Serratia savalmannii capable of improving phosphorus utilization capacity of tobacco and application of serratia savalmannii

Abstract

The invention discloses a serratia saumannii strain capable of improving phosphorus utilization capacity of tobacco and application of the serratia saumannii strain, the strain is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation address is Beijing, the preservation center number is CGMCC No.36319, the preservation date is October 23, 2025, and the strain is classified and named as serratia saumannii. The strain serratia salumannii is an efficient inorganic phosphorus decomposing bacterium, inorganic phosphorus which is fixed in soil and cannot be utilized by plants can be effectively activated and converted into soluble available phosphorus, and the process directly improves the effectiveness of phosphorus in the soil, so that the phosphorus absorption efficiency of tobacco is remarkably enhanced, the growth and development of the tobacco are promoted, and the yield of the tobacco is increased. Wide application prospects are realized.

Description

Technical Field

[0001] This invention relates to a strain of Serratia sarumanii that enhances phosphorus utilization in tobacco and its application, belonging to the field of Serratia sarumanii application. Background Technology

[0002] Phosphorus, as a crucial nutrient component essential for plants, plays an irreplaceable role in plant growth, development, and physiological processes. However, most phosphorus resources in natural soils exist in stable chemical states with extremely low bioavailability. Research data shows that the proportion of soluble phosphorus directly available to crops in arable soil is typically less than 5%, with the remainder fixed by soil colloids in various insoluble phosphate forms. To maintain agricultural productivity, traditional farming methods have long relied on excessive input of chemical phosphate fertilizers, which has not only led to a continuous decline in fertilizer utilization and rising planting costs but also resulted in ecological and environmental risks such as eutrophication of water bodies. Against this backdrop, developing green activation technologies that can efficiently release the soil's inherent phosphorus pool has become a core breakthrough for the transformation and upgrading of modern agriculture.

[0003] Microbial-mediated activation technology for inorganic phosphorus resources exhibits unique advantages. This technology utilizes the metabolic activity of specific functional microorganisms to convert insoluble phosphates fixed in the soil into forms that can be absorbed by plants, effectively opening up the conversion channel between soil phosphorus pools and crop absorption. These microorganisms with highly efficient inorganic phosphorus-dissolving capabilities significantly improve the bioavailability of soil phosphorus, providing an innovative solution to the "phosphorus fixation" problem. Therefore, the development and application of highly efficient inorganic phosphorus-dissolving agents can not only improve the phosphorus nutrition status of tobacco but also serve as an important technological pathway to achieve reduced application and increased efficiency, as well as sustainable development in the tobacco industry. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a strain of *Serratia sarumanica* with high activity in efficiently activating insoluble inorganic phosphorus in soil and significantly improving phosphorus bioavailability. Serratia sarumanii IA-3 strain and its application.

[0005] Technical solution: This invention provides a strain of Serratia sarumanii ( Serratia sarumanii Strain IA-3, deposited at the China General Microbiological Culture Collection Center (CGMCC), Beijing, China, with accession number CGMCC No. 36319, deposited on October 23, 2025, and classified as *Serratia sarumanii*. Serratia sarumanii .

[0006] The strain was isolated from the root surface of healthy tobacco plants and has a good affinity for tobacco.

[0007] The present invention also provides a microbial agent or preparation containing the *Serratia sarumanii* (…). Serratia sarumanii ) IA-3 or its fermentation broth, sterile supernatant or one or more of them.

[0008] The present invention also provides the aforementioned Serratia sarumanii ( Serratia sarumanii The application of IA-3 or the aforementioned microbial agents or preparations in enhancing soil phosphorus availability.

[0009] The present invention also provides a method for improving tobacco plant growth using the aforementioned strain or inoculum, comprising the following steps: applying an effective dose of the aforementioned *Serratia sarumanii* to the tobacco plant. Serratia sarumanii )IA-3 or the aforementioned bacterial agent or preparation.

[0010] The promotion of tobacco growth is achieved by converting insoluble inorganic phosphorus in the soil into soluble phosphorus.

[0011] Among them, the Saluman's Serratia ( Serratia sarumanii The dosage of IA-3 is 5 × 10⁻⁶. 6 ~6×10 6 One per plant.

[0012] Among them, the bacterial agent or preparation contains Serratia sarumanii ( Serratia sarumanii The concentration of IA-3 is 1×10 8 cfu / ml ~2×10 8 cfu / ml.

[0013] Among them, Serratia sarumanii ( Serratia sarumanii The application temperature of IA-3 or the aforementioned microbial agent or preparation is 15-20 ℃.

[0014] The present invention also provides a method for obtaining the phosphate-solubilizing strain from tobacco roots: (1) Abandoned tobacco-rice planting plots were selected in Guiyang County, Hunan Province (longitude: 112.737503; latitude: 25.741275). The plots had not been planted with crops or phosphate fertilizer for the past 5 years. Soil samples were collected from the plots for indoor pot experiments. The surrounding soil was carefully removed 30 days after tobacco sowing and transplanting, while keeping the root structure intact, for the extraction of tobacco root surface microorganisms.

[0015] (2) Use sterile forceps to remove the complete root system and stir vigorously in an Erlenmeyer flask containing PBS buffer to ensure that all soil on the root surface is submerged in the buffer. Rinse the roots thoroughly with PBS buffer and place them in a test tube containing surfactant (0.5 mmol / L Tween 80), ensuring the surfactant just covers the roots. Sonicate the roots twice for 30 seconds each time, then remove the roots to obtain a tobacco root surface microbial suspension.

[0016] (3) 1 mL of tobacco rhizosphere microbial suspension was taken and diluted to 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 series of gradients, respectively, 200 μL of 10 -4 , 10 -5 , 10 -6 concentration of the suspension was uniformly coated on inorganic phosphorus solid medium, each concentration gradient was repeated twice, and 20°C dark culture was carried out for 5 days. According to the bacterial morphology, color and size, single colonies were picked from the plates of suitable concentration gradients, and purified culture was carried out in turn.

[0017] (4) The DNA of the test strain was extracted, and 16S rRNA sequencing was carried out to determine the taxonomic status of the strain.

[0018]

[0019] The application also provides effect verification of the inorganic phosphorus solubility of the phosphorus solubilizing strain: (1) The obtained strain is inoculated into inorganic phosphorus solid culture medium respectively, and after 2 days of culture, single colonies are picked and transferred to test tubes containing inorganic phosphorus liquid culture medium, and after the colonies grow to the logarithmic growth phase, the precipitate is obtained by centrifugal shaking, sterile water is added and mixed with the precipitate to obtain a bacterial suspension; meanwhile, single colonies in the solid culture medium are picked and transferred to another test tube containing 5 mL of inorganic phosphorus liquid culture medium, and a control without the above strain is set, and after 2 days of shaking table shaking, the supernatant is obtained by centrifugation.

[0020] (2) 10 μL of the above bacterial suspension is dropped onto the center of the inorganic phosphorus solid culture medium, and after 2 days, the size of the phosphorus solubilizing ring is observed. Meanwhile, the supernatant in another test tube is taken, and the OD value of the bacterial liquid is measured by the molybdenum-antimony anti-colorimetric method to further estimate the phosphorus solubilizing ability, and the results show that the phosphorus solubilizing strain appears an obvious phosphorus solubilizing ring in the inorganic phosphorus solid culture medium; compared with the bacterial liquid in the inorganic phosphorus liquid culture medium without inoculation of the strain, the selected strain has a certain phosphorus solubilizing ability.

[0021] The application also provides effect verification of the inorganic phosphorus solubility of the phosphorus solubilizing strain: (1) Sample preparation: soil samples and brown soil sample bottles are taken from the experimental sample plot, sterilized in sequence, and prepared for use.

[0022] (2) Strain culture: the Salmonella salamanca strain is initially inoculated into inorganic phosphorus solid culture medium, and after 2 days of culture, single colonies are picked and transferred to a conical flask containing inorganic phosphorus liquid culture medium, and the shaking table is cultured for 2 days, the bacterial liquid is extracted, the supernatant is discarded by centrifugation, the precipitate is added with sterile water and mixed uniformly by shaking, and the OD value is adjusted to 1. 600 =1.

[0023] (3) Strain inoculation: the above phosphorus solubilizing strain is inoculated into the soil in the soil sample bottle (12 mL of bacterial liquid is added to 100 g of soil), and the water content is maintained at 20%.

[0024] (4) After 7 days of culture in the dark, the molybdenum-antimony anti-colorimetric method is used for phosphorus determination to compare the available phosphorus content, and compared with the control group, the available phosphorus content after inoculation is increased by 52.7%.

[0025] The application also determines the inorganic phosphorus solubilizing effect of the strain through sterile seedling inoculation test: (1) MS medium configuration: 20 times MS macroelement mother liquor is configured, and 3.4g of soluble potassium dihydrogen phosphate in the 20 times MS macroelement mother liquor is replaced by 3.8g of insoluble calcium phosphate, 1.8g of potassium chloride is further added to supplement the missing potassium element, 100 times MS microelement mother liquor and 100 times iron salt mother liquor are configured. 15mL of 20 times macroelement mother liquor, 6mL of 100 times microelement mother liquor, 6mL of 100 times iron salt mother liquor, 1g of anhydrous glucose, 1g of sucrose, 14g of agar powder are mixed, and then distilled water is added to make up to 1L, the pH is adjusted to 5.8, and the mixture is divided into tissue culture bottles, 80mL per bottle, and sterilized at 121 DEG C for 15min.

[0026] (2) Tobacco seedling culture: the tobacco seeds are surface sterilized with 75% ethanol (30s) and 3% NaClO (15min), and then placed in a culture dish with only sterilized filter paper. After germination for 7d, the seedlings are transferred to the tissue culture bottles with the MS medium, 2 seedlings per bottle. The light is on for 16h and off for 8h at 22 DEG C, and the process is continued for 7d.

[0027] (3) Preparation of phosphate solubilizing bacteria fermentation liquor: the above strain is initially inoculated into inorganic phosphorus solid medium, and after 2d of culture, single colonies are picked into a test tube containing inorganic phosphorus liquid medium, and cultured again for 2d. The bacterial liquid is extracted, centrifuged, and the supernatant is discarded. The precipitate is mixed uniformly with sterile water, and the concentration of the bacterial liquid is adjusted to OD600=0.1 as a bacterial suspension. 50ul of the bacterial suspension is inoculated near the roots of each tobacco plant, so that the concentration of Serratia marcescens (I-A-3) is 1×10 Serratia sarumanii ) I-A-3 is 1×10 8 cfu / ml~2×10 8 cfu / ml, and the amount is 5×10 6 ~6×10 6 per plant.

[0028] (4) Phosphate solubilizing effect verification: after 45d of inoculation of the strain, the tissue culture bottles of tobacco are destructively sampled, and the growth conditions of tobacco are compared to verify the phosphate solubilizing effect.

[0029] Beneficial effects: Compared with the prior art, the phosphate solubilizing bacteria agent has the following advantages: 1. The phosphate solubilizing bacteria agent is aimed at the problem of phosphorus fixation in the soil of the tobacco planting area, and the selected inorganic phosphorus strain can efficiently activate the insoluble calcium phosphate in the soil, convert a large amount of ineffective phosphorus in the soil into a form available to plants, and directly improve the concentration of available phosphorus in the soil, thereby providing a sustained and stable phosphorus nutrient source for the growth of tobacco; 2. The bacteria have strong phosphate solubilizing ability and good rhizosphere colonization effect, and effectively overcome the shortcomings of traditional phosphorus fertilizer, such as easy fixation and low utilization rate. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Serratia sarumani ( Serratia sarumanii The phosphorus-solubilizing zone of IA-3 preliminarily confirms its phosphorus-solubilizing ability; Figure 2 Serratia sarumani ( Serratia sarumanii After IA-3 was cultured in inorganic phosphorus liquid medium for 2 days, the phosphorus concentration in the supernatant was measured by colorimetry. Figure 3 To compare the available phosphorus content in soil using the molybdenum-antimony colorimetric method for phosphorus determination; Figure 4 Serratia sarumani ( Serratia sarumanii Comparison of the phosphorus solubilization effect of IA-3 after 45 days of inoculation in tissue culture flask experiments; Figure 5 To compare the phosphorus-solubilizing effects of inoculated and uninoculated tobacco plants in a pot experiment, the measured indicators were seedling height, ground diameter, number of leaves, and upper fresh weight. Detailed Implementation

[0031] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0032] Example 1: Isolation and identification of phosphate-solubilizing microorganisms on tobacco root surfaces (1) Soil sampling: Fields with a history of rice and tobacco rotation were selected in Guiyang County, Hunan Province (longitude: 112.737503; latitude: 25.741275). These fields had not been planted with crops or phosphate fertilizer applied for the past 5 years. Soil samples were collected for greenhouse pot experiments. Samples were collected 30 days after tobacco sowing and transplanting. The above-ground parts of the plants were cut off, and the roots were held with sterile forceps and vigorously stirred in a conical flask containing PBS buffer to ensure that all the soil on the root surface was submerged in the buffer. The roots were then rinsed with PBS buffer and placed in a test tube containing surfactant (0.5 mmol / L Tween 80), just submerging the roots. The tubes were sonicated twice for 30 seconds each time, and the roots were removed to obtain a suspension of tobacco root surface microorganisms.

[0033] (2) Coating, culture and purification: Take 1 mL of tobacco root surface microbial suspension and dilute to 10. -2 10 -3 10 -4 10 -5 and 10 -6 A series of gradients, each using 200 μL of 10 -4 10 -5 10 -6The suspension of each concentration was uniformly coated on the inorganic phosphorus solid medium, and each concentration gradient was repeated twice, and cultured at 20°C in the dark for 5d. Single colonies were picked from the appropriate concentration gradient plate according to bacterial morphology, color, and size, and sequentially purified and cultured.

[0034] (3) Classification position determination: The genetic material DNA of the bacteria was extracted using a DNA extraction kit (TIANamp Bacteria DNA Kit, TIANGEN). For these DNA samples, 16S rDNA PCR amplification was performed, with primers 27F-1492R, sequences AGRGTTYGATYMTGGCTCAG and RGYTACCTTGTTACGACTT; the total reaction volume was 50 μL, TransStart Fastpfu DNA Polymerase was used; the program was pre-denaturation: 95°C, 5 minutes (1 cycle), amplification: 27 cycles of 95°C, 30 seconds, 55°C, 30 seconds, 72°C, 45 seconds, final extension: 72°C, 10 minutes, storage: 10°C, until removal. In order to verify the fragment size of the amplification product, 1% agarose gel electrophoresis technology was used for detection. Then the PCR amplification product was sent to Shanghai Ling'en Biological Engineering Technology Service Co., Ltd. for sequencing, and the sequencing results were compared and analyzed by Blast on NCBI, and the homology was compared with other strain gene sequences in GenBank, and the sequencing results are shown in SEQ ID No: 1.

[0035] (4) The strain belongs to the phylum of Proteobacteria, the class of γ-Proteobacteria, the order of Enterobacteriales, the family of Enterobacteriaceae, and the genus of Serratia. It was preserved at the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Beijing, China, on October 23, 2025, with the preservation number CGMCC No. 36319, and the classification name Serratia saruman. Serratia saruman .

[0036] Example 2: Preliminary screening of strains by phosphorus ring and molybdenum-antimony anti-colorimetric method The obtained strains were inoculated into inorganic phosphorus solid medium (total phosphorus content about 1.01 g / L), and single colonies were picked after 2d of culture and transferred to test tubes containing inorganic phosphorus liquid medium. After the colonies grew to the logarithmic growth phase, they were centrifuged and shaken, and the sterile water and precipitate were mixed uniformly as a bacterial suspension. 10 μL of bacterial solution was dropped onto the center of the inorganic phosphorus solid medium, and the size of the phosphorus ring was observed after 2d to verify the phosphorus solubilizing ability. The specific results are as follows Figure 1As shown, the phosphorus-solubilizing strain appeared obvious phosphorus-solubilizing circle in inorganic phosphorus solid medium, indicating that the strain could exhibit phosphorus-solubilizing ability. Meanwhile, single colony in inorganic phosphorus solid medium was picked up on a clean bench and placed in a test tube containing 5 mL liquid medium, and after 2 days of shaking table shaking, the supernatant was obtained by centrifugation, and the OD value of the bacterial liquid was measured by molybdenum-antimony anti-colorimetric method. The calculation method is Y = 0.2757x + 0.0621 (Y is the mass concentration of phosphorus, and x is the absorbance). Further estimate the phosphorus-solubilizing ability, the results are shown in Table 1. Figure 2 As shown, compared with the bacterial liquid of inorganic phosphorus liquid medium without inoculating the strain, it indicates that the selected strain has a certain phosphorus-solubilizing ability.

[0037] Example 3: Further verify the phosphorus-solubilizing ability of the strain by the difference of soil available phosphorus before and after The experimental sample soil and brown soil culture bottles were taken, and then sterilized. The obtained strain was preliminarily inoculated into inorganic phosphorus solid medium, and after 2 days of culture, it was transferred to a conical flask containing liquid medium for shaking culture for 2 days. Then, the bacterial liquid was extracted, the supernatant was discarded by centrifugation, and the precipitate was added with sterile water and mixed uniformly by shaking. The OD value of the bacterial suspension was adjusted to 1. 600 As a bacterial suspension. The bacterial suspension was inoculated into the soil in the soil sample bottle at a ratio of 12 mL bacterial suspension per 100 g soil. Specifically, 12 mL bacterial liquid was added to 100 g soil, and the water content was maintained at 20%. After 7 days of dark culture, the change of available phosphorus in the soil before and after was measured, and the results are shown in Table 2. Figure 3 The results show that the strain increases the content of available phosphorus in the soil. Compared with the control group, the content of available phosphorus after inoculation is increased by 52.7%.

[0038] Example 4: Tobacco-microorganism interaction test to test the phosphorus-solubilizing ability of the strain 1. Prepare 20 times MS macroelement mother liquor, remove the soluble phosphorus source potassium dihydrogen phosphate 3.4 g, replace it with 3.8 g of insoluble calcium phosphate, and add 1.8 g of potassium chloride to supplement the missing potassium element. Prepare 100 times MS trace element mother liquor and 100 times MS iron salt mother liquor. Take 15 mL of 20 times macroelement mother liquor, 6 mL of 100 times trace element mother liquor, 6 mL of 100 times iron salt mother liquor, 1 g of anhydrous glucose, 1 g of sucrose, and 14 g of agar powder. Mix the above and add distilled water to 1 L. Pour the prepared medium into tissue culture bottles, and generally fill each bottle with about 1 / 3-1 / 2 of the volume of the bottle to facilitate subsequent operation and gas exchange.

[0039] 2. Surface sterilize tobacco seeds with 75% ethanol (30 s) and 3% NaClO (15 min), and then place them in a culture dish with only sterilized filter paper. After 7 days of culture and germination, the seedlings are transferred to the above MS medium-containing tissue culture bottles, with 2 seedlings per bottle. The culture is carried out at 22 °C with 16 h light and 8 h darkness for 7 days.

[0040] 3. The above-mentioned strains were initially inoculated onto inorganic phosphorus solid medium and cultured for 2 days. Single colonies were then picked and transferred to test tubes containing inorganic phosphorus liquid medium. The culture was incubated again for 2 days. The bacterial suspension was extracted, centrifuged, and the supernatant was discarded. The precipitate was mixed thoroughly with sterile water by shaking. The bacterial suspension concentration was adjusted to OD using sterile water. 600 =0.1 as bacterial suspension.

[0041] 4. Inoculate near the tobacco roots with 50 μL of bacterial suspension per plant. After 45 days of growth, the specific results are as follows: Figure 4 As shown, the phosphate-solubilizing strains promote the growth of tobacco biomass.

[0042] Example 5: Pot experiment to verify the phosphorus solubilization effect of the strain on tobacco plants The above-mentioned strains were initially inoculated onto inorganic phosphorus solid medium, and then transferred to liquid medium for shaking culture for 2 days. The bacterial culture was then extracted, the supernatant was discarded by centrifugation, and the precipitate was mixed thoroughly with sterile water by shaking. The OD was then adjusted. 600 1 was used as the bacterial suspension. Uniformly grown tobacco seedlings were selected and planted in pots containing sterile vermiculite and sand (6:1, v / v). On day 10, 10 mL of the bacterial suspension was inoculated, while the control group was inoculated with an equal volume of sterile water. Tobacco biomass was measured 45 days after planting, and the results are shown below. Figure 5 The results showed that *Serratia sarumanii* (…) Serratia sarumanii IA-3 significantly promotes the increase of tobacco biomass.

Claims

1. A strain of Serratia marcescens (S. marcescens) I-A-3, characterized in that, Serratia sarumanii )I-A-3, characterized in that, The Saluman Serratia ( Serratia sarumanii The deposit date of IA-3 is October 23, 2025. It is deposited at the China General Microbiological Culture Collection Center (CGMCC), Beijing, China, with accession number CGMCC No. 36319, and its taxonomical name is *Serratia sarumanii*. Serratia sarumanii .

2. The Serratia marcescens of claim 1, Serratia sarumanii ) I-A-3, characterized in that, The Salmonella salamae (S. salamae) Serratia sarumanii ) I-A-3 was isolated from the rhizoplane of tobacco.

3. The Serratia marcescens of claim 1, Serratia sarumanii )I-A-3 characterized in that, The 16S rDNA base sequence thereof is shown as SEQ ID No.

1.

4. A microbial inoculant or formulation, characterized in that, The bacterial agent or preparation contains one or several of Serratia marcescens ( Serratia sarumanii ) I-A-3 or a fermentation broth, a sterile supernatant thereof according to any one of claims 1 to 3.​ 5. The Serratia marcescens of any one of claims 1 to 3 ( Serratia sarumanii ) I-A-3 or the microbial inoculant or preparation of claim 4 for use in increasing the content of soluble phosphates in soil.​ 6. The Serratia marcescens of any one of claims 1 to 3 ( Serratia sarumanii ) I-A-3 or the microbial inoculant or formulation of claim 4 for use in promoting the growth of tobacco.​ 7. Use according to claim 5, characterized in that, The promotion of tobacco growth is obtained by converting soil-insoluble inorganic phosphorus into soluble phosphorus.

8. Use according to claim 6 or 7, characterized in that, The Salmonella salamae (S. salamae) Serratia sarumanii The amount of S. salamae (S. salamae) I-A-3 was 5 x 10 6 6 x 10 6 per strain.

9. Use according to claim 6 or 7, characterized in that, S. salumannii (ATCC 14771) Serratia sarumanii The concentration of S. salumannii (ATCC 14771) I-A-3 was 1 x 10 8 cfu / ml ~ 2 x 10 8 cfu / ml.

10. Use according to claim 6 or 7, characterized in that, the Salmonella salamae Serratia sarumanii The application temperature of the bacterial agent or preparation of claim 1-A-3 or claim 4 is 15-20°C.

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