Marinobacter strain and application thereof

Abstract

The invention provides a marinobacter strain, which is named as Marinobacter kunyu, is preserved in the China General Microbiological Culture Collection Center (CGMCC), and has the preservation number of CGMCC No.29757. The marinobacter strain is named as Marinobacter kunyu. The preservation date is January 25, 2024, and the 16S rRNA nucleotide sequence is as shown in SEQ ID NO: 1. The invention also provides an application for improving saline-alkaline resistance of plants, promoting plant growth, reducing salt and alkali of soil, increasing nitrogen fixation capability of plants and increasing the content of effective iron and phosphorus elements in soil. The strain disclosed by the invention has the functions of resisting salt and alkali, reducing salt and alkali, fixing nitrogen, decomposing organic phosphorus, dissolving inorganic phosphorus, producing iron carriers, producing IAA, producing amylase, producing volatile acidic substances, producing oxidase and promoting plant growth. The compound fertilizer can be used for improving saline-alkali resistance of plants, reducing saline-alkali of soil, increasing nitrogen fixation capacity of the plants, increasing the content of effective iron and phosphorus nutrient elements in the soil and promoting plant growth, and can also be prepared into a functional fertilizer with plant growth promoting and saline-alkali soil improving effects for use.

Description

Technical Field

[0001] This invention belongs to the field of microbial application technology, specifically relating to a strain of the genus *Hymenobacter* and its application. Background Technology

[0002] Soil salinization is one of the major abiotic stress factors affecting global crop production, and its causes can be divided into primary (natural causes) and secondary (human-caused causes). Currently, the expanding area of ​​soil salinization globally has become one of the major abiotic stress factors affecting crop production, seriously impacting the sustainable development of the agricultural economy.

[0003] Salt stress affects plant growth primarily in two ways: firstly, increased salt concentration lowers root osmotic pressure, complicating water absorption and causing osmotic stress; secondly, salt stress leads to the accumulation of ions within the plant, causing ion toxicity. Bio-based soil improvement technology, widely recognized in the industry as a low-cost and effective method for remediating saline-alkali land, can effectively promote the reuse of such land. Microbial fertilizers, as a novel type of fertilizer, have been proven to improve soil salinity, enhancing fertilizer utilization while also promoting plant growth and improving crop quality.

[0004] Therefore, inoculating saline-alkali land with microorganisms to improve saline-alkali land and promote plant growth is a low-cost, long-lasting, green and pollution-free biological improvement method. Screening functional strains is an important prerequisite for microbial treatment methods, which is of great significance for the rational development and utilization of saline-alkali land resources, ensuring sustainable agricultural development, and improving the ecological environment. Summary of the Invention

[0005] The technical problem this invention aims to solve is to address the shortcomings of the prior art by providing a *Hymenobacter* strain and its applications. This strain possesses functions such as salt and alkali tolerance, salt and alkali reduction, nitrogen fixation, organic phosphorus solubilization, inorganic phosphorus solubilization, iron carrier production, IAA production, amylase production, volatile acidic substance production, oxidase production, and plant growth promotion. It can be used to improve plant salt and alkali tolerance, reduce soil salinity, increase plant nitrogen fixation capacity, increase the content of available iron and phosphorus nutrients in the soil, and promote plant growth. It can also be prepared into a functional fertilizer with plant growth-promoting and saline-alkali soil improvement effects.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a strain of the genus *Marinobacter*, named *Marinobacterkunyu*, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 29757; the deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; the deposit date is January 25, 2024; the 16S rRNA nucleotide sequence of *Marinobacterkunyu* is shown in SEQ ID NO: 1.

[0007] The present invention also provides the application of the above-mentioned Marinobacter genus strain, which is used to improve the salt and alkali tolerance of plants, promote plant growth, reduce soil salinity and alkalinity, increase the nitrogen fixation capacity of plants, and increase the content of available iron and phosphorus in the soil.

[0008] Preferably, the plants include Arabidopsis thaliana, tomato, and corn.

[0009] Preferably, when the plant is Arabidopsis thaliana, the Marinobacterkunyu is used to promote the increase of the number of lateral roots, root length, fresh weight, and number of leaves of the Arabidopsis thaliana; When the plant is a tomato, the Marinobacterkunyu is used to promote an increase in the number of lateral roots and fresh weight of the tomato. When the plant is corn, the Marinobacterkunyu is used to promote an increase in the fresh weight of the leaves, the fresh weight of the stems, and the plant height of the corn.

[0010] Preferably, the Marinobacterkunyu is used in the preparation of microbial fertilizers that promote plant growth and improve saline-alkali land.

[0011] Preferably, when the Marinobacterkunyu is used as a microbial fertilizer, it has at least one application in reducing soil salinity, fixing nitrogen, producing iron carriers, decomposing organic phosphorus, dissolving inorganic phosphorus, producing IAA, producing amylase, producing oxidase, and producing volatile acidic substances.

[0012] Compared with the prior art, the present invention has the following advantages: The *Marinobacter kunyu* strain of this invention possesses functions such as salt and alkali tolerance, salt and alkali reduction, nitrogen fixation, organic phosphorus solubilization, inorganic phosphorus solubilization, iron carrier production, IAA production, amylase production, volatile acidic substance production, oxidase production, and plant growth promotion. It can be used to improve plant salt and alkali tolerance, reduce soil salinity, increase plant nitrogen fixation capacity, increase the content of available iron and phosphorus nutrients in the soil, and promote plant growth. It can also be prepared into a functional fertilizer with plant growth-promoting and saline-alkali soil improvement effects.

[0013] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0014] Figure 1 This is the colony morphology of the Marinobacterkunyu KY-590 strain from Example 1 of this invention.

[0015] Figure 2 This is the Gram staining result of the Marinobacterkunyu KY-590 strain from Example 1 of this invention.

[0016] Figure 3 This is the result of amylase production by the Marinobacterkunyu KY-590 strain in Example 1 of this invention.

[0017] Figure 4 This is the phylogenetic tree of the Marinobacterkunyu KY-590 strain from Example 1 of this invention.

[0018] Figure 5 This is a genomic circle diagram of the Marinobacterkunyu KY-590 strain from Example 1 of this invention.

[0019] Figure 6 These are the growth curves of the Marinobacterkunyu KY-590 strain under different conditions in Example 2 of this invention.

[0020] Figure 7 This is a diagram showing the nitrogen fixation results of the Marinobacterkunyu KY-590 strain in Example 3 of this invention.

[0021] Figure 8 This is a diagram showing the results of organophosphate decomposition by the Marinobacterkunyu KY-590 strain in Example 3 of this invention.

[0022] Figure 9 This is a graph showing the results of inorganic phosphorus dissolution by the Marinobacterkunyu KY-590 strain in Example 3 of this invention.

[0023] Figure 10 This is a graph showing the results of siderogenic vector production by the Marinobacterkunyu KY-590 strain in Example 3 of this invention.

[0024] Figure 11 This is the IAA standard curve of Embodiment 3 of the present invention.

[0025] Figure 12This is a diagram showing the results of inoculating the Marinobacterkunyu KY-590 strain of the present invention on a medium containing neutral red indicator for 48 hours, as described in Example 3 of the present invention.

[0026] Figure 13 This is a graph showing the results of placing the Marinobacterkunyu KY-590 strain on neutral red medium for 24 hours in an open container, as described in Example 3 of this invention.

[0027] Figure 14 This is a graph showing the results of neutral red culture medium containing the Marinobacterkunyu KY-590 strain of Example 3 of the present invention, after being kept uncovered for 24 hours.

[0028] Figure 15 The figures show the growth-promoting effect of the Marinobacterkunyu KY-590 strain of Example 4 of this invention on Arabidopsis thaliana, along with a bar chart.

[0029] Figure 16 The figures show the growth-promoting effect of the Marinobacterkunyu KY-590 strain of Example 4 of this invention on tomatoes, along with a bar chart.

[0030] Figure 17 The figures show the growth-promoting effect of the Marinobacterkunyu KY-590 strain of Example 4 of this invention on maize, along with a bar chart. Detailed Implementation

[0031] Example 1 This example describes the isolation, purification, and identification of the Marinobacterkunyu KY-590 strain.

[0032] Isolation and purification of Marinobacterkunyu KY-590 strain The strain in this embodiment was isolated from the rhizosphere soil of wild Tamarix chinensis in Kunyu, Xinjiang Uygur Autonomous Region. After removing the surface soil, large clods of soil and decaying leaves, it was placed in a sterile bag and transported back to the laboratory.

[0033] (1) Weigh 5.0 g of soil sample in a clean bench, add 45 mL of water and vortex mix. After standing for 2-5 min, the original soil solution is obtained. (2) Take 1 mL of the original soil solution and add it to 9 mL of water to obtain a concentration gradient of 10. -1 The soil suspension was then subjected to gradient dilution to obtain 10... -2 10 -3 10 -4 Soil gradient suspension; (3) After the soil suspensions of different concentration gradients were vortexed and mixed, 100 μL of each suspension was spread on high-salt and high-alkali LB solid medium (NaCl 7.5%, pH 9), and each condition was repeated 3 times. (4) Invert the petri dish and place it in a 30℃ constant temperature incubator for 48 h; (5) Select a single colony for at least three streak isolation cultures to obtain a pure culture of microorganisms that can survive in a high-salt and high-alkali environment, and record the colony morphology; (6) The microorganisms obtained in (5) above were inoculated into LB liquid medium (pH=9, NaCl 75g / L) and cultured in a constant temperature shaking incubator at 30℃ and 180 r / min. The resulting bacterial solution was added to 50% glycerol at a ratio of 1:1 and stored at -80℃ for later use. The bacterial solution was then inoculated onto LB slant medium and cultured at 30℃ for 7 days before being stored at 4℃ for later use.

[0034] II. Identification of Marinobacterkunyu KY-590 strain (a) Morphological identification The KY-590 strain was inoculated onto LB solid medium (pH=9, NaCl 75g / L), and colony morphology was observed. The colony characteristics of the KY-590 strain on the medium were: small colonies, milky white and translucent, moist surface with raised areas, and neat edges. The results are shown in Table 1 and 2. Figure 1 The KY-590 strain turned red after Gram staining, indicating that it is a Gram-negative bacterium. The results are as follows: Figure 2 As shown.

[0035] Table 1. Colony morphology of strain KY-590 (II) Identification of the physiological and biochemical characteristics of Marinobacterkunyu KY-590 strain Micro-fermentation tubes and oxidase test strips were purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd. KY-590 strain was inoculated into these tubes or separately into corresponding functional identification media to obtain the physiological and biochemical characteristics of KY-590 strain. The identification results are shown in Table 2. KY-590 strain can grow using inulin, muscarin, arabinose, galactose, menobiose, and fructose as its sole carbon source. It can also produce amylase to hydrolyze starch as an energy source and produce oxidase, which provides a foundation for the production and application of this strain. The amylase production capacity of KY-590 strain is shown in Table 2. Figure 3 . Table 2 Physiological and biochemical indicators of strain KY-590 Note: "+" indicates a positive result; "-" indicates a negative result. Identification of the 16S rRNA gene of strain KY-590 The 16S rRNA gene sequence of a single colony was amplified by PCR using the universal primers for bacterial 16S rRNA gene: 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTT ACGACTT-3′).

[0036] PCR reaction system (25 μL): 12.5 µL of 2×Rapid Taq Master Mix, 1 µL each of upstream and downstream primers (10 µmol / L), 0.5 µL of DNA template, and 10 µL of ddH2O; PCR reaction conditions: pre-denaturation 95℃ for 5 min; denaturation 95℃ for 30 s, annealing 60℃ for 30 s, extension 72℃ for 2 min, 35 cycles; final extension 72℃ for 5 min.

[0037] The PCR products were sequenced (Sangon Biotech (Shanghai) Co., Ltd.) for verification. The sequencing results were BLASTed using NCBI (https: / / www.ncbi.nlm.nih.gov / ) and registered in the NCBI database. The constructed phylogenetic tree is as follows: Figure 4 As shown, strain KY-590 is most closely related to the type strains of the genus *Marinobacter alinus*, strain Hb8 (NR_156086.1) and *Marinobacter lipolyticus* strain SM-19 (NR_025671.1), with a 16S rRNA gene sequence similarity of 98.33%. Therefore, this strain is a potential new species of the genus *Marinobacter*.

[0038] (iv) Identification of the whole genome sequence of strain KY-590 After extracting the genome from KY-590, whole-genome sequencing was performed. The genome map is shown below. Figure 5 The complete genome characteristics of KY-590 are as follows: the genome is 5,471,767 bp in length, with a G+C content of 55.59%. Strain KY-590 contains one plasmid with a full length of 940,139 bp. It is predicted to encode 5,370 genes, accounting for 89.8% of the total genome. Among the non-coding RNA genes, there are 3 16S rRNA genes, 3 23S rRNA genes, 3 5S rRNA genes, and 118 tRNA genes.

[0039] Based on the genome sequencing results, the closest relatives to the strain KY-590 of this invention are the type strains Marinobactersalinus strain Hb8 (NR_156086.1) and Marinobactersimilis strain A3d10. T (CP007151.1). Strains KY-590 and Hb8 T The dDDH value was 16.03–21.47%, similar to that of strain A3d10. T The dDDH values ​​ranged from 16.03% to 21.50%, all below the threshold for describing a new species (<70%). KY-590 and the type strain Hb8 T The ANI value was 76.56%, which was similar to that of the model strain A3d10. T The ANI value was 76.86%, significantly lower than the interspecific ANI standard (95%–96%), as shown in Table 3. The low ANI and dDDH values ​​provide stronger support for KY-590 as a new species.

[0040] Table 3. Genomic comparison between strain KY-590 and the model strain In summary, strain KY-590 differs significantly from the type strain of the most homologous *Gymnema*, possessing low levels of ANI and dDDH values, which is sufficient to prove that strain KY-590 is a new species of *Gymnema*.

[0041] Based on molecular biological results, it was named *Marinobacterkunyu* and deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 29757; the deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; the deposit date is January 25, 2024; the 16S rRNA nucleotide sequence of the *Marinobacterkunyu* KY-590 strain is shown in SEQ ID NO: 1. The discovery and identification of this strain further enriches the resources of beneficial microbial strains, especially salt-tolerant and growth-promoting strains.

[0042] Example 2 This example illustrates the growth characteristics of the MarinobacterkunyuKY-590 strain.

[0043] I. Growth curve of Marinobacterkunyu KY-590 strain Strain strain KY-590 was inoculated into liquid LB medium and cultured until OD. 600The pH range was 0.6–0.8. Bacterial suspensions were inoculated at 1% into liquid LB medium at pH 9 with NaCl concentrations of 0‰, 1‰, 2‰, 3‰, 6‰, 9‰, 15‰, and 7.5%. The absorbance of the bacterial suspensions at 600 nm was measured every 3 hours, and growth curves were plotted based on the absorbance values. The growth curves of strain KY-590 in media with different salt concentrations are shown below. Figure 6 This provides theoretical data for the production of this strain.

[0044] II. Salt tolerance of Marinobacterkunyu KY-590 strain Prepare LB solid medium with pH 9 and NaCl concentrations ranging from 0% to 20% (in 2% intervals). Inoculate the strain into the liquid medium and culture until OD... 600 When the pH is 0.6–0.8, 100 μL of bacterial culture is spread onto culture dishes with different salt concentrations, with three replicates for each salt concentration. The culture dishes are incubated upside down at 30°C in a constant temperature incubator, and the microbial growth is observed. The salt tolerance of strain KY-590 is shown in Table 4. The results show that at pH 9, strain KY-590 can tolerate a salt concentration range of 0–200 g / L, indicating that this strain has excellent salt tolerance.

[0045] Table 4. Salt tolerance test of strain KY-590 Note: +: bacterial growth, −: bacterial non-growth.

[0046] III. Alkali tolerance of Marinobacterkunyu KY-590 strain Prepare LB solid medium with 7.5% NaCl and pH values ​​ranging from 7.0 to 13.0 (in increments of 1.0). Inoculate the strain into the liquid medium and culture until OD... 600 When the pH is 0.6–0.8, 100 μL of bacterial culture is spread onto culture dishes with different salt concentrations, with three replicates for each salt concentration. The culture dishes are incubated upside down at 30 °C in a constant temperature incubator, and the growth of microorganisms is observed. The alkali tolerance of strain KY-590 is shown in Table 5. At a salt concentration of 75 g / L, strain KY-590 can grow normally between pH 7 and 13, exhibiting excellent alkali tolerance.

[0047] Table 5. Alkali tolerance test of strain KY-590 Note: +: bacterial growth, −: bacterial non-growth.

[0048] IV. Salt-reducing ability of Marinobacterkunyu KY-590 strain Strains of KY-590 were inoculated into LB liquid medium with pH values ​​of 8, 9, and 10 and a NaCl concentration of 75‰. Every 12 hours, 200 μL of bacterial culture was taken, and 7.5 μL of 20% potassium chromate was added as an indicator. Titration was performed with 0.1 M AgNO3 solution. The titration endpoint was defined as a brick-red color that did not fade upon shaking. The salt-reducing capacity of strain KY-590 was calculated and shown in Table 6. The results indicate that strain KY-590 achieved the highest salt-reducing rate (15.47%) at pH 10. This demonstrates that strain KY-590 can effectively improve the properties of saline-alkali soil, help plants resist the saline-alkali environment, and provide microbial strain resources for future plant growth and soil improvement in saline-alkali areas.

[0049] Perform three parallel determinations and calculate the average value. Substitute the titration result into the formula to calculate Cl. - Quantity C2: In the formula: C1 is the concentration of the AgNO3 solution, in mol / L; V1 is the volume of AgNO3 solution consumed in the titration, in mL; V2 is the volume of the bacterial solution during titration, in mL.

[0050] Substitute C2 into the formula to calculate the salt reduction rate η: In the formula: C represents the Cl in the culture medium. - The initial concentration, mol / L; Table 6 Results of salt reduction capacity determination of KY-590 strain V. Alkali-reducing ability of Marinobacterkunyu KY-590 strain The bacterial strain was inoculated into LB liquid medium at pH 9 with NaCl concentrations of 3‰, 6‰, 9‰, 15‰, and 75‰. Every 12 hours, 3 mL of bacterial culture was collected, centrifuged at 5000 g for 5 min, and the supernatant was collected. The pH value of the bacterial culture was measured using a pH meter. The measurements were performed in triplicate, and the average value was calculated. The results were then used to calculate the alkalinity reduction rate: Alkalinity reduction rate = (pH - pH1) ÷ pH × 100% In the formula: pH refers to the initial pH value of the culture medium; pH1 is the pH value measured after inoculation with the strain.

[0051] The alkali reduction capacity of strain KY-590 after calculation is shown in Table 7. When the salt concentration is 6‰ and 9‰, strain KY-590 has the best alkali reduction effect, with an alkali reduction rate of 12.78%. This proves that strain KY-590 has the function of reducing soil pH, which can effectively alleviate the stress caused by alkali to plants. Therefore, it can be applied to promote plant growth and improve saline-alkali land.

[0052] Table 7 Results of alkali-reducing ability determination of KY-590 strain VI. Temperature tolerance of Marinobacterkunyu KY-590 strain Strain strain KY-590 was inoculated into LB liquid medium and cultured until OD500. 600 When the concentration was 0.6–0.8, 100 μL of bacterial culture was spread onto petri dishes with different salt concentrations, with three petri dishes per salt concentration as biological replicates. The petri dishes were placed in a constant temperature incubator and incubated upside down at 4–45 °C (1 °C intervals) for 24 h, and the growth of microorganisms was observed. The results showed that the temperature tolerance range of strain KY-590 was 5–41 °C.

[0053] Example 3 This example demonstrates the identification of the growth-promoting function of the Marinobacterkunyu KY-590 strain.

[0054] I. Nitrogen fixation capacity of Marinobacterkunyu KY-590 strain Strain strain KY-590 was inoculated into liquid Assumption nitrogen-free medium and passaged twice. The second passage was then inoculated at three equal intervals on Assumption nitrogen-free solid medium, with 5.0 μL inoculated at each site, for a total of three culture dishes as biological replicates. The dishes were incubated upside down in a 30 ℃ incubator for 3 days to observe whether the strain could grow. Results are as follows... Figure 7 As shown, strain KY-590 can grow on nitrogen fixation function identification medium, indicating that the strain has nitrogen fixation function, thereby increasing the nitrogen fixation capacity of plants.

[0055] II. Organophosphate solubilizing ability of Marinobacterkunyu KY-590 strain Strain strain KY-590 was inoculated into liquid LB medium and cultured until the OD of the bacterial culture reached a certain level. 600 The values ​​ranged from 0.6 to 0.8. Three equal-spaced inoculations of bacterial suspension were performed on the *Montagna montana* identification medium, with 5.0 μL inoculated at each site. Three culture dishes were inoculated as biological replicates and incubated upside down at 30 ℃ for 8 days. The formation of a clear zone was observed. Results are as follows: Figure 8As shown, when the KY-590 strain grows on the Monkina identification medium, a clear zone is formed around the colony, indicating that the KY-590 strain has the function of solubilizing organic phosphorus, which can convert organic phosphorus in the soil that is difficult to utilize into available phosphorus that can be absorbed and utilized by plants, thereby increasing the content of available phosphorus in the soil and promoting plant growth.

[0056] III. The inorganic phosphorus solubility of Marinobacterkunyu KY-590 strain Strain strain KY-590 was inoculated into liquid LB medium and cultured until the OD of the bacterial culture reached a certain level. 600 The values ​​ranged from 0.6 to 0.8. Three equal-spaced inoculations of bacterial suspension were performed on inorganic phosphorus identification medium, with 5.0 μL inoculated at each site. Three culture dishes were inoculated as biological replicates and incubated upside down at 30 ℃ for 8 days. The formation of a clear zone was observed. Results are as follows: Figure 9 As shown, when the KY-590 strain grows on an inorganic phosphorus medium, a transparent zone is formed around the colony, indicating that the KY-590 strain has the function of soluble inorganic phosphorus, which can convert inorganic phosphorus in the soil that is difficult to utilize into available phosphorus that can be absorbed and utilized by plants, thereby increasing the content of available phosphorus in the soil and promoting plant growth.

[0057] IV. Siderophore production capacity of Marinobacterkunyu KY-590 strain Strain strain KY-590 was inoculated into liquid LB medium and cultured until the OD of the bacterial culture reached a certain level. 600 The values ​​ranged from 0.6 to 0.8. Three equal-spaced inoculations of bacterial suspension were performed on CAS identification medium, with 5.0 μL inoculated at each site, for a total of three culture dishes as biological replicates. The dishes were incubated upside down at 30 ℃ for 8 days, and the presence of an orange reaction zone was observed. Results are as follows: Figure 10 As shown, when KY-590 strain grows on CAS medium, a color reaction zone is generated around the colony, indicating that KY-590 strain has the function of producing iron carriers, which can convert iron in the soil that is difficult to use into available iron elements that plants can use, increase the available iron content in the soil, and promote plant growth.

[0058] V. IAA production capacity of Marinobacterkunyu KY-590 strain Weigh 10 mg of IAA standard, dissolve it in a small amount of anhydrous ethanol, and then dilute to 100 mL with distilled water to prepare a 100 μg / mL IAA solution as a stock solution. Then, dilute the stock solution to prepare gradient standard solutions with concentrations of 0, 10, 20, 30, 40, and 50 μg / mL. Take six clean test tubes and add 2 mL of the standard solution and 2 mL of Salkowski colorimetric solution to each tube. Place them in the dark and allow the color reaction to proceed for 30 min. Measure the absorbance at 530 nm using a spectrophotometer. Plot a standard curve with absorbance on the x-axis and the concentration of IAA in the fermentation broth on the y-axis, as shown below. Figure 11 As shown, the standard curve equation was finally constructed: y = 95.054x – 10.07.

[0059] Strain KY-590 was inoculated into sterile LB broth and cultured on a shaker at 30°C and 180 r / min. Every 48 h, 2 mL of the bacterial suspension was transferred to a centrifuge tube and centrifuged at 14000 r / min for 10 min. The supernatant was transferred to a clean test tube, and an equal volume of Salkowski chromogenic solution was added. The tube was placed in the dark for 30 min, and the color reaction was allowed to proceed in triplicate. The A value was measured and recorded using a spectrophotometer. 530 The absorbance was measured. The standard curve equation y = 95.054x – 10.07 was used to calculate the maximum IAA concentration produced by strain KY-590 after 13 days of culture, which was 38 ± 2 μg / mL. The results are shown in Table 8.

[0060] Table 8. Average IAA concentration of strain KY-590 after 13 days. VI. The ability of Marinobacterkunyu KY-590 strain to produce volatile acidic metabolites Neutral red is known to indicate a pH range of 6.8–8.0, displaying orange for alkaline conditions and red for acidic conditions. LB solid medium supplemented with neutral red was added to both sides of an isolated culture dish. One side was inoculated with strain KY-590, while the other side remained uninoculated. The dishes were then incubated upside down at 30 °C for 48 h. The results are as follows: Figure 12As shown: both the inoculated and uninoculated sides of the culture medium turned red, indicating that strain KY-590 produced acidic metabolites, leading to a decrease in the pH of the medium. Since the uninoculated side also changed color, indicating a decrease in pH, the strain produced volatile acidic products under the experimental conditions. One culture medium was then left uncovered for 24 hours, and the other closed for 24 hours. The uncovered dish returned to orange. (See attached image). Figure 13 The petri dishes that were not exposed to the open did not change color; the results are shown in the figure. Figure 14 This further illustrates that strain KY-590 produces volatile acidic metabolites.

[0061] Example 4 This example demonstrates the identification of the plant growth-promoting function of the Marinobacterkunyu KY-590 strain.

[0062] Effects of Marinobacterkunyu KY-590 strain on Arabidopsis growth The five salt-alkali stress conditions used for culturing Arabidopsis thaliana in 1 / 2 MS solid medium were: pH 5.8, pH 8, pH 8 + 2 mM NaHCO3, pH 8 + 100 mM NaCl, and pH 8 + 2 mM NaHCO3 + 100 mM NaCl. The pH of all five media was adjusted with 1 mol / L NaOH and sterilized at 115°C for 30 min.

[0063] This experiment used a self-made composite culture medium: 1 / 2 MS solid medium was added to a 90 mm round culture dish. After it was completely solidified, the medium 1.5 cm from the bottom of the dish was cut off, and 2.0 mL LB solid medium was added. 20 treated Arabidopsis thaliana seeds were inoculated at 6.0 cm from the bottom of the dish. The LB solid medium was partially inoculated with the strain (the control group was not inoculated). The culture dishes were sealed and placed in an incubator for vertical culture. Three biological replicates were set up for each condition.

[0064] Arabidopsis thaliana growth conditions: 22 ℃, light intensity 12,000 Lux, 16 h light intensity, 8 h darkness, relative humidity 50%, vertical culture for 10 days. After 10 days, the root length, number of leaves, number of lateral roots, and fresh weight of Arabidopsis thaliana seedlings were recorded.

[0065] The growth-promoting effect of Marinobacterkunyu strain KY-590 on Arabidopsis thaliana is shown in [reference needed]. Figure 15 And Table 9, Figure 15In the table, 'a' represents pH 5.8, 'b' represents pH 8, 'c' represents pH 8 + 2 mM NaHCO3, 'd' represents pH 8 + 100 mM NaCl, and 'e' represents pH 8.0 + 2 mM NaHCO3 + 100 mM NaCl. '*' indicates p < 0.05, '**' indicates p < 0.01, '***' indicates p < 0.001, and '****' indicates p < 0.0001. 'ns' indicates no significant difference. Table 9 shows the statistical results of various indicators of Arabidopsis thaliana after inoculation with strain KY-590 under different salt stress conditions.

[0066] The results showed that, compared with the uninoculated control group, the number of lateral roots, root length, and fresh weight of Arabidopsis thaliana were significantly increased (p < 0.0001), and the number of leaves were significantly increased (p < 0.05) at pH 5.8; the number of lateral roots, fresh weight, and root length of Arabidopsis thaliana were significantly increased (p < 0.01), and the number of leaves and lateral roots of Arabidopsis thaliana were significantly increased (p < 0.01) at pH 8 + 2 mM NaHCO3. The statistical results in Table 9 also showed that strain KY-590 significantly promoted root growth in Arabidopsis thaliana.

[0067] Table 9. Statistical results of various indicators of Arabidopsis thaliana after inoculation with strain KY-590 at different salt concentrations. Note: " / " indicates that there is no corresponding physiological indicator growth in this group.

[0068] Effects of Marinobacterkunyu KY-590 strain on tomato growth Tomato seed cleaning: Remove tomato seeds from a 4°C refrigerator. Place the seeds in a sterile Erlenmeyer flask in a clean bench and soak in sterile deionized water for 20 minutes, then drain the water. Add 75% ethanol to the flask, soak for 1 minute while shaking continuously. Drain the ethanol and rinse three times with sterile deionized water. Add 50% sodium hypochlorite solution to the flask, soak for 15-20 minutes while shaking continuously until the surface of the tomato seeds turns golden yellow. Drain the sodium hypochlorite solution and rinse three times with sterile deionized water.

[0069] Tomato seed propagation: Add 1 / 2 MS medium (pH 5.8) to a 130 mm × 130 mm square petri dish. Use tweezers to transfer the treated tomato seeds onto the 1 / 2 MS medium, placing 5 rows of 10 seeds per row on each medium. Place the petri dishes vertically in a light incubator for 5 days, until the tomatoes have developed a taproot of about 1 cm.

[0070] Growth promotion of tomato by Marinobacterkunyu strain KY-590: This section sets up five stress conditions: pH 5.8, pH 8, pH 8 + 2 mM NaHCO3, pH 8 + 3‰ NaCl, and pH 8 + 2 mM NaHCO3 + 3‰ NaCl. The pH of all culture media was adjusted using 1 mol / L NaOH. Half MS solid medium was added to a 130 mm × 130 mm square Petri dish. After complete solidification, the medium was removed 1 cm from the bottom of the dish. 2.0 mL of LB solid medium was added, and 10 pre-treated tomato seeds were inoculated 8.5 cm from the bottom of the dish. The LB solid medium was partially inoculated with Marinobacterkunyu KY-590 strain (the control group was not inoculated). The Petri dishes were sealed and placed vertically in an incubator for 7 days.

[0071] Tomato cultivation conditions: photoperiod 14 h / 10 h, light intensity 12000 Lux / 0 Lux, temperature 25 ℃ / 18 ℃, relative humidity 70%. After 7 days, physiological indicators such as fresh weight, root length, and number of lateral roots of tomato seedlings were recorded.

[0072] The growth-promoting effect of strain KY-590 on tomatoes can be found in [reference needed]. Figure 16 As shown, Figure 16 In the table, 'a' represents pH 5.8, 'b' represents pH 8, 'c' represents pH 8 + 2 mM NaHCO3, 'd' represents pH 8 + 3‰ NaCl, and 'e' represents pH 8 + 2 mM NaHCO3 + 3‰ NaCl. '*' indicates p < 0.05, '**' indicates p < 0.01, '***' indicates p < 0.001, '****' indicates p < 0.0001, and 'ns' indicates no significant difference.

[0073] The results showed that, compared with the uninoculated control group, strain KY-590 increased the fresh weight and average number of lateral roots per tomato seedling to varying degrees. Specifically, the number of lateral roots and the fresh weight of tomato seedlings increased significantly (p < 0.0001) under pH 8 and pH 8 + 2 mM NaHCO3 conditions; the number of lateral roots and the fresh weight of tomato seedlings increased significantly (p < 0.001) under pH 8 + 3‰ NaCl conditions; and the number of lateral roots increased significantly (p < 0.001) under pH 8 + 2 mM NaHCO3 + 3‰ NaCl conditions. In conclusion, strain KY-590 can significantly promote the growth of tomatoes, especially root growth.

[0074] Effects of Marinobacterkunyu KY-590 strain on maize growth Corn seed cleaning: Place corn seeds in an Erlenmeyer flask, add 95% ethanol and soak for 0.5-1 minute. Pour out the ethanol and wash the seeds 3-4 times with sterile deionized water. Add 0.1% sodium hypochlorite solution to the Erlenmeyer flask and soak for 15 minutes while shaking continuously. Pour out the sodium hypochlorite. Wash the seeds 4 times with sterile deionized water. Add sterile deionized water to the Erlenmeyer flask until the liquid level covers the seeds, and soak overnight in the dark.

[0075] Growth-promoting effect of strain KY-590 on maize: Nutrient soil and vermiculite were mixed in a 1:1 ratio. NaCl, NaHCO3, NaCO3, and NaSO4 were mixed in a 1:1:1:1 molar ratio and added to the nutrient soil according to the following mass ratios: 0 g / kg, 6 g / kg, 9 g / kg, and 15 g / kg of mixed salts. Three maize seeds were sown in each pot at a depth of 1 cm, with six biological replicates for each condition. Strain KY-590 was inoculated into LB liquid medium and cultured at 30℃ and 180 r / min until OD... 600 0.6~0.8, dilute the bacterial solution 10 times with sterile deionized water to prepare a bacterial suspension for later use.

[0076] Once the corn plants had developed two cotyledons, 5 mL of the prepared bacterial suspension was applied to the roots of each plant in the experimental group, while 5 mL of LB medium was applied to the control group. Watering was carried out as needed during the corn's growth to maintain soil moisture. The plants were cultured for 14 days. The corn culture conditions were: photoperiod 14 h / 10 h, light intensity 20000 Lux / 0 Lux, temperature 28 ℃ / 22 ℃, and relative humidity 70%. After 14 days, the corn roots were washed clean, and leaf weight, stem weight, root weight, and plant height were measured.

[0077] The growth-promoting effect of strain KY-590 on maize is shown in [reference needed]. Figure 17 As shown in the figure, "*" indicates p < 0.05, "**" indicates p < 0.01, "***" indicates p < 0.001, "****" indicates p < 0.0001, and ns indicates no significant difference.

[0078] The results showed that inoculation with strain KY-590 had different promoting effects on maize after salt-alkali stress. Specifically, at a salt concentration of 6‰, the fresh weight of maize stems was significantly increased (p < 0.0001), and the fresh weight of leaves and plant height were also significantly increased (p < 0.05); at a salt concentration of 9‰, the plant height of maize was significantly increased (p < 0.0001).

[0079] Example 5 The *Marinobacter kunyu* KY-590 strain provided by this invention was isolated from the rhizosphere soil of *Tamarix chinensis*, a salt-tolerant plant, in Kunyu, Xinjiang Uygur Autonomous Region. It exhibits strong salt and alkali tolerance, providing a foundation for its colonization. Strain KY-590 possesses multiple characteristics, including salt reduction, nitrogen fixation, organic phosphorus solubilization, inorganic phosphorus solubilization, siderophore production, IAA production, amylase production, volatile acidic substance production, and oxidase production. It can effectively improve the soil microenvironment of plant roots, increase the content of available iron, phosphorus, nitrogen, and other nutrients in the roots, effectively help plants resist salt and alkali stress, and ultimately promote plant growth. This strain can use inulin, muscarin, arabinose, galactose, menobiose, and fructose as its sole carbon source and can grow at pH 7.0–13.0, salt concentrations of 0–200 g / L, and temperatures of 5–41℃, exhibiting strong environmental tolerance, which provides a basis for its resistance to harsh environments. Therefore, the KY-590 strain of *Hymenobacter* of this invention has great application value in promoting plant growth, improving plant resistance to salt and alkali stress, and improving saline-alkali land.

[0080] The *Hymenobacter* strain KY-590 of this invention can also be used in the preparation of fertilizers that promote plant growth and improve saline-alkali soil.

[0081] Fertilizer can be prepared using the KY-590 strain of the genus *Hymenobacter*. Specific components may include rice straw, stalks, animal manure, chitin, yeast extract, phosphates, potassium fertilizer, potassium humate, vermiculite, and *Hymenobacter* KY-590.

[0082] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A strain of the genus *Gynostemma*, characterized in that, The *Marinobacter* strain was named *Marinobacterkunyu* and deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 29757 on January 25, 2024. The 16S rRNA nucleotide sequence of *Marinobacterkunyu* is shown in SEQ ID NO:

1.

2. The application of a *Hymenobacter* strain as described in claim 1, characterized in that, The Marinobacterkunyu is used to improve the salt and alkali tolerance of plants, promote plant growth, reduce soil salinity and alkalinity, increase the nitrogen fixation capacity of plants, and increase the content of available iron and phosphorus in the soil.

3. The application according to claim 2, characterized in that, The plants mentioned include Arabidopsis thaliana, tomato, and corn.

4. The application according to claim 3, characterized in that, When the plant is Arabidopsis thaliana, the Marinobacterkunyu is used to promote the increase of the number of lateral roots, root length, fresh weight and number of leaves of the Arabidopsis thaliana; When the plant is a tomato, the Marinobacterkunyu is used to promote an increase in the number of lateral roots and fresh weight of the tomato. When the plant is corn, the Marinobacterkunyu is used to promote an increase in the fresh weight of the leaves, the fresh weight of the stems, and the plant height of the corn.

5. The application according to claim 2, characterized in that, The Marinobacterkunyu is used in the preparation of microbial fertilizers that promote plant growth and improve saline-alkali land.

6. The application according to claim 5, characterized in that, When Marinobacterkunyu is used in microbial fertilizers, it has at least one application in reducing soil salinity, fixing nitrogen, decomposing organic phosphorus, dissolving inorganic phosphorus, producing iron carriers, producing IAA, producing amylase, producing volatile acidic substances, and producing oxidase.

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