Burkholderia pyrrocinia for improving phosphorus utilization capacity of tobacco and application of burkholderia pyrrocinia

Abstract

The invention discloses a Burkholderia pyrrocinia capable of improving phosphorus utilization capacity of tobacco and application of the Burkholderia pyrrocinia, the Burkholderia pyrrocinia is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation address is Beijing, the preservation center number is CGMCC No.36320, the preservation date is October 23, 2025, and the Burkholderia pyrrocinia is classified and named as Burkholderia pyrrocinia. The strain Burkholderia pyrrocinia is an efficient inorganic phosphorus decomposing bacterium, can significantly increase the effectiveness of insoluble inorganic phosphorus in soil and promote phosphorus absorption and healthy growth of tobacco, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to a strain of Burkholderia pyrrole that improves phosphorus utilization in tobacco and its application, belonging to the field of tobacco production technology. Background Technology

[0002] Phosphorus is one of the three essential nutrients for plant growth and development, playing a crucial role in physiological processes such as root development, flower and fruit formation, and seed maturation. However, the vast majority of phosphorus in soil exists in insoluble forms, making it difficult for plants to directly absorb and utilize it. Statistics show that only about 1% to 5% of the phosphorus in soil is available to plants, with the vast majority fixed in the soil as insoluble phosphates (Li Tingxuan et al., 2017, Journal of Plant Nutrition and Fertilizers: Research Progress on Plant Response Characteristics to Different Forms of Phosphorus). To ensure crop yields, agricultural production has long relied on the application of chemical phosphate fertilizers, but this not only leads to low phosphate fertilizer utilization and increased production costs but also easily causes environmental pollution problems. Therefore, developing efficient and green soil phosphorus activation technologies has become a key issue for sustainable agricultural development.

[0003] Against this backdrop, microbial phosphorus solubilization technology has become a research hotspot both domestically and internationally due to its environmentally friendly and resource-efficient characteristics. The core of this technology lies in utilizing phosphorus-solubilizing microorganisms (especially strains with highly efficient inorganic phosphorus solubilization capabilities) to convert fixed, insoluble inorganic phosphorus in the soil into soluble phosphates through their metabolic processes, thereby enhancing the bioavailability of soil phosphorus and alleviating the contradiction between "soil phosphorus fixation" and "plant phosphorus demand." Therefore, discovering and applying highly efficient inorganic phosphorus-solubilizing strains to promote tobacco growth under low phosphorus stress is of great significance for the sustainable development of tobacco production. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a strain of Burkholderia pyrrole, which has high activity in activating insoluble inorganic phosphorus in soil and significantly improving the bioavailability of phosphorus. Burkholderia puraquae IB-3-1 strain and its application.

[0005] Technical solution: This invention provides a strain of Burkholderia pyrrole (… Burkholderia puraquae IB-3-1, this strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), Beijing, China, with accession number CGMCC No. 36320, deposited on October 23, 2025, and its taxonomical name is *Burkholderia pyrrole*. Burkholderia puraquae .

[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 aforementioned Burkholderia pyrroloides (…). Burkholderia puraquae IB-3-1 or its fermentation broth, sterile supernatant or one or more thereof.

[0008] The present invention also provides the aforementioned Burkholderia pyrrole ( Burkholderia puraquae The application of IB-3-1 or the aforementioned microbial agent or preparation in enhancing soil phosphorus availability, namely, converting insoluble inorganic phosphorus in the soil into soluble phosphate.

[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 Burkholderia pyrrologensis (B. pyrrologensis) to the tobacco plant. Burkholderia puraquae IB-3-1 or microbial agents or preparations.

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

[0011] Among them, the *Burkholderia pyrrole* ( Burkholderia puraquae The dosage of IB-3-1 is 5 × 10⁻⁶. 6 ~6×10 6 One per plant.

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

[0013] Among them, Burkholderia pyrrole ( Burkholderia puraquae The application temperature of IB-3-1 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) Take 1 mL of tobacco root surface microbial suspension and dilute it 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 -6 Suspensions of varying concentrations were evenly spread onto inorganic phosphorus solid medium, with each concentration gradient repeated twice, and incubated in the dark at 20°C for 5 days. Single colonies were picked from plates of appropriate concentration gradients based on bacterial morphology, color, and size, and then purified and cultured sequentially.

[0017] (4) Extract DNA from the tested strains and perform 16S rRNA sequencing to determine the taxonomic position of the strains.

[0018]

[0019] This invention also provides verification of the effect of the phosphate-solubilizing strain on the solubility of inorganic phosphorus: (1) The obtained strains were inoculated into inorganic phosphorus solid medium and cultured for 2 days. Single colonies were picked and transferred to test tubes containing inorganic phosphorus liquid medium. After the colonies grew to the logarithmic growth phase, they were centrifuged and the precipitate was collected. Sterile water was added and the precipitate was shaken and mixed evenly to form a bacterial suspension. At the same time, single colonies in solid medium were picked into test tubes containing 5 mL of inorganic phosphorus liquid medium in a clean bench and a control without the above strains was set up. After shaking on a shaker for 2 days, the supernatant was collected by centrifugation.

[0020] (2) Take 10 μL of the above bacterial suspension and drop it into the center of the inorganic phosphorus solid medium. Observe the size of the phosphorus-solubilizing zone after 2 days. At the same time, take the supernatant from another test tube and measure the OD value of the bacterial culture using the molybdenum antimony colorimetric method to further estimate the phosphorus-solubilizing ability. The results show that the phosphorus-solubilizing strains show obvious phosphorus-solubilizing zones in the inorganic phosphorus solid medium. Compared with the bacterial culture in the inorganic phosphorus liquid medium without inoculation, the selected strains have a certain phosphorus-solubilizing ability.

[0021] This invention also provides verification of the phosphorus-solubilizing efficacy of the aforementioned phosphorus-solubilizing strain: (1) Sample preparation: Take soil samples and brown soil sample bottles from the experimental site, sterilize them in sequence, and set them aside.

[0022] (2) Culture of bacterial strain: Burkholderia pyrrole was initially inoculated into inorganic phosphorus solid medium and cultured for 2 days. After that, a single colony was picked and transferred to an Erlenmeyer flask containing inorganic phosphorus liquid medium and cultured on a shaker for 2 days. The bacterial culture was extracted, the supernatant was discarded by centrifugation, and the precipitate was mixed with sterile water by shaking and the OD was adjusted. 600 The value is 1.

[0023] (3) Inoculation of strains: Inoculate the above-mentioned phosphate-solubilizing strains into the soil in the soil sample bottle (take 12 mL of bacterial solution and add it to 100 g of soil), and keep the moisture content at 20%.

[0024] (4) After 7 days of incubation in the dark, phosphorus was determined by the molybdenum antimony colorimetric method to compare the effective phosphorus content. Compared with the control group, the effective phosphorus content increased by 52.3% after inoculation.

[0025] This invention also determined the inorganic phosphorus solubilizing effect of the strain through aseptic seedling inoculation tests: (1) Preparation of MS medium: Prepare a 20-fold stock solution of MS macroelements, remove 3.4g of soluble phosphorus source potassium dihydrogen phosphate and replace it with 3.8g of insoluble calcium phosphate, and add 1.8g of potassium chloride to supplement the missing potassium element. Prepare a 100-fold stock solution of MS microelements and a 100-fold stock solution of MS iron salts. Take 15mL of the 20-fold stock solution of macroelements, 6mL of the 100-fold stock solution of microelements, 6mL of the 100-fold stock solution of iron salts, 1g of anhydrous glucose, 1g of sucrose, and 14g of agar powder. Mix the above and add distilled water to make up to 1L. Adjust the pH to 5.8, dispense into tissue culture flasks, 80mL per dish, and autoclave at 121 °C for 15 minutes.

[0026] (2) Tobacco seedling culture: Tobacco seeds were surface sterilized with 75% ethanol (30s) and 3% NaClO (15min), and then placed in petri dishes with only sterilized filter paper. After germination for 7 days, the seedlings were transferred to tissue culture bottles containing MS medium, with 2 seedlings per bottle. They were then kept at 22 °C for 16 h of light and 8 h of darkness for 7 days.

[0027] (3) Preparation of phosphate-solubilizing bacteria fermentation broth: The above strains were initially inoculated into inorganic phosphorus solid medium and cultured for 2 days. Single colonies were picked and transferred to test tubes containing inorganic phosphorus liquid medium and cultured again for 2 days. The bacterial broth was extracted, centrifuged and the supernatant was discarded. The precipitate was mixed with sterile water by shaking. The bacterial broth concentration was adjusted to OD using sterile water. 600 =0.1 as the bacterial suspension. Inoculate each tobacco plant near the root system with 50 μL of the bacterial suspension to allow *Burkholderia pyrrolochia* (… Burkholderia puraquae The concentration of IB-3-1 is 1×10 8 cfu / ml ~2×10 8 cfu / ml, dosage 5×10 6 ~6×10 6 One per plant.

[0028] (4) Verification of phosphorus solubilization effect: 45 days after inoculation, tobacco in the tissue culture bottle was destructively sampled and the growth status of tobacco was compared to verify the phosphorus solubilization effect.

[0029] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The phosphorus-solubilizing bacterial agent of the present invention addresses the phosphorus fixation problem that is common in tobacco planting soil. The selected inorganic phosphorus-solubilizing bacterial strain can efficiently activate insoluble calcium phosphate in the soil, ensuring that tobacco roots can directly absorb soluble phosphorus. Moreover, the bacteria have strong phosphorus-solubilizing ability and good rhizosphere colonization effect, which can provide a continuous and stable source of phosphorus nutrition for tobacco growth, effectively overcoming the shortcomings of traditional phosphate fertilizers that are easily fixed and have low utilization rate. Attached Figure Description

[0030] Figure 1Burkholderia pyrrole ( Burkholderia puraquae Preliminary evidence of phosphorus solubilization ability was obtained from the phosphorus-solubilizing zone of IB-3-1. Figure 2 Burkholderia pyrrole ( Burkholderia puraquae After IB-3-1 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 Burkholderia pyrrole ( Burkholderia puraquae Comparison of the phosphorus solubilization effect of IB-3-1 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 -6Suspensions of varying concentrations were evenly spread onto inorganic phosphorus solid medium, with each concentration gradient repeated twice, and incubated in the dark at 20°C for 5 days. Single colonies were picked from plates of appropriate concentration gradients based on bacterial morphology, color, and size, and then purified and cultured sequentially.

[0034] (3) Determination of taxonomic position: Bacterial genetic material DNA was extracted using a DNA extraction kit (TIANamp Bacteria DNA Kit, TIANGEN). PCR amplification of 16S rDNA was performed on these DNA samples using primers 27F-1492R with sequences AGRGTTYGATYMTGGCTCAG and RGYTACCTTGTTACGACTT. The total reaction volume was 50 μl, and TransStart Fastpfu DNA Polymerase was used. The program was as follows: pre-denaturation: 95℃, 5 minutes (1 cycle); amplification: 27 cycles of 95℃, 30 seconds, 55℃, 30 seconds, 72℃, 45 seconds; final extension: 72℃, 10 minutes; storage: 10℃ until removal. To verify the fragment size of the amplified products, 1% agarose gel electrophoresis was used for detection. The PCR amplification products were then sent to Shanghai Lingen Biotechnology Service Co., Ltd. for sequencing. The sequencing results were compared with BLAST on NCBI and compared with the gene sequences of other strains in GenBank for homology. The sequencing results are shown in SEQ ID No: 1.

[0035] (4) This strain belongs to the phylum Pseudomonas, class Gamma-Proteobacteria, order Burkholderia, family Burkholderia, genus Burkholderia. It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, China. The deposit date is October 23, 2025, and the accession number is CGMCC No. 36320. The classification name is *Burkholderia pyrrole*. Burkholderia puraquae .

[0036] Example 2: Preliminary screening of strains using the phosphorus solubilization zone and molybdenum-antimony colorimetric method The obtained bacterial strains were inoculated into inorganic phosphorus solid medium (total phosphorus content approximately 1.01 g / L). After culturing for 2 days, single colonies were picked and transferred to test tubes containing inorganic phosphorus liquid medium. After the colonies reached the logarithmic growth phase, they were centrifuged, and the sterile water and precipitate were mixed thoroughly to form a bacterial suspension. 10 μL of the suspension was dropped into the center of the inorganic phosphorus solid medium. After 2 days, the size of the phosphate-solubilizing zone was observed to verify the phosphate-solubilizing ability. Specific results are shown below. Figure 1As shown, the phosphorus-solubilizing strain exhibits a distinct phosphorus-solubilizing zone in inorganic phosphorus solid medium, indicating that the strain can demonstrate phosphorus-solubilizing ability.

[0037] Simultaneously, on a clean bench, a single colony from the inorganic phosphorus solid culture medium was transferred to a test tube pre-filled with 5 mL of liquid culture medium. After shaking for 2 days, the supernatant was collected by centrifugation. The OD value of the bacterial culture was measured using the molybdenum antimony colorimetric method, calculated as: Y = 0.2757x + 0.0621 (where Y is the mass concentration of phosphorus and x is the absorbance) to further estimate the phosphorus solubility. The results are as follows: Figure 2 As shown, compared with the bacterial culture in inorganic phosphorus liquid medium without inoculation, the selected strain has a certain phosphorus solubilizing ability.

[0038] Example 3: Further verification of the strain's phosphorus solubilization ability by examining the difference in available phosphorus in the soil before and after processing. Soil samples from the experimental site and brown soil culture bottles were sterilized sequentially. The obtained bacterial strain was initially inoculated onto inorganic phosphorus solid medium and cultured for 2 days. Then, it was transferred to an Erlenmeyer flask containing liquid medium and cultured with shaking for another 2 days. The bacterial suspension was extracted, the supernatant was discarded by centrifugation, and the precipitate was mixed thoroughly with sterile water by shaking. The OD was adjusted. 600 1 was used as the bacterial suspension. The bacterial suspension was inoculated into the soil in soil sample bottles at a ratio of 12 mL bacterial suspension / 100 g soil. Specifically, 12 mL of bacterial suspension was added to 100 g of soil, maintaining a moisture content of 20%. After incubation in the dark for seven days, the change in available phosphorus in the soil before and after incubation was measured using the molybdenum-antimony colorimetric method. The results are shown in [Figure 1]. Figure 3 The results showed that the strain increased the available phosphorus content in the soil. Compared with the control group, the available phosphorus content increased by 52.3% after inoculation.

[0039] Example 4: Tobacco-microbe interaction test to examine the phosphate solubilization ability of strains 1. Prepare a 20x stock solution of MS macronutrients, removing 3.4g of the soluble phosphorus source potassium dihydrogen phosphate and replacing it with 3.8g of insoluble calcium phosphate. Add 1.8g of potassium chloride to supplement the missing potassium. Prepare a 100x stock solution of MS micronutrients and a 100x stock solution of MS iron salts. Take 15mL of the 20x macronutrient stock solution, 6mL of the 100x micronutrient stock solution, 6mL of the 100x iron salt stock solution, 1g of anhydrous glucose, 1g of sucrose, and 14g of agar powder. Mix these together and add distilled water to a final volume of 1L. Pour the prepared culture medium into tissue culture flasks, filling each flask with an appropriate amount of liquid culture medium, generally about 1 / 3 to 1 / 2 of the flask's volume, to facilitate subsequent operations and gas exchange.

[0040] 2. Tobacco seeds were surface-sterilized with 75% ethanol (30 s) and 3% NaClO (15 min), and then placed in petri dishes containing only sterilized filter paper. After germination for 7 days, the seedlings were transferred to tissue culture flasks containing MS medium, two seedlings per flask. They were then incubated at 22 °C for 16 h in light and 8 h in darkness for 7 days.

[0041] 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.

[0042] 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.

[0043] 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 Burkholderia pyrrole (…) Burkholderia puraquae IB-3-1 significantly promoted the increase of tobacco biomass.

Claims

1. A strain of Burkholderia pyrrole ( Burkholderia puraquae )IB-3-1, characterized in that, The pyrrolizobacterium purpureus ( Burkholderia puraquae IB-3-1 is deposited at the China General Microbiological Culture Collection Center (CGMCC), Beijing, China, on October 23, 2025, with accession number CGMCC No. 36320, and is classified as *Burkholderia pyrrole*. Burkholderia puraquae .

2. The Burkholderia pyrrole-Holder as described in claim 1 ( Burkholderia puraquae )IB-3-1, characterized in that, The pyrrolizobacterium purpureus ( Burkholderia puraquae IB-3-1 was obtained by isolating tobacco root epidermis.

3. Burkholderia pyrrole as described in claim 1 ( Burkholderia puraquae )IB-3-1, characterized in that, Its 16S rDNA base sequence is shown in SEQ ID No.

1.

4. A microbial agent or preparation, characterized in that, The bacterial agent or preparation contains *Burkholderia pyrogallol* as described in any one of claims 1 to 3. Burkholderia puraquae IB-3-1 or its fermentation broth, sterile supernatant or one or more thereof.

5. The Burkholderia pyrrole-Holder as described in any one of claims 1 to 3 ( Burkholderia puraquae The application of the microbial agent or preparation described in IB-3-1 or claim 4 in increasing the soluble phosphate content in soil.

6. The Burkholderia pyrrole-Holder as described in any one of claims 1 to 3 ( Burkholderia puraquae The application of the microbial agent or preparation as described in claim IB-3-1 or claim 4 in promoting tobacco growth.

7. The application according to claim 6, characterized in that, The method of promoting tobacco growth is achieved by converting insoluble inorganic phosphorus in the soil into soluble phosphorus.

8. The application according to claim 6 or 7, characterized in that, The pyrrolizobacterium purpureus ( Burkholderia puraquae The dosage of IB-3-1 is 5 × 10⁻⁶. 6 ~6×10 6 One per plant.

9. The application according to claim 6 or 7, characterized in that, The pyrrolizobacterium purpureus ( Burkholderia puraquae The concentration of IB-3-1 is 1×10 8 cfu / ml ~2×10 8 cfu / ml.

10. The application according to claim 6 or 7, characterized in that, The pyrrolizobacterium purpureus ( Burkholderia puraquae The application temperature of the microbial agent or preparation described in claim IB-3-1 or claim 4 is 15-20 ℃.

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