the control (CK), the composite bacterial agent can significantly increase the hole ear number, hole ear weight and yield of rice; and compared with SM13, the composite bacterial agent has no significant difference. In the yield component analysis, for the primary branch (Table 14), compared with the control (CK), the composite bacterial agent significantly increased the primary branch filled grain number and the primary branch filled grain weight, and compared with SM13, the composite bacterial agent significantly increased the primary branch number, primary branch filled grain number and primary branch filled grain weight; for the secondary branch (Table 15), the empty and empty number of the secondary branch of the composite bacterial agent was significantly higher than that of the control (CK) and the single bacterium SM13.

[0109] In summary of the above Example 3, the composite bacterial solution significantly increased the tiller number of rice at the tillering stage, significantly increased the ear weight of rice at the grain filling stage which was the key stage to determine the yield of rice, and also found that the composite bacterial solution could increase the yield of rice at the mature stage, increased the yield by 12.48% compared with the control, significantly increased the primary branch filled grain number and the primary branch filled grain weight. The above data can provide data support for the composite bacteria to increase the yield of rice.

[0110] Table 13 Results of ear number, ear weight, ear length and yield of rice potting under each treatment

[0111]

[0112] Table 14 Results of primary branch related traits of rice potting under each treatment

[0113]

[0114] Table 15 Results of secondary tiller related traits of rice pot experiment of each treatment

[0115]

[0116] SEQ ID NO: 1

[0117]

Claims

1. Acidovorax walii, characterized in that: The classification of the Acidovorax walii is named Acidovorax wautersii D2 was deposited in the China Center for Type Culture Collection with the deposit number CCTCC M 2025811 and the deposit date of April 17, 2025. The 16S rRNA gene sequence is shown in SEQ ID NO:

1.

2. The Acidovorax walii according to claim 1, characterized in that: The Acidovorax walii has the ability to secrete IAA, dissolve inorganic phosphorus and organic phosphorus, fix nitrogen, and tolerate soda salts at a concentration of 0-150 mmol / L, and significantly improves the yield-increasing ability of Bacillus subtilis on crops.

3. A use of the Acidovorax walii of claim 1 in promoting rice growth and yield, characterized in that: The application is specifically: compounding Acidovorax walii and Bacillus subtilis to form a composite bacterial liquid.

4. The use according to claim 3, characterized in that: The classification of the Bacillus subtilis is named Bacillus subtilis SM13, deposited in China Center for Type Culture Collection, with the deposit number CCTCC M 2025812 and the deposit date of April 17, 2025.

5. The use according to claim 3, characterized in that: The ratio of the number of viable bacteria of the compounded Acidovorax walii and Bacillus subtilis is 1:

1.

6. The use according to claim 3, characterized in that: The composite bacterial liquid has the abilities of no antagonism between strains, producing IAA, dissolving inorganic phosphorus and organic phosphorus, fixing nitrogen, and being resistant to soda salts at a concentration of 0-200 mmol / L.

7. The use according to claim 3, characterized in that: The specific application is: during the rice greening period, add 22.8L / mu of composite bacterial solution (D2 strain and SM13 strain in a ratio of 1:1) with irrigation water, and the bacterial concentration reaches 10 9 CFU / mL.

8. The use according to claim 3, characterized in that: The composite bacterial liquid can increase the number of rice tillers, panicles, panicle weight and panicle length, and the rice yield increases by 12.48%.

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