Romboutsia sp. and application thereof

Abstract

The application provides a Romboutsia sp. and an application thereof, the classification name of the Romboutsia sp. is Romboutsia sp. LXY-2, the preservation number is CGMCC No. 41194, the preservation date is June 19, 2024, and the preservation unit is the China General Microbiological Culture Collection Center. The application also provides an application of the Romboutsia sp. in microbial phosphorus release and an application of the Romboutsia sp. as a microbial fertilizer for promoting crop growth in a salinization area.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and relates to the application of plant growth promotion technology and microbial fertilizers in production, specifically to a strain of Rombutz bacterium and its application. Background Technology

[0002] Against the backdrop of increasingly scarce global arable land resources, soil salinization has become a key factor restricting sustainable agricultural development. According to the latest statistics from the Food and Agriculture Organization of the United Nations (FAO), more than 100 countries and regions worldwide are affected by soil salinization, leading to a significant deterioration in soil physical and chemical properties: increased soil bulk density, decreased porosity, and decreased saturated hydraulic conductivity. At the same time, high NaCl concentrations reduce the availability of micronutrients such as phosphorus, iron, and zinc, severely restricting crop growth and development.

[0003] Phosphorus plays an irreplaceable and crucial role in plant growth and development, and its content and distribution significantly affect plant growth potential, metabolic efficiency, and ability to cope with adversity. As a key component of adenosine triphosphate (ATP), phosphorus ensures the stable storage and efficient transfer of energy within plant cells by constructing high-energy phosphate bonds, providing core power support for anabolic metabolic processes such as protein synthesis and cell division, as well as growth activities. In the photosynthetic system, phosphorus participates in photophosphorylation, promoting the directional conversion of light energy into chemical energy. This not only accelerates the accumulation of plant biomass but also optimizes the physiological processes of flowering and fruit setting, resulting in improved flower development quality and a significant increase in fruit yield. Furthermore, phosphorus regulates the expression of genes related to root development, stimulating taproot elongation and lateral root branching, significantly enhancing the plant's absorption efficiency of water and mineral nutrients. It also forms a synergistic absorption mechanism with nutrients such as nitrogen and potassium, achieving overall optimization of plant nutrient utilization efficiency. In terms of adaptation to adversity, phosphorus effectively enhances the plant's resistance to drought, salinity, and biotic stress by inducing increased activity of antioxidant enzyme systems and the synthesis of osmotic regulators, providing an important guarantee for maintaining stable and high crop yields.

[0004] From an ecosystem function perspective, the microbial community serves as the core regulatory hub of the soil-plant system, with phosphate-solubilizing bacteria attracting significant attention due to their unique phosphorus conversion capabilities. These microorganisms, by secreting low-molecular-weight organic acids (such as citric acid and oxalic acid) and bioactive substances like phosphatases, can specifically dissolve insoluble phosphorus sources in the soil (including mineral phosphates and organic phosphorus compounds), converting them into orthophosphate forms that can be directly absorbed and utilized by plants. This biotransformation process not only significantly increases the available phosphorus pool in the soil but also improves soil aggregate structure through organic acid-metal ion chelation, promoting increased soil microbial community diversity. Notably, the mutually beneficial symbiotic system formed between phosphate-solubilizing bacteria and plant roots has a dual effect: on the one hand, it directly transports phosphorus nutrients through mycorrhizal channels; on the other hand, it indirectly promotes the bioavailability of micronutrients such as iron and zinc by altering the pH and enzyme activity of the rhizosphere microenvironment. This phosphorus management strategy based on the synergistic effect of microorganisms and plants has significant advantages over the application of traditional chemical phosphate fertilizers. Its environmental benefits are reflected in reducing non-point source pollution caused by phosphate fertilizer runoff, and at the same time, by maintaining the balance of soil phosphorus cycling, it provides an innovative path for building a resource-saving and sustainable agricultural system.

[0005] Therefore, screening a salt-tolerant phosphate-solubilizing bacterium is an urgent problem to be solved in saline-alkali areas. Summary of the Invention

[0006] To address the aforementioned problems, the present invention aims to provide a strain of Rombutz bacterium that is resistant to high concentrations of NaCl.

[0007] Another object of the present invention is to provide the application of the above-mentioned Rombutz bacillus.

[0008] To achieve the above objectives, the present invention provides a strain of Romboutsia sp. LXY-2, with the following classification name: CGMCC No. 41194; deposit date: June 19, 2024; depositing institution: China General Microbiological Culture Collection Center.

[0009] The present invention also provides the application of the above-mentioned Rombutz bacillus in microbial phosphate solubilization.

[0010] The present invention also provides the application of the above-mentioned Rombutz bacillus in areas with high concentrations of NaCl.

[0011] The present invention provides the application of the above-mentioned Rombutz bacillus as a microbial fertilizer to promote crop growth in areas with high concentrations of NaCl.

[0012] The above-mentioned method transforms insoluble phosphates into soluble phosphate ions that can be efficiently absorbed and utilized by plants under high-concentration NaCl conditions, providing a large amount of nutrients for crop production and showing great potential in the production of high-efficiency bio-organic fertilizers.

[0013] The beneficial effects of this invention are as follows:

[0014] This invention provides a *Rhombus* bacterium, which shows great promise for the remediation of saline-alkali sites with high concentrations of NaCl and for agricultural production. It can convert insoluble phosphates into soluble phosphate ions that can be efficiently absorbed and utilized by plants, providing abundant nutrients for crop production. Through the controlled large-scale production of this *Rhombus* bacterium, it can be applied to the preparation of microbial fertilizers. This microbial fertilizer based on *Rhombus* bacteria not only increases the content of soluble phosphorus in the soil but also promotes crop growth and development, thereby improving crop yield and quality. Furthermore, because its production process is relatively controllable and easily scaled up, it has high economic value and practicality in real-world applications. Attached Figure Description

[0015] Figure 1 This is a diagram showing the growth status of Rombutz bacterium LXY-2 on a culture medium, as provided by the present invention.

[0016] Figure 2 The phylogenetic tree of Rombutz bacterium LXY-2 based on 16S rDNA provided by the present invention.

[0017] Figure 3 The OD values ​​of Rombutz LXY-2 provided by this invention in LB medium containing different NaCl concentrations 600 Change diagram.

[0018] Figure 4 The graph shows the changes in phosphorus solubility of Rombutz bacillus LXY-2 in Monkina inorganic phosphorus medium containing different NaCl concentrations, as provided in this invention. Detailed Implementation

[0019] The embodiments of the present invention will now be described in detail and comprehensively so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0020] Material

[0021] 1. The DNA extraction kit was purchased from Beijing Jinsha Biotechnology Co., Ltd., product number: DE703-50.

[0022] Preparation of culture medium:

[0023] The microbial enrichment medium (1L) formula is as follows: 2g anhydrous magnesium sulfate, 5g sodium citrate dihydrate, 1g calcium sulfate dihydrate, 1g ammonium chloride, 0.5g dipotassium hydrogen phosphate, 3.5g sodium lactate, and 1g yeast extract. The preparation method is as follows: Mix the above ingredients and add water to 1L. Adjust the pH to 7.2 with 1mol / L dilute sulfuric acid. Dispense the mixture into anaerobic tubes in an anaerobic workbench and sterilize in a high-pressure steam autoclave at 121℃ for 20min. Cool before use.

[0024] The formula for solid culture medium (1L) is as follows: 2g anhydrous magnesium sulfate, 5g sodium citrate dihydrate, 1g calcium sulfate dihydrate, 1g ammonium chloride, 0.5g dipotassium hydrogen phosphate, 3.5g sodium lactate, 1g yeast extract, and 15g agar. The preparation method is as follows: Mix the above ingredients and add water to 1L. Adjust the pH to 7.2 with 1mol / L dilute sulfuric acid. After venting the bottle containing the culture medium with 95% high-purity nitrogen in an anaerobic environment, seal the bottle and autoclave at 121℃ for 20 minutes. While still hot, pour the mixture into sterile petri dishes. After cooling, the solid culture medium plates are obtained.

[0025] LB liquid medium (1L): 5g yeast extract, 10g sodium chloride, 10g tryptone. After thorough mixing, adjust the pH to 7 using NaOH or HCl, autoclave at 121℃ for 20 minutes, and then cool for later use.

[0026] LB solid culture medium plates are made by adding 15g of agar powder to LB liquid culture medium, adjusting the pH to 7, autoclaving at 121℃ for 20min, pouring the hot mixture into sterile petri dishes, and cooling to obtain LB solid culture medium plates.

[0027] LB medium is used for the large-scale culture of phosphate-solubilizing bacteria.

[0028] The formula for Monkina Inorganic Phosphorus Liquid Culture Medium (1L) is as follows: 10.0g glucose, 0.5g ammonium sulfate, 0.5g yeast extract, 0.3g sodium chloride, 0.3g potassium chloride, 0.3g magnesium sulfate, 0.03g ferrous sulfate, 0.03g manganese sulfate, and 5.0g tricalcium phosphate. After thorough mixing, adjust the pH to 7 using NaOH or HCl, autoclave at 121℃ for 20 minutes, and then cool to obtain Monkina Inorganic Phosphorus Liquid Culture Medium for later use.

[0029] The Monkina Inorganic Phosphorus Medium Plate is prepared by adding 15g of agar powder to Monkina Inorganic Phosphorus Liquid Medium, adjusting the pH to 7, autoclaving at 121℃ for 20min, and then pouring the hot liquid into sterile Petri dishes. After cooling, the Monkina Inorganic Phosphorus Medium Plate is obtained.

[0030] The Monkina Inorganic Phosphate Liquid Medium and Monkina Inorganic Phosphate Plate are used to isolate and detect the ability of screened phosphate-solubilizing bacteria to dissolve tricalcium phosphate.

[0031] Example 1: Isolation and Identification of Strains

[0032] Soil samples were collected from a wastewater treatment plant in Beijing using sterilized shovels or samplers at a depth of 40-100 cm. The samples were placed in sterile plastic bags or containers and labeled with information. The collected samples were then sent to the laboratory at low temperature for microbial isolation and screening.

[0033] Take 5g of soil sample and add 45mL of sterile physiological saline. Shake thoroughly for 10 minutes to fully suspend the soil particles. Let the soil mixture stand for 10 minutes to allow solid particles to settle. The supernatant is the bacterial suspension used for screening. Add 100μL of the bacterial suspension to 900μL of sterile physiological saline and mix thoroughly to form a 10... -1 Diluent. Take 100 μL of 10 -1 The diluent was added to 900 μL of sterile physiological saline to form a 10... -2 Diluent, and so on, up to 10 -6 Dilution. Take 200 μL of 10... -4 10 -5 and 10 -6 The serially diluted solutions were added to the Monkina inorganic phosphorus agar plates, spread evenly with a sterile spreader, and then inverted and placed in a 30°C incubator for 48 hours. The growth of the colonies was then observed.

[0034] After clear single colonies have grown on the Monkina inorganic phosphorus agar plate, select representative and well-grown colonies and inoculate them using a sterile inoculation loop. Streak the colonies on fresh Monkina inorganic phosphorus agar plates to ensure the isolation of single colonies. Invert the plate and incubate at 30°C for 48 hours. Observe the growth of the colonies and repeat the streaking process until a purified strain is obtained. Figure 1 The image shown is a photograph of the growth status of the strain on a solid culture medium plate.

[0035] Genomic DNA was extracted from the purified strain using a DNA extraction kit and amplified by PCR. The PCR products were then subjected to agarose gel electrophoresis to confirm the amplification effect. The 16S rRNA of this bacterium was amplified by PCR and sequenced, yielding the sequence shown in SeqID No. 1. Comparison with the Ezbiocloud database (https: / / www.ezbiocloud.net / ) showed that the maximum full-length similarity of the 16S rRNA gene sequence of this strain with all standard strains in the genus *Romboutsia* was 97.54%, which is below the 98.5% threshold for new species classification. Figure 2 As shown, based on physiological and biochemical characteristics, this bacterium was identified as a new species of the genus *Romebutzia*, and named...

[0036] Romboutsia sp. LXY-2, abbreviated as Romboutsia LXY-2, has been deposited for preservation with accession number CGMCC No. 41194; the deposit date is June 19, 2024, and the depositary institution is the China General Microbiological Culture Collection Center.

[0037] Example 2: Detection of Phosphate-Solubilizing Ability of Strains

[0038] The *Rombutzella* LXY-2 strain obtained in Example 1 was inoculated into LB liquid medium and cultured at 30°C and 180 rpm for 24 h. The OD of the bacterial culture was adjusted. 600 =1, to obtain seed culture. Take 10 μL of seed culture and inoculate it onto a Monkina inorganic phosphorus medium plate, place it in an incubator at 30℃ and invert it for 3 days, then measure the colony diameter and the diameter of the phosphate-solubilizing ring.

[0039] The seed culture was divided into three parallel inoculations at a volume percentage of 1%, each containing 1 mL of seed culture in 100 mL of Monkina inorganic phosphorus liquid medium. The inoculations were carried out in an incubator at 30°C for 3 days. The water-soluble phosphorus content in the culture medium was determined by the molybdenum antimony colorimetric method.

[0040] The colony diameter, phosphate-solubilizing ring diameter, and water-soluble phosphorus content are shown in Table 1.

[0041] Table 1. Determination of phosphorus solubilization ability of Rombutz bacillus strain LXY-2

[0042]

[0043] As shown in Table 1, after 3 days of cultivation, a distinct phosphate-solubilizing ring appeared around the colony, indicating that this bacterium has the ability to dissolve insoluble tricalcium phosphate into soluble phosphorus, suggesting that it can provide the necessary and absorbable phosphorus for plant growth. Simultaneously, the dissolved phosphorus content in the supernatant of the liquid culture medium was measured. After 3 days of cultivation, the phosphorus content in the supernatant reached 169.1 mg / L, and the pH of the culture medium reached 4.4, indicating that *Romebutzella* LXY-2 dissolves phosphorus by secreting organic acids.

[0044] Example 3: Adaptability of Rombutz bacillus strain LXY-2 under high concentration NaCl conditions

[0045] Different concentrations of NaCl were used to simulate the salinity of saline-alkali areas to observe the adaptability of Rombutz bacterium LXY-2 to high-salt environments.

[0046] Six concentration gradients of NaCl were obtained by adjusting the mass percentage concentration of NaCl in LB medium to 0%, 2%, 4%, 6%, 8%, and 10%, respectively, to obtain LB medium containing different concentrations of NaCl.

[0047] OD 600 The seed culture of 1 was inoculated at a volume percentage of 1% into LB medium containing different concentrations of NaCl. After incubation at 30°C and 160 rpm for 24 h with shaking, the OD of the bacterial suspension was measured. 600 Value, result as Figure 3 As shown.

[0048] from Figure 3 It can be seen that after 24 hours of shaking culture, the OD of Rombutz LXY-2 at NaCl concentrations of 0% and 2% was... 600 The value is 0.36; at a NaCl concentration of 4%, OD 600 The value is 0.31; at a NaCl concentration of 6%, OD 600 The value is 0.23; at a NaCl concentration of 8%, OD 600 The value is 0.09; at a NaCl concentration of 10%, OD 600 The value is 0.05. From Figure 3 It can be seen that *Romebutzella* LXY-2 grows well under 0-6% NaCl conditions, exhibits some growth ability at 8% NaCl concentration, and shows almost no growth at 10% NaCl concentration. This indicates that *Romebutzella* LXY-2 has a good adaptability to high concentrations of NaCl, suggesting its potential for application in saline-alkali areas.

[0049] Example 4: Phosphate-solubilizing ability of strain under high NaCl conditions

[0050] The environment of salinized areas was simulated by preparing liquid culture medium containing different NaCl concentrations from *Montagna lanceolata*. Six concentration gradients of NaCl were established, with mass percentages of 0%, 2%, 4%, 6%, 8%, and 10%, resulting in liquid culture media containing different NaCl concentrations. Each concentration was prepared in triplicate. The pH was adjusted to 7, and the media were autoclaved at 121°C and then cooled for later use. OD... 600 The seed culture of 1 was inoculated into the above LB medium at a volume percentage of 1%, and cultured at 30°C and 160 rpm for 24 h with shaking. The OD of the bacterial suspension was then measured. 600 Value, result as Figure 4 As shown. From Figure 4It can be seen that after 24 hours of shaking culture, the phosphorus solubility of Rombutz bacillus LXY-2 under different NaCl concentrations showed significant differences: when the NaCl concentration was 0%, the phosphorus solubility reached 167 mg / L; when the concentration increased to 2%, the phosphorus solubility increased slightly to 173 mg / L; at a concentration of 4%, the phosphorus solubility remained at a high level of 171 mg / L; however, when the NaCl concentration climbed to 6%, the phosphorus solubility decreased to 112 mg / L; at a concentration of 8%, the phosphorus solubility further decreased sharply to 80 mg / L; and when the NaCl concentration reached the extreme condition of 10%, the phosphorus solubility completely dropped to 0 mg / L.

[0051] As can be seen from Examples 3 and 4 above, within the NaCl concentration range of 0% to 6%, *Romebutzinus* LXY-2 exhibited excellent phosphorus solubilization efficiency, maintaining a high level of phosphorus solubilization. This fully demonstrates the strong phosphorus solubilization potential of this strain in mildly to moderately salinized soils. Even under severe salinization conditions with a NaCl concentration of 8%, *Romebutzinus* LXY-2 still retained a certain phosphorus solubilization ability. Although the phosphorus solubilization decreased, this performance still highlighted its strong environmental adaptability and phosphorus solubilization stability. However, when the NaCl concentration climbed to the extreme salinization level of 10%, the phosphorus solubilization function of *Romebutzinus* LXY-2 was completely lost. This indicates that the threshold of salinization degree for *Romebutzinus* LXY-2 needs to be considered in applications. In summary, Rombutz bacterium LXY-2 exhibited varying degrees of phosphorus solubilization ability within a NaCl concentration range of 0% to 8%, with particularly significant effects under low to moderate salinization conditions. This provides a solid scientific basis and broad application prospects for its application in the fields of salinized soil improvement and sustainable agricultural development.

[0052] Example 5: The ability of strains to promote plant growth under saline conditions.

[0053] Rombutz bacillus LXY-2 was cultured in LB liquid medium at 30℃ and 150 rpm until the logarithmic growth phase. The bacterial culture was then transferred to sterile centrifuge tubes in a laminar flow hood and centrifuged at 8000 rpm for 5 min. The supernatant was discarded, and the culture was washed with sterile water and the concentration was adjusted to 10. 8 CFU / ml. Take 100g of air-dried soil and inoculate the bacterial suspension into the soil at an inoculation rate of 10. 6 CFU / g dry soil; a control group (CK) was set up with no inoculation, and a substitute group was set up with an equal volume of sterile water added to the bacterial suspension. Each group had 5 replicates.

[0054] Simulated salinization environment: When the salt content in the soil reaches 0.3-0.8 g / kg, it will lead to the deterioration of the soil's physical and chemical properties. Here, we use 1%, which is slightly higher than the above standard, to simulate the salinization environment.

[0055] Rice seeds with uniform germination were selected and inoculated into planting pots. The temperature was 25℃, the light duration was 16 h / d, and the dark duration was 8 h / d. Two days after transplanting, the control group was irrigated with 10 mL of sterile water near the root zone; the test group was irrigated with 10 mL of 1.0% NaCl solution near the root zone to simulate a salinization environment. Irrigation was performed every 2 days. Five parallel groups were set up for each rice group, with 6 rice plants per pot. After 7 days of growth, the root zone was irrigated a second time as described above. The rice was harvested after 20 days of salt stress. Six rice plants were randomly selected from each group, and the aboveground fresh weight, aboveground dry weight, plant height, and root length were measured. The roots were washed in tap water to remove soil, then dried with filter paper, and the length of the stems and roots were measured, and their fresh and dry weights were also measured. The washed and dried rice roots were blanched in a hot air oven at 105℃ for 30 minutes, and then dried at 80℃ for 72 hours. The dry weight was then measured, and the results are shown in Table 2.

[0056] Table 2. Determination of plant growth-promoting ability of strains

[0057]

[0058] Table 2 shows that the germination rate of rice seeds in the control group was 78.1%, while the germination rate treated with *Rhizoctonia solani* LXY-2 was 90.2%, an increase of 15.5% compared to the control group. Regarding the effects on growth and development, the above-ground fresh weight, above-ground dry weight, plant height, and root length of rice seedlings in the LXY-2 group increased by 100%, 40%, 28.3%, and 60.7% respectively compared to the control group. These results indicate that strain LXY-2 significantly improves plant germination rate, increases fresh and dry weight, plant height, and root length, demonstrating a good effect on promoting plant growth.

[0059] As can be seen from the above embodiments, the *Rhombus bacterium* LXY-2 provided by this invention exhibits significant plant growth-promoting characteristics. This ability enables *Rhombus bacterium* LXY-2 to grow effectively in the Mongkina inorganic phosphorus culture medium environment and exert its unique phosphorus-solubilizing effect. Through reasonable cultivation and regulation, the controlled large-scale production of this bacterium can be achieved, and it can then be applied to the preparation of microbial fertilizers.

[0060] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A strain of Rombutzium, characterized in that, The taxonomic name of this Romboutsia sp. is: Romboutsia sp.; the accession number is CGMCC No. 41194; the accession date is June 19, 2024; and the depositary institution is the China General Microbiological Culture Collection Center.

2. A microbial fertilizer, characterized in that, It contains Rombutz bacillus as described in claim 1.

3. The application of Rombutz bacillus as described in claim 1 in microbial phosphate solubilization.

4. The application of *Rhombus bacillus* as a microbial fertilizer to promote crop growth in saline-alkali areas as described in claim 1, characterized in that... The NaCl concentration in the soil of the salinized area is 0-8% by mass, and the crop is rice.

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