Rose Kocuria LAT6 and application thereof

Abstract

The invention discloses rosa rugosa LAT6, which is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number of the rosa rugosa LAT6 is CGMCC No.33523, and the preservation date of the rosa rugosa LAT6 is February 13, 2025. The invention further discloses the application of the rosa rugosa LAT6 in promoting plant growth under salt stress. A preparation method of the rosa rugosa LAT6 comprises the following steps: step S1, collecting a sample; step S2, strain separation; and S3, strain identification. According to the invention, plant rhizosphere microorganisms growing in saline-alkali soil are separated, a strain LAT6 with strong salt and alkali resistance and a growth-promoting effect is screened out, molecular biology identification shows that the strain LAT6 is rose kocuria, and tests prove that the strain LAT6 still has a remarkable growth-promoting effect on plants under the condition of saline-alkali adversity stress; and a feasible solution is provided for improving the salt tolerance of plants.

Description

Technical Field

[0001] The present invention relates to the technical field of agriculture, and in particular to Coxiella rosea LAT6 and applications thereof. Background Art

[0002] Currently, salinity stress affects over 6% of arable land worldwide, posing a significant threat to sustainable agricultural development and global food security. To address this challenge, researchers have explored various strategies to improve salt tolerance in crops, including the introduction of salt-tolerant genes, genotypic selection, and improvements to traditional breeding methods such as multigenerational hybridization and trait screening. While these approaches have achieved some success, they remain limited by high costs, long breeding cycles, and the complexity of quantitative trait improvement.

[0003] In this context, rhizosphere growth-promoting bacteria have been considered as a potential solution to alleviate saline-alkali stress in crops in recent years due to their sustainability and high cost-effectiveness. Plant growth-promoting rhizobacteria are a type of bacteria with multiple growth-promoting functions that are widely present in the rhizosphere of plants, referred to as PGPR. These microorganisms can promote plant growth and increase crop yields in saline-alkali environments, becoming one of the important strategies for coping with saline-alkali stress in the agricultural field. Rhizosphere growth-promoting bacteria exert their effects through multiple mechanisms, including regulating ion balance, enhancing nutrient absorption, promoting the synthesis of antioxidants, and reducing oxidative damage caused by the accumulation of reactive oxygen species. In addition, they can also improve the tolerance of crops to biotic and abiotic stresses, optimize physiological metabolic processes, and provide new ideas for sustainable agricultural development.

[0004] However, research on PGPR to improve saline-alkali soil and promote salt tolerance of wheat is still in its infancy. There is a lack of strains that have strong tolerance to saline-alkali soil and can still significantly promote the growth of wheat and other plants under saline-alkali stress conditions. Summary of the Invention

[0005] The purpose of the present invention is to provide Coxiella rosea LAT6 and its application to address the above problems, and to provide a practical solution for improving plant salt tolerance.

[0006] To achieve the above object, the present invention discloses Coxiella rosea ( Kocuria rosea ) LAT6, the rose Coxiella LAT6 was deposited with the General Microbiology Center of the China National Center for Microbiological Culture Collection under the deposit number CGMCC No. 33523, and the deposit date was February 13, 2025. Due to the large amount of full genome sequence data for the rose Coxiella LAT6 gene, a portion of the nucleotide sequence related to nitrogen fixation was extracted and shown in SEQ ID NO. 1.

[0007] The preparation method of Coxiella rosea LAT6 as described above comprises the following steps: Step S1, sample collection; Step S2, strain isolation; Step S3, strain identification.

[0008] Preferably, in step S1, the rhizosphere of the pioneer plant in the saline-alkali land is collected, sealed in a sterile bag, placed in an ice box and transported back to the laboratory, and the soil 1-2 mm away from the root system is taken as the rhizosphere soil, and the soil sample is stored in a refrigerator at 4°C; In step S2, 1.0 g of soil sample was weighed and placed in a container containing 10 mL of sterile water. The sample was shaken evenly and then placed on a shaker at 28°C and 150-250 rpm for 25-35 min to prepare a soil suspension. The stepwise gradient dilution method was used. 1 mL of soil suspension was placed in a container containing 9 mL of sterile water using a pipette and gradient dilution was performed in sequence to obtain 10 -2 -10 -5 gradient soil suspension; Take 10 respectively -3 , 10 -4 , 10 -5 100 μL of each dilution was spread on TSB, R2A and Gao's No. 1 solid medium, with 3 replicates per gradient. The cells were cultured in an incubator at 28°C for 3-4 days, and colonies of different morphologies and colors were selected for three-zone streaking. Inoculate a single colony into a shake tube containing 4 mL of liquid LB medium and culture overnight. When the bacterial solution becomes turbid, preserve the strain with 50% glycerol by volume. Repeat three times for each strain and store the strain in a -80°C ultra-low temperature freezer. In step S3, the glycerol bacterial suspension stored in a -80°C ultra-low temperature freezer is taken out, and the strain with a single colony is inoculated into liquid LB medium and cultured in a shaker at 28°C and 150-250 rpm for 14-18 hours. The bacterial suspension is kept for subsequent use. Take eight tubes in a row and add 6 μL of bacterial solution, then add 10 μL of buffer I containing 25 mM NaOH, 0.2 mM EDTA, pH 12, shake and mix evenly, heat at 95°C for 25-35 minutes, cool, and then add 10 μL of buffer II containing 40 mM Tris-HCl, pH 7.5, which is the bacterial solution DNA template; Universal primers 27F and 1492R were used to amplify 16S rRNA. The PCR amplification reaction system was 25 μL, including 3 μL DNA template, 12.5 μL 2× mixTaq enzyme, and 7.5 μL ddH2O. The PCR amplification reaction procedure was as follows: 30 cycles of pre-denaturation at 94°C for 2.5–3.5 min, denaturation at 94°C for 25–35 s, annealing at 55°C for 0.8–1.2 min, and extension at 72°C for 1.2–1.8 min, followed by a final extension at 72°C for 8–12 min. The PCR products were then stored at 4°C and detected by 1% agarose gel electrophoresis before sequencing. The LAT6 oxidase activity, urease activity, gelatin liquefaction ability and starch hydrolysis ability were tested, or VP test, MR test and Gram staining were performed.

[0009] Use of the above-mentioned Coxiella rosea LAT6 in the preparation of indoleacetic acid.

[0010] The use of Coxiella rosea LAT6 in nitrogen fixation as described above.

[0011] Use of Coxiella rosea LAT6 as described above in producing siderophores.

[0012] The use of the above-mentioned Coxiella rosea LAT6 in promoting plant growth under salt stress.

[0013] The application of Coxiella rosea LAT6 as described above in promoting the accumulation of photosynthetic pigments in plants under salt stress.

[0014] The use of the above-mentioned Coxiella rosea LAT6 in reducing the effects of oxidative stress on plants under salt stress.

[0015] As mentioned above, the application of Coxiella rosea LAT6 in promoting wheat growth under low nitrogen conditions.

[0016] Preferably, the application of rose Coxiella LAT6 as described above is to inoculate the rose Coxiella LAT6 strain into a culture medium and culture at 28°C to obtain a culture solution. After a single colony grows on the culture medium plate, the single colony is picked and placed in a shaking tube containing 4 mL of liquid LB, and cultured overnight at 28°C and 200 rpm for 16 hours. The bacterial solution is inoculated into 400 mL of liquid LB culture medium according to an inoculum size of 1% (v / v), and cultured overnight at 28°C and 200 rpm for 16 hours. The bacterial solution is centrifuged at 6000 rpm for 10 minutes, and then the bacteria are resuspended twice with sterile water and centrifuged at 6000 rpm for 10 minutes. The bacterial precipitate is collected, and the bacteria are mixed with soil or soaked in seeds to promote plant growth under salt stress.

[0017] Preferably, the bacterial cells are prepared into a LAT6 treatment solution using a standard Hoagland nutrient solution, the absorbance value of the LAT6 treatment solution is OD600=1.0, and the LAT6 treatment solution is mixed with soil or soaked in seeds.

[0018] In summary, the beneficial effects of the present invention are as follows: by isolating plant rhizosphere microorganisms grown in saline-alkali land, the present invention screened out a strain LAT6 with strong salt and alkali resistance and a growth-promoting effect. Molecular biological identification of strain LAT6 was conducted to identify Coxiella rosea. Experiments have shown that strain LAT6 still has a significant growth-promoting effect on plants under saline-alkali stress, providing a practical solution for improving plant salt tolerance.

[0019] Cultures and preparations of Coxiella rosea LAT6 are used as plant growth promoters, plant growth regulators, plant rooting agents, plant salt-resistance agents, plant salt-tolerance growth promoters, microbial fertilizers, fertilizer synergists, soil conditioners, or saline-alkali soil conditioners. They promote plant growth, particularly under salt stress conditions. The culture and preparations may include carriers, adjuvants, dispersants, stabilizers, penetrants, spreaders, and defoamers, with the carrier being either a solid or liquid carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a morphological diagram of the roseola LAT6 strain of the present invention.

[0021] Figure 2 This is a phylogenetic tree analysis diagram of Coxiella rosea LAT6 of the present invention.

[0022] Figure 3 This is a graph showing the test results of the nitrogen fixation ability of Coxiella rosea LAT6 of the present invention.

[0023] Figure 4 This is a diagram showing the PCR amplification results of nitrogen cycle-related genes in the genome of Coxiella rosea LAT6 of the present invention.

[0024] Figure 5a This is a graph showing the test results of the effect of the rose bacteria LAT6 of the present invention on wheat plants under salt stress when nitrogen is deficient.

[0025] Figure 5b This is a statistical diagram showing the effect of the roseola bacteria LAT6 of the present invention on wheat plants under salt stress when nitrogen is deficient.

[0026] Figure 6 This is a graph showing the detection results of the siderophore production ability of Coxiella rosea LAT6 of the present invention.

[0027] Figure 7a This is a graph showing the test results of the effect of the rose bacteria LAT6 of the present invention on wheat plants under salt stress.

[0028] Figure 7b This is a graph showing the test results of the effects of the roseola bacteria LAT6 of the present invention on physiological and biochemical indices of wheat under salt stress.

[0029] Figure 7c This is a graph showing the test results of the effect of the roseola LAT6 strain of the present invention on promoting the growth of rapeseed seedlings under salt stress.

[0030] Figure 8 This is a graph showing the detection results of the effect of the rose bacteria LAT6 of the present invention on the accumulation of photosynthetic pigments in plants under salt stress.

[0031] Figure 9This is a graph showing the detection results of the effect of Coxiella rosea LAT6 of the present invention on oxidative stress in salt-stressed wheat. DETAILED DESCRIPTION

[0032] The following embodiments of the present invention are described in further detail with reference to the accompanying drawings and examples. The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0033] Example 1, Coxsackie rosea ( Kocuria rosea ) LAT6, the roseola LAT6 was deposited with the General Microbiology Center of the China National Center for Microbiological Culture Collection under the deposit number CGMCC No. 33523, and the deposit date was February 13, 2025. Due to the large amount of full genome sequence data for the roseola LAT6 gene, a portion of the nucleotide sequence related to nitrogen fixation was extracted and shown in SEQ ID NO. 1.

[0034] The preparation method of Coxiella rosea LAT6 as described in Example 1 comprises the following steps: Step S1, sample collection; In step S1, the rhizosphere of the pioneer plant in the saline-alkali land is collected, sealed in a sterile bag, placed in an ice box and transported back to the laboratory, and the soil 1-2 mm away from the root system is taken as the rhizosphere soil. The soil sample is stored in a 4 ° C refrigerator. Specifically, the soil sample is collected from the rhizosphere of the salt-alkali land Suaeda salsa.

[0035] Step S2, strain isolation; In step S2, 1.0 g of soil sample was weighed and placed in a container containing 10 mL of sterile water, shaken evenly, and then placed on a shaker at 28°C and 150-250 rpm for 25-35 min to prepare a soil suspension; The stepwise gradient dilution method was used. 1 mL of soil suspension was placed in a container containing 9 mL of sterile water using a pipette and gradient dilution was performed in sequence to obtain 10 -2 -10 -5 gradient soil suspension; Take 10 respectively -3 , 10 -4 , 10 -5 100 μL of each dilution was spread on TSB, R2A and Gao's No. 1 solid medium, with 3 replicates per gradient. The cells were cultured in an incubator at 28°C for 3-4 days, and colonies of different morphologies and colors were selected for three-zone streaking. A single colony was inoculated into a shake tube containing 4 mL of liquid LB medium and cultured overnight. When the bacterial solution became turbid, the strain was preserved in 50% by volume glycerol. Each strain was replicated three times and stored in a -80°C ultra-low temperature freezer.

[0036] Step S3, strain identification; In step S3, take out the glycerol bacterial solution stored in the -80°C ultra-low temperature refrigerator, inoculate the strain with a single colony into liquid LB medium and culture it in a shaker at 28°C and 150-250 rpm for 14-18 hours, and the bacterial solution is ready for subsequent use; Take eight tubes in a row and add 6 μL of bacterial solution, then add 10 μL of buffer I containing 25 mM NaOH, 0.2 mM EDTA, pH 12, shake and mix evenly, heat at 95°C for 25-35 minutes, cool, and then add 10 μL of buffer II containing 40 mM Tris-HCl, pH 7.5, which is the bacterial solution DNA template; Universal primers 27F and 1492R were used to amplify 16S rRNA. The PCR amplification reaction system was 25 μL, including 3 μL DNA template, 12.5 μL 2× mixTaq enzyme, and 7.5 μL ddH2O. The PCR amplification reaction procedure was as follows: 30 cycles of pre-denaturation at 94°C for 2.5–3.5 min, denaturation at 94°C for 25–35 s, annealing at 55°C for 0.8–1.2 min, and extension at 72°C for 1.2–1.8 min, followed by a final extension at 72°C for 8–12 min. The PCR products were then stored at 4°C and detected by 1% agarose gel electrophoresis before sequencing. The LAT6 oxidase activity, urease activity, gelatin liquefaction ability and starch hydrolysis ability were tested, and VP test, MR test and Gram staining were performed.

[0037] The results showed that the oxidase activity, starch hydrolysis ability, gelatin liquefaction ability, VP and MR test results of LA1T6 were negative; the Gram staining result was positive. The 16SrRNA sequence of the strain was compared with the NCBI database, and the phylogenetic tree of the strain was constructed using MEGA software. At the same time, combined with morphological characteristics, it was identified as Coxiella rosea ( Kocuria rosea ).

[0038] Coxiella rosea ( Kocuria rosea ) Salt tolerance assay of LAT6: 1. Test method: Bacteria were inoculated into TSB liquid medium and incubated on a shaker (28°C, 200 rpm) for 16 hours. Then, 100 μL of TSB liquid medium was added to each well of a 96-well cell culture plate. For the control treatments (CK), an equal volume of TSB liquid medium was added. 2.5 μL of bacterial suspension was added to each well of the treated area, with six replicates for each bacterial strain. A salt concentration of 500 mM NaCl was used. The plates were sealed with parafilm and incubated on a shaker at 28°C, 200 rpm. The OD600 absorbance of the bacterial suspension was measured at 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 60, and 72 hours to assess the strain's tolerance to salt stress.

[0039] 2. Test results: As shown in the table, Coxiella rosea ( Kocuria rosea ) The OD600 absorbance value of LAT6 grown in 500mM NaCl liquid culture medium within 72h was higher than that of the control group, and exceeded 1.8 times that of the control group during 24-36h.

[0040]

[0041] Example 2: Use of Coxiella rosea LAT6 as described in Example 1 in the preparation of indoleacetic acid.

[0042] Coxiella rosea ( Kocuria rosea )LAT6 quantitative detection of IAA (indoleacetic acid) production test: 1. Test method: Prepare Salkowski colorimetric solution: 150 mL of concentrated H2SO4, 7.5 mL of FeCl3 (molar concentration is 0.5 mol / L), and 250 mL of H2O. Be sure to mix well and prepare it before use.

[0043] Preparation of IAA standard solution: Prepare IAA stock solution with a concentration of 100 μg / mL, accurately weigh 10.0 mg of IAA standard, dissolve it in a small amount of ethanol, and then dilute to 100 mL with distilled water. Prepare standard solutions with concentrations of 0, 10, 20, 30, 40, and 50 mg / L from the stock solution. 2 When the value is greater than 0.99, the data is reliable. The IAA standard curve is drawn with the IAA concentration as the horizontal axis and the absorbance value OD530 as the vertical axis.

[0044] Quantitative determination of the IAA production capacity of the strain: pick a single colony and inoculate it into a shaking tube containing 5 mL of R2A liquid, shake and culture at 28°C and 200 rpm for 4 days, centrifuge at 8000 rpm for 10 minutes, take 2 mL of the bacterial suspension supernatant and 2 mL of the colorimetric solution and put them into a test tube. Treat 3 parallels for each strain, place them in the dark for color development, and measure the absorbance OD530 after 30 minutes.

[0045] 2. Test results: IAA is a type of plant auxin that promotes plant cell elongation and division, thereby promoting plant growth. Therefore, the IAA production capacity of a strain can be used as an indicator of its potential to promote plant growth. As shown in the table, strain LAT6 produced 4.18 mg / L of IAA after culturing at 28°C for 4 days, demonstrating its IAA production capacity.

[0046]

[0047] Example 3: Application of Coxiella rosea LAT6 in nitrogen fixation as described in Example 1.

[0048] Coxiella rosea ( Kocuria rosea )LAT6 nitrogen fixation ability test: 1. Test method: Ashby solid medium is a selective medium lacking bound inorganic or organic nitrogen sources. When a carbon source (such as glucose or mannitol) and inorganic nutrients are available, autotrophic nitrogen-fixing bacteria can utilize free nitrogen in the air to synthesize their own nitrogen-containing organic matter. Most other microorganisms lack autotrophic nitrogen fixation, thus selectively enriching nitrogen-fixing bacteria. LAT6 was seeded onto Ashby solid medium using a toothpick. The medium was incubated upside down at 28°C for one week. The strain was observed for growth and its ability to fix nitrogen was determined.

[0049] 2. Test results: like Figure 3 As shown, LAT6 grows well on Ashby medium, demonstrating its ability to fix nitrogen. Nitrogen fixation is an important foundation for a strain's growth-promoting properties. Azotobacteria convert nitrogen from the air into a form that can be used by plants, increasing nitrogen levels in the soil and thus promoting plant growth. Nitrogen fixation can enhance a strain's adaptability to saline-alkali environments. By fixing nitrogen from the air, diazotobacteria provide a nitrogen source for themselves and other microorganisms, helping to maintain the stability of the soil microbial community and thereby enhancing the adaptability of the entire community to saline-alkali environments.

[0050] Example 4: Detection of the nitrogen-fixing gene LAT6 of Kocuria rosea.

[0051] PCR amplification reaction program: 35 cycles of pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 2 min, and final extension at 72°C for 7 min. The PCR products were detected by 1% agarose gel electrophoresis and then sequenced. The amplification results are shown in the table below. Figure 4 As shown in Figure 3, nitrogen transport-related genes can be amplified in the T6 genome, indicating that the LAT6 strain itself can encode nitrogen transport-related proteins and play a role in nitrogen transport.

[0052] PCR amplification primers are as follows:

[0053] NRT,narK,nrtP,nasA The nucleotide sequence of primer F is shown in SEQ ID NO.2; NRT,narK,nrtP,nasA The nucleotide sequence of the R primer is shown in SEQ ID NO.3; NRT,narK,nrtP,nasA The nucleotide sequence of primer F is shown in SEQ ID NO.4; NRT,narK,nrtP,nasA The nucleotide sequence of the R primer is shown in SEQ ID NO.5; narI,narV The nucleotide sequence of primer F is shown in SEQ ID NO.6; narI,narV The nucleotide sequence of the R primer is shown in SEQ ID NO.7; narH、narY,nxrB The nucleotide sequence of primer F is shown in SEQ ID NO.8; narH、narY,nxrB The nucleotide sequence of the R primer is shown in SEQ ID NO.9; narG、narZ,nxrA The nucleotide sequence of primer F is shown in SEQ ID NO.10; narG、narZ,nxrA The nucleotide sequence of the R primer is shown in SEQ ID NO.11.

[0054] Example 5: Application of Kocuria rosea LAT6 in promoting wheat growth under low nitrogen conditions.

[0055] Fielder wheat seeds were surface disinfected with 2% sodium hypochlorite solution for 10 minutes, then rinsed three times with sterile water, and grown hydroponically for about 5 days. Seedlings with consistent growth were selected for the following experimental treatments. The experiment set up two nitrogen concentration gradients: low nitrogen and normal nitrogen. All experimental groups contained 250mM NaCl. The experimental group was added with LA1T6 bacterial solution with OD=1.0, and the volume was 1 / 10 of the volume of the hydroponic nutrient solution. After the experimental materials grew for two weeks, the lengths of the underground and above-ground parts were counted respectively. The statistical results are as follows Figure 5a 、 Figure 5b LAT6 can still alleviate salt stress in wheat under nitrogen deficiency conditions, indicating that the LAT6 strain can better help wheat absorb and utilize nitrogen.

[0056] Normal nitrogen nutrient solution formula: 1mM Ca(NO3)2, 0.2mM KH2PO4, 0.5mM MgSO4, 1.5mM KCl, 1.5mM CaCl2, 1×10 −3 mM H3BO3, 5×10 −5 mM (NH4)6Mo7O 24 ,5×10 −4 mM CuSO4, 1×10 −3 mM ZnSO4, 1×10 −3 mM MnSO4, 0.1 mM Fe(III)–EDTA.

[0057] Low nitrogen nutrient solution formula: Based on the normal nitrogen nutrient solution formula, except for replacing 0.02mM Ca(NO3)2 and 2.48mM CaCl2 (to compensate for the Ca2+ concentration in the nutrient solution), other ingredients remain unchanged.

[0058] Example 6: Use of Coxiella rosea LAT6 as described in Example 1 in producing siderophores.

[0059] Coxiella rosea ( Kocuria rosea ) LAT6 siderophore production capacity test: 1. Test method: Use a toothpick to pick up a single colony on CAS (Citrate-Ammonium-Sulfate) solid medium, place the inoculated medium in a 28°C incubator and incubate it upside down for 1 week, and observe whether a transparent circle appears to infer whether it can produce siderophores. When the strain grows on CAS solid medium, if it can produce siderophores, the siderophores will react with Fe in the medium. 3+ Specific binding. 3+ The affinity of CAS is much higher than that of CAS, so it will be transferred from CAS-Fe 3+ Extraction of Fe from the complex 3+ , leading to the release of CAS. Free CAS appears red, forming a yellow or orange-red halo around the colony. The size of the halo is related to the strain's ability to produce siderophores; a larger halo indicates a stronger siderophore-producing strain.

[0060] 2. Test results: like Figure 6 As shown, LAT6 grows well on CAS medium and produces a clear zone, indicating its ability to produce siderophores. Iron is one of the trace elements essential for plant growth and development and is involved in many important physiological processes, such as photosynthesis, respiration, and nitrogen metabolism. Siderophore-producing strains can promote root development, chlorophyll synthesis, and overall growth by increasing iron absorption. In saline-alkali soils, iron availability is low, making it difficult for plants to absorb sufficient iron to meet their growth needs. Strains capable of producing siderophores can secrete siderophores, which bind to iron ions in the environment to form soluble iron complexes, thereby increasing the plant's iron absorption efficiency and helping plants grow better in saline-alkali environments.

[0061] Example 7: Use of Coxiella rosea LAT6 as described in Example 1 in promoting plant growth under salt stress.

[0062] Specifically, the plant is wheat or rapeseed. Wheat is one of the most important food crops in the world and plays a vital role in ensuring world food security. However, its yield is facing severe challenges due to various environmental stresses, among which soil salinization is one of the most threatening factors. Salt-alkali stress can seriously hinder wheat growth and disrupt key physiological and metabolic processes such as protein synthesis, lipid metabolism, photosynthetic efficiency, water absorption and ion balance, thereby affecting seedling development and reducing crop yield.

[0063] Under salt stress, Coxsackie rosea ( Kocuria rosea )Effects of LAT6 on physiological and biochemical parameters of wheat: 1. Experimental setup: A total of four treatments were set up in this experiment, namely CK (Hoagland's nutrient solution, no bacterial solution inoculation, no salt addition), LAT6 (Hoagland's nutrient solution inoculated with bacterial solution, no salt addition), NaCl (Hoagland's nutrient solution without bacterial solution inoculation, 200mM salt addition), and NaT6 (Hoagland's nutrient solution inoculated with bacterial solution, 200mM salt addition).

[0064] 2. Coxiella rosea ( Kocuria rosea ) Preparation of LAT6 bacterial suspension: The glycerol bacterial suspension in the -80°C ultra-low temperature freezer was activated and placed in an inverted culture in a 28°C biochemical incubator. After a single colony grew on the culture plate, the single colony was picked and placed in a shaking tube containing 4 mL of liquid LB. The culture was shaken overnight at 28°C and 200 rpm for 16 h. The bacterial suspension was inoculated into 400 mL of liquid LB medium at a 1% (v / v) inoculum size and cultured again overnight under the same culture conditions. The bacterial suspension that had been cultured for 16 h was centrifuged (6000 rpm, 10 min) and resuspended twice in sterile water under the same centrifugation conditions. The bacterial pellet was collected and the absorbance of the bacterial suspension was adjusted to OD600 = 1.0 using standard Hoagland's nutrient solution and Hoagland's nutrient solution containing 400 mM NaCl, respectively, to prepare T6 and NaT6 treatment solutions.

[0065] 3. Salt stress growth promotion test: First, select wheat seeds with full grains and uniform appearance size and treat them in the dark at 4℃ for 48 hours. Rinse with clean water 2-3 times to remove impurities on the surface of the seeds, then disinfect the wheat seeds with 20% sodium hypochlorite solution. Finally, rinse the wheat seeds with clean water 3-4 times and place them in a culture room at 25℃ with 16h light and 8h dark for 3-4 days. Mix equal volumes of vermiculite and nutrient soil in a black square basin (9cm×9cm×10cm), and thoroughly mix the prepared bacterial suspension with the soil. Wheat seedlings with consistent growth were selected for transplanting, with 5 wheat seedlings placed in each pot, 6 pots per treatment, and a total of 30 seedlings. The wheat seedlings were cultured in a growth chamber at 25°C, a light intensity of 6000 lx, and a photoperiod of 16 h light / 8 h dark. The seeds were treated once a week according to the experimental settings (CK: Hoagland's nutrient solution, no inoculation of bacterial solution, no salt treatment; LAT6: Hoagland's nutrient solution inoculated with bacterial solution, no salt treatment; NaCl: Hoagland's nutrient solution not inoculated with bacterial solution, with 200 mM salt treatment; NaT6: Hoagland's nutrient solution inoculated with bacterial solution, with 200 mM salt treatment). After 2 weeks, the growth phenotypes of wheat (plant height, aboveground fresh weight, aboveground dry weight, underground fresh weight, and underground dry weight) were evaluated and counted.

[0066] 4. Test results: like Figure 7a 、 Figure 7b As shown in Figure 2, inoculation of bacterial suspension under salt stress can significantly increase the biomass of wheat ( P <0.05), wheat plant height, aboveground fresh weight, aboveground dry weight, underground fresh weight, and underground dry weight increased by 40.0%, 30.38%, 29.44%, 6.28%, and 20.0%, respectively. The relative electrical conductivity of wheat leaves increased sharply under salt stress, reaching 4.28 times that of the control treatment ( P <0.05). Wheat leaves inoculated with strain LAT6 under salt stress showed a significant decrease in relative electrical conductivity. These results indicate that inoculation with strain LAT6 can significantly increase wheat biomass under salt stress and alleviate salt stress damage.

[0067] Under salt stress, Coxsackie rosea ( Kocuria rosea ) Effects of LAT6 on the Growth Promotion of Rapeseed Seedlings: 1. Experimental setup: Three treatments were set up in this experiment, namely CK (Hoagland's nutrient solution, no bacterial solution inoculation, no salt treatment), NaCl (Hoagland's nutrient solution without bacterial solution inoculation, 200mM salt treatment), and NaT6 (Hoagland's nutrient solution inoculated with bacterial solution, 200mM salt treatment).

[0068] 2. Coxiella rosea ( Kocuria rosea ) Preparation of LAT6 bacterial suspension: The bacterial solution cultured for 16 h was centrifuged (6000 rpm, 10 min), and the cells were resuspended twice in sterile water under the same centrifugation conditions as above. The bacterial pellet was collected and resuspended in Hoagland's nutrient solution containing 200 mM NaCl. The absorbance of the bacterial solution was adjusted to OD600 = 1.0 to prepare the NaT6 treatment solution.

[0069] 3. Salt stress growth promotion test: First, select rapeseed seeds with plump, uniform grains and an apparent size and rinse them 2-3 times with clean water to remove surface impurities. The seeds are then disinfected with a 20% sodium hypochlorite solution and rinsed 3-4 times with clean water. The seeds are then placed in a dark environment at 25°C for 3 days to germinate. Selected rapeseed seedlings with consistent growth are transferred to petri dishes for treatment. Eight rapeseed seedlings are placed per dish, with three replicates (24 seedlings) per treatment, for a total of 72 rapeseed seedlings across the three treatments. These seedlings are cultured in a growth chamber at 25°C, a light intensity of 6000 lx, and a photoperiod of 16 h light / 8 h dark. After 10 days, the rapeseed growth phenotypes (fresh weight, number of root tips, number of root branch points, total root length, root branch frequency, and root network area) are evaluated and analyzed.

[0070] 4. Test results: like Figure 7c As shown in the figure, whole rapeseed seedlings were weighed and statistically analyzed after treatment. The results showed that LAT6 addition significantly increased the fresh weight of rapeseed seedlings under salt stress, with an increase of 34.81% compared to the salt treatment without LAT6. Furthermore, root scanning and statistical analysis of rapeseed seedling roots revealed that compared to the salt-stress-free treatment without LAT6, the number of root tips, root branch points, total root length, root branch frequency, and root network area increased by 82.81%, 112.19%, 39.16%, 55.18%, and 36.86%, respectively, in the LAT6-treated rapeseed seedlings. In summary, LAT6 significantly promotes the growth of rapeseed seedlings under salt stress.

[0071] Example 8: Use of Coxiella rosea LAT6 as described in Example 1 in promoting the accumulation of photosynthetic pigments in plants under salt stress.

[0072] Specifically, plant photosynthetic pigments include chlorophyll a, chlorophyll b or carotenoids.

[0073] Specifically, the plant includes wheat or rapeseed.

[0074] Under salt stress, Coxsackie rosea ( Kocuria rosea )Effects of LAT6 on photosynthetic pigment content in wheat leaves: 1. Test method: The photosynthetic pigment content of wheat leaves treated with different treatments (same as in Example 7) was measured to evaluate the effects of the inoculated strain LAT6 on wheat photosynthesis. Two weeks after treatment, 0.05 g of fresh wheat leaves were weighed and placed in a 1.5 mL centrifuge tube. The leaves were thoroughly ground on ice and then incubated overnight in the dark at 4°C until the leaves completely faded. The photosynthetic pigment content of the wheat leaves was then measured. The following formula was used to calculate the content of the photosynthetic pigment: C chlorophyll a = 13.95 × A665 - 6.88 × A649; C chlorophyll b = 24.96 × A649 - 7.32 × A665; C carotenoids = (1000 × A470–2.05 × Ca-114.8 × Cb) / 245; C total chlorophyll = C chlorophyll a + C chlorophyll b; Pigment content (mg / g) = pigment concentration (mg / L) × extract volume (mL) × 10-3 × dilution factor / sample mass (g); 2. Test results: like Figure 8 As shown in the figure, the analysis of photosynthetic pigment content in wheat under different treatments showed that the photosynthetic pigment content in wheat under salt stress was significantly reduced ( P <0.05). After inoculation with strain LAT6 under salt stress, the contents of chlorophyll a, chlorophyll b, chlorophyll ab, and carotenoids in wheat leaves increased by 33.3%, 20.0%, 19.3%, and 20.8%, respectively. Among them, the content of chlorophyll a was significantly different between the NaCl treatment and the NaT6 treatment, indicating that the inoculation strain LAT6 can significantly increase the content of chlorophyll a in leaves under salt stress ( P <0.05). Chlorophyll a directly participates in light reactions, converting light energy into chemical energy. It is the core pigment of the light reaction center of photosynthesis. This suggests that strain LAT6 can enhance the photosynthetic capacity of wheat and improve its salt tolerance.

[0075] Example 9: Use of Coxiella rosea LAT6 as described in Example 1 in reducing the effects of oxidative stress on plants under salt stress.

[0076] Specifically, the plant includes wheat or rapeseed.

[0077] Under salt stress, Coxsackie rosea ( Kocuria rosea ) Effects of LAT6 on oxidative stress in wheat: 1. Test method: The activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in wheat leaves under different treatments (the treatments were the same as those in Example 7) were determined using the kits provided by Suzhou Grace Biotechnology Co., Ltd. to explore the effects of inoculation with strain LAT6 on the production of plant antioxidant enzyme systems under salt stress.

[0078] Superoxide dismutase (SOD) assay method: (1) Sample preparation: Take approximately 0.1 g of tissue (0.25 g for samples with sufficient moisture), add 1 mL of extract, and homogenize at 4°C or in an ice bath (or use a common homogenizer). Centrifuge at 4°C × 12,000 rpm for 10 min, and collect the supernatant as the test solution.

[0079] (2) On-machine testing: ① Preheat the microplate reader for more than 30 minutes and adjust the wavelength to 450nm.

[0080] ② Before the test, thaw reagents 1, 2, 3, and 4 to room temperature (25°C), or place them in a water bath at 25°C for 5-15 minutes.

[0081] ③ Use a row gun to reduce the error caused by the time of adding reagents between wells.

[0082] ④ Add reagents to the 96-well plate in the order shown in the table below:

[0083] (3) Calculation of results: Calculation of inhibition percentage: Inhibition percentage = [(A 空白管1 -A 空白管2 )-(A 样本管 -A 样本对照管* )] / (A 空白管1 -A 空白管2 )×100%; If no sample A control tube was prepared, substitute the value of 0 into the formula to calculate the inhibition percentage; maintain the inhibition percentage within the range of 30-80%. 1: If less than 30%, increase the sample mass W (e.g., to 0.2g) or increase the sample volume V1 (e.g., from 20μL to 50μL or more, reduce the amount of Reagent 1 accordingly, keeping the total volume unchanged). 2: If greater than 80%, dilute the crude sample extract with distilled water or extraction solution. Substitute the modified W and V1 and the dilution factor D into the formula for calculation.

[0084] Calculation of SOD enzyme activity: SOD enzyme activity unit: When the inhibition percentage in the above xanthine oxidase coupled reaction system is 50%, the SOD enzyme activity in the reaction system is defined as one enzyme activity unit (U / mL).

[0085] SOD activity (U / g fresh weight) = [inhibition percentage ÷ (1-inhibition percentage) × V2] ÷ (W × V1 ÷ V) × D = 10 × inhibition percentage ÷ (1-inhibition percentage) ÷ W × D; V-volume of extract added, 1 mL; V1-volume of sample added to the reaction system, 0.02 mL; V2-total volume of the reaction system, 0.2 mL; D-sample dilution factor, undiluted is 1; W-sample mass, g.

[0086] Peroxidase (POD) assay method: (1) Sample preparation: Weigh approximately 0.1 g of tissue, add 1 mL of extraction solution, and homogenize on ice. Centrifuge at 4°C × 12,000 rpm for 10 min, remove the supernatant, and place on ice for testing.

[0087] (2) On-machine testing: ① Preheat the microplate reader for more than 30 minutes and adjust the wavelength to 470nm.

[0088] ② Thaw reagents 1, 2, and 3 to room temperature (25°C) before measurement.

[0089] ③Add the following into the 96-well plate:

[0090] (3) Calculation of results: Definition of enzyme activity: One enzyme activity unit (U) is the amount of absorbance at 470 nm that increases by 1 per gram of tissue per minute in the reaction system.

[0091] POD (△OD470 / min / g fresh weight) = ΔA ÷ (W × V1 ÷ V) ÷ 1 ÷ T × D = 100 × ΔA ÷ W × D; V---the volume of the extract added, 1 mL; V1---the volume of the sample added, 0.01 mL; T---reaction time, 1 min; W---sample mass, g; D---dilution multiple, undiluted is 1; Catalase (CAT) assay method: (1) Sample preparation: Weigh approximately 0.1 g of tissue, add 1 mL of extraction solution, and homogenize on ice. Centrifuge at 4°C × 12,000 rpm for 10 min, remove the supernatant, and place on ice for testing.

[0092] (2) On-machine testing: ① Preheat the microplate reader for more than 30 minutes and adjust the wavelength to 510nm.

[0093] ② Prepare reagent 2 in advance according to the reagent preparation requirements, and then perform the following operations.

[0094] ③ Test the blank tube first (do this only once): 80μL Reagent 1 + 20μL Reagent 2 + 100μL Reagent 3, mix immediately, take 10μL, and immediately add the sample and test according to the color development reaction in step ⑥. The absorbance value is the A blank.

[0095] ④Suggestion: Since the reaction time is 5 minutes, if there are many samples to be tested at one time, the samples can be tested in batches.

[0096] ⑤Add the following into the EP tube in sequence:

[0097] ⑥ Color reaction:

[0098] (3) Calculation of results: Unit definition: At 25°C, one enzyme activity unit (U) is defined as the amount of enzyme activity that catalyzes the decomposition of 1 μmolH2O2 per gram of tissue per minute.

[0099] CAT (μmoL / min / g fresh weight) = [(ΔA + 0.0137) ÷ 0.1412] ÷ (W × V1 ÷ V) ÷ T × D = 141.6 × (ΔA + 0.0137) ÷ W × D; V---the volume of the extract added, 1mL; V1---sample volume added, 0.01mL; T---reaction time, 5min; W---sample mass, g; D---dilution multiple, undiluted is 1; Appendix: Standard curve preparation process: ①Prepare standard stock solution (250mM): ② Dilute the stock solution to the following concentrations: 0, 50, 100, 150, 200, 250 mM.

[0100] ③20μL standard + 80μL reagent 1 + 100μL reagent 3, mix well, take 10μL of the mixture, and operate according to the measurement tube sample addition system in the color reaction stage. The standard curve can be prepared based on the results.

[0101] 2. Test results: like Figure 9 As shown in Figure 2, compared with the control, the activities of SOD and POD in wheat leaves under salt stress were significantly reduced by 31.4% and 29.41% ( P<0.05). Inoculation with strain LAT6 under salt stress increased SOD, POD, and CAT activities by 48.19%, 37.66%, and 21.13%, respectively. Malondialdehyde (MDA) is a metabolite of membrane lipid peroxidation, reflecting the degree of plant damage under adverse conditions. MDA content in wheat leaves inoculated with strain LAT6 under salt stress decreased by 24.44%. These results suggest that inoculation with strain LAT6 can enhance antioxidant enzyme activity in wheat and promote the removal of excess reactive oxygen species in the body.

[0102] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and substitutions can be made without departing from the technical principles of the present invention. These improvements and substitutions should also be regarded as the scope of protection of the present invention.

Claims

1. Coxiella rosea ( Kocuria rosea )LAT6, characterized in that The rose Coxsackie species LAT6 is deposited in the General Microbiology Center of the China Culture Collection Administration, with the deposit number CGMCC No. 33523 and the deposit date being February 13, 2025.

2. Use of Coxiella rosea LAT6 as claimed in claim 1 in the preparation of indoleacetic acid.

3. Use of Coxiella rosea LAT6 as claimed in claim 1 in nitrogen fixation.

4. Use of Coxiella rosea LAT6 as claimed in claim 1 in producing siderophores.

5. Use of Coxiella rosea LAT6 as claimed in claim 1 in promoting plant growth under salt stress.

6. Use of Coxiella rosea LAT6 as claimed in claim 1 in promoting the accumulation of photosynthetic pigments in plants under salt stress. 7 . Use of Coxiella rosea LAT6 according to claim 1 in reducing the effects of oxidative stress on plants under salt stress.

8. Use of Coxiella rosea LAT6 according to any one of claim 1 in promoting wheat growth under low nitrogen conditions.

9. The use of Coxiella rosea LAT6 according to any one of claims 5 to 7, characterized in that The rose Coxiella LAT6 strain is inoculated into a culture medium and cultured at 28°C to obtain a culture solution. After a single colony grows on a culture medium plate, the single colony is picked and placed in a shaking tube containing 4 mL of liquid LB, and cultured overnight at 28°C and 200 rpm for 16 hours. The bacterial solution is inoculated into 400 mL of liquid LB culture medium according to an inoculum volume of 1% (v / v), and cultured overnight at 28°C and 200 rpm for 16 hours. The bacterial solution is centrifuged at 6000 rpm for 10 minutes, and the bacteria are resuspended twice with sterile water and centrifuged at 6000 rpm for 10 minutes. The bacterial precipitate is collected and mixed with soil or used for soaking seeds to promote plant growth under salt stress.

10. The use of Coxiella rosea LAT6 according to claim 9, characterized in that The bacteria are prepared into LAT6 treatment solution using standard Hoagland nutrient solution, the absorbance value of the LAT6 treatment solution OD600 = 1.0, and the LAT6 treatment solution is mixed with soil or soaked in seeds.

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