Burkholderia new strain and application thereof in tobacco growth promotion

Abstract

The invention discloses a novel Burkholderia sp. Strain and application thereof in tobacco growth promotion, and belongs to the technical field of microorganisms. The new strain is preserved in the China Center for Type Culture Collection (CCTCC), the preservation address is Wuhan University, Wuhan, China, the preservation number is CCTCC NO: M 20252947, and the preservation time is December 18, 2025. The new Burkholderia sp. BP5 is obtained through separation for the first time, the new Burkholderia sp. BP5 has good phosphate solubilizing capacity, seed germination is promoted by remarkably improving the germination rate, germination potential, germination number and root length of seeds, plant growth can be remarkably promoted, and the yield of plants is increased. Meanwhile, the new strain BP5 has a relatively good prevention and treatment effect on plant pathogenic bacterial diseases such as rice bacterial streak, so that the new strain BP5 has a relatively good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a new species of Burkholderia and its application in tobacco growth promotion. Background Technology

[0002] Phosphate-solubilizing microorganisms (PSMs) are a class of plant rhizosphere growth-promoting bacteria (PGPRs) with great development potential. They generally refer to bacteria that can convert insoluble inorganic phosphorus (such as Ca-P, Fe-P, Al-P) and organic phosphorus (such as phytic acid and phospholipids) in the soil into available phosphorus (H2PO4) that can be directly absorbed and utilized by plants through mechanisms such as acidification, chelation, redox reactions, or enzymatic reactions. - / HPO4 2- PSMs are a general term for microorganisms that promote phosphate dissolution. During their growth and reproduction, PSMs secrete organic acids (such as gluconic acid, citric acid, oxalic acid, etc.) to lower the pH of the rhizosphere and promote the dissolution of phosphate minerals. On the other hand, they synthesize extracellular enzymes such as acid / alkaline phosphatases and phytases to mineralize organic phosphorus compounds. In addition, PSMs stimulate the development of lateral roots and root hairs and enhance nutrient absorption efficiency by producing plant hormones such as indole-3-acetic acid (IAA), cytokinins (CKs), and 1-aminocyclopropane-1-carboxylic acid deaminase (ACC deaminase), exhibiting a typical dual function of "promoting growth and dissolving phosphorus".

[0003] In recent years, research on PSMs has expanded from their single function of phosphorus solubilization to multiple dimensions, including heavy metal contaminated soil remediation, innovation of microbial fertilizer carriers, and rhizosphere microbiome regulation. Studies have shown that Burkholderia (… Burkholderia sp. Cd can be fixed through bioprecipitation and bioadsorption mechanisms. 2+ Pb 2+ This reduces its bioavailability. Meanwhile, an environmentally friendly microbial fertilizer prepared by encapsulating phosphate-solubilizing bacteria in sodium alginate-biochar composite microspheres increased phosphate fertilizer utilization by 31.5% and grain yield by 18.4% in a maize field trial. Furthermore, the Institute of Urban Environment, Chinese Academy of Sciences, used single-cell Raman-heavy water labeling technology to identify and quantify active phosphate-solubilizing bacteria in situ in nine different soil types, revealing the significant impact of soil properties and fertilization management on PSMs activity, providing a theoretical basis for the precise application of microbial fertilizers.

[0004] Phosphorus is an essential element for plant growth, directly determining plant productivity and ecological functions. The total phosphorus content in soil is approximately (0.05-3.00) g / kg, but most phosphorus exists primarily in the form of insoluble organic or inorganic phosphorus, limiting direct absorption and utilization by plants. Therefore, the amount of available phosphorus that can be directly absorbed and utilized by plants is relatively small. Thus, increasing the available phosphorus content in soil through the application of phosphate fertilizers is a major method in current agricultural production. However, excessive fertilization can lead to a decrease in fertilizer utilization efficiency.

[0005] Therefore, developing new strains of highly efficient phosphate-solubilizing bacteria is of great significance for enriching the variety of phosphate-solubilizing bacteria and expanding the new uses of strains. Summary of the Invention

[0006] The purpose of this invention is to provide a new species of Burkholderia and its application in tobacco growth promotion. The new Burkholderia species BP5 provided by this invention has good phosphorus solubilization, seed germination promotion, and plant growth promotion abilities, and also shows good control effects against plant pathogenic bacterial diseases such as rice bacterial leaf streak; therefore, it has good application prospects in plant growth promotion and / or control of plant pathogenic bacterial diseases.

[0007] In a first aspect, the present invention provides a new species of Burkholderia (… Burkholderia sp. BP5, a new species of Burkholderia ( Burkholderia sp. The accession number for BP5 is CCTCC NO:M 20252947.

[0008] The novel strain BP5 was isolated from rhizosphere soil from the tobacco field experimental base in Baiyangba Town, Lichuan City, Enshi Tujia and Miao Autonomous Prefecture, Hubei Province (30°17′37.5″N, 108°55′56.3″E). On solid culture plates, colonies of BP5 are milky white, with raised surfaces and regular edges. SEM analysis showed that BP5 is a typical short rod-shaped cell with a long axis diameter of 1.5–3.0 µm, a short axis diameter of 0.5–0.7 µm, and an aspect ratio of approximately 3.0–4.0. Its cell wall surface is smooth, and no obvious flagella or cilia were observed.

[0009] Furthermore, the 16S rDNA of the novel strain BP5 was sequenced, and its 16S rDNA sequence was BLASTed in NCBI (https: / / www.ncbi.nlm.nih.gov / ). A phylogenetic tree was constructed using MEGA 6.0 software. The results showed that the novel strain BP5 had high homology with Burkholderia and... Burkholderia sp. strains 1-9 are in the same branch; furthermore, by constructing a draft bacterial genome framework, a new species strain BP5 was found to be related to... Burkholderia cepacia , Burkholderia cenocepacia , Burkholderia ambifaria The protein sequence similarity of the three strains was 30.43%, 13.82%, and 13.55%, respectively. The average nucleotide identity (ANI) and DNA hybridization homology (dDDH) of the novel strain BP5 were compared with those of the three strains. The results showed that the ANIs of the novel strain BP5 with the three strains were 89.88%, 90.25%, and 92.42%, respectively; the dDDHs were 39.3%, 40.4%, and 47.4%, respectively. These values ​​were below the threshold for identifying a novel strain (ANI below 95% and dDDH below 70%). Based on the morphological characteristics and molecular biological identification results, the novel strain BP5 was named *Burkholderia burkermansia*. Burkholderia sp. BP5.

[0010] In a second aspect, the present invention provides a biological agent comprising the aforementioned new species of Burkholderia (… Burkholderia sp. BP5.

[0011] In some implementations, the biological agent is a liquid agent.

[0012] Understandably, biological agents can be selected from conventional formulations in the prior art according to actual usage needs. For example, they can also be solid agents, and solid agents can be prepared using conventional methods in the art.

[0013] In a third aspect, the present invention provides a method for preparing the above-described biological agent, comprising the following steps: preparing the above-described new species of Burkholderia (… Burkholderia sp. BP5 was inoculated into liquid culture medium and cultured. The resulting culture was separated and resuspended in a solvent to obtain a biological agent.

[0014] The preparation method of the biological agent provided by this invention is simple, environmentally friendly, and has good biosafety.

[0015] Understandably, the liquid culture medium can be any common culture medium in the field, as long as it meets the requirements of this new species of Burkholderia (…). Burkholderia sp. Normal growth of BP5 is sufficient. Conventional culture conditions can be selected. In this invention, the liquid culture medium is preferably LB medium.

[0016] In some implementations, the solvent includes at least one of PBS buffer and sterile water.

[0017] In a fourth aspect, the present invention provides a new species of Burkholderia as described above ( Burkholderia sp. BP5. Application of any of the above-mentioned biological agents or biological agents prepared by the above-mentioned methods in phosphorus solubilization.

[0018] In a fifth aspect, the present invention provides a new species of Burkholderia as described above ( Burkholderia sp. BP5. The application of any of the above-mentioned biological agents or biological agents prepared by the above-mentioned methods in promoting seed germination.

[0019] In some implementations, the seeds include flue-cured tobacco seeds.

[0020] Understandably, the seeds can be conventional plant seeds in the prior art; for example, in this invention, the seeds preferably include flue-cured tobacco seeds.

[0021] In a sixth aspect, the present invention provides a new species of Burkholderia as described above ( Burkholderia sp. BP5. Application of any of the above-mentioned biological agents or biological agents prepared by the above-mentioned methods in promoting plant growth.

[0022] In some implementations, the plant includes tobacco.

[0023] It is understood that the plant can be a conventional plant in the prior art, for example, in this invention, the plant preferably includes tobacco.

[0024] In a seventh aspect, the present invention provides a new species of Burkholderia as described above ( Burkholderia sp. BP5. Application of any of the above-mentioned biological agents or biological agents prepared by the above-mentioned methods in the prevention and control of plant diseases.

[0025] In some implementations, plant diseases include bacterial leaf streak of rice.

[0026] It is understood that the plant diseases can be conventional plant diseases in the prior art. For example, in this invention, the plant diseases preferably include bacterial leaf streak of rice.

[0027] In some implementations, the pathogen of bacterial leaf streak in rice includes bacterial leaf streak bacterium RH3.

[0028] The beneficial effects of this invention are: unlike the prior art, this invention is the first to isolate a new species of Burkholderia (…). Burkholderia sp. BP5, this new strain has good phosphorus solubilization ability. It promotes seed germination by significantly improving seed germination rate, germination potential, number of germinations and root length, and can also significantly promote plant growth. At the same time, it has a good control effect on plant pathogenic bacterial diseases such as rice bacterial leaf streak. Therefore, this new strain BP5 has good application prospects. Attached Figure Description

[0029] Figure 1 This is a colony morphology diagram of the novel strain BP5 in Example 1 of the present invention;

[0030] Figure 2 This is a SEM image of the novel strain BP5 in Example 1 of the present invention; Figure 3 This is a gel electrophoresis image of the 16S rDNA fragment of the novel strain BP5 in Example 1 of the present invention; Figure 4 This is a phylogenetic tree diagram constructed based on the novel strain BP5 in Example 1 of the present invention; Figure 5 This is a draft of the bacterial genome framework constructed based on the novel strain BP5 in Example 1 of the present invention; Figure 6 This is a growth curve of the new strain BP5 in Example 2 of the present invention; Figure 7 This is a plate graph showing the phosphate-solubilizing ability of the novel strain BP5 in Example 3 of the present invention; Figure 8 This is a graph showing the pH value and soluble phosphorus content of the novel strain BP5 in Example 3 of the present invention after being cultured in a phosphorus-solubilizing liquid medium. Figure 9 The figures show the germination potential, germination rate, number of germinated seeds, root length, and growth results on a plate after tobacco seeds were treated with the new strain BP5 inoculant in Example 4 of this invention. Figure 10 This is a graph showing the inhibitory effect of the novel strain BP5 on bacterial streak causal agent RH3 in Example 5 of the present invention.

[0031] Biological Preservation The strain provided by this invention is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20252947, deposited on December 18, 2025, and classified as a new species of Burkholderia. Burkholderia sp. BP5. Detailed Implementation

[0032] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0033] Experimental methods not specifically described in the examples are generally performed according to conventional experimental methods in the field of molecular biology, including but not limited to those described in *Molecular Cloning: A Laboratory Manual* by M.R. Green and *Molecular Biology* by Robert F. Weaver, or according to the experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, all reagents and biological materials used in the examples are commercially available.

[0034] The culture medium formula used in this invention is as follows: The solid culture medium for isolating phosphate-solubilizing bacteria consists of the following components: glucose 10 g / L, sodium chloride 0.3 g / L, magnesium sulfate 0.3 g / L, ammonium sulfate 0.5 g / L, ferrous sulfate 0.03 g / L, manganese sulfate 0.03 g / L, potassium chloride 0.3 g / L, calcium phosphate 5 g / L, yeast 0.5 g / L, and agar 15 g / L, with the pH adjusted to approximately 7.0.

[0035] The solid culture medium for screening phosphate-solubilizing bacteria consisted of the following components: glucose 10 g / L, magnesium sulfate 0.25 g / L, magnesium chloride 0.3 g / L, ammonium sulfate 0.1 g / L, and agar 15 g / L, with the pH adjusted to approximately 7.0.

[0036] The LB liquid culture medium consists of the following components: 10 g / L peptone, 5 g / L yeast, 10 g / L NaCl, and natural pH.

[0037] The NA solid culture medium consists of the following components: 5 g / L peptone, 10 g / L sucrose, 1 g / L yeast, 3 g / L beef extract, and 15 g / L agar, with the pH adjusted to approximately 7.0.

[0038] Example 1: Screening and Identification of New Strains BP5 1.1 Screening of the new strain BP5 10g of rhizosphere soil sample from the tobacco field experimental base (30°17′37.5″N, 108°55′56.3″E) in Baiyangba Town, Lichuan City, Enshi Tujia and Miao Autonomous Prefecture, Hubei Province was placed into an Erlenmeyer flask containing 90mL of sterile water. The sample was shaken at 180r / min for 30min, serially diluted, and spread onto a solid medium for phosphate-solubilizing bacteria isolation. The sample was then incubated at 37℃ for 3-4 days. After colonies emerged, the best-growing colonies were selected and inoculated onto solid medium plates for phosphate-solubilizing bacteria screening. The plates were then incubated at 30℃ for 7 days. Strains with good growth were selected from the screening medium plates and inoculated onto slant agar (containing components of the solid medium for phosphate-solubilizing bacteria isolation) for preservation. A strain with phosphate-solubilizing ability was obtained and named the new strain BP5.

[0039] The colonies of this new strain BP5 on solid culture plates are milky white, with raised surfaces and neat edges (e.g., ...). Figure 1 As shown in the image); SEM testing revealed that the new strain BP5 exhibits a typical short rod shape, with a major axis diameter of 1.5-3.0 µm, a minor axis diameter of 0.5-0.7 µm, and an aspect ratio of approximately 3.0-4.0; its cell wall surface is smooth, and no obvious flagellar or ciliate structures were observed (e.g., ...). Figure 2 (As shown).

[0040] 1.2 Biological identification of the new strain BP5 The novel strain BP5 obtained in step 1.1 was inoculated into LB liquid medium and cultured at 30°C and 150 rpm for 24 h. After culture, the bacterial cells were collected by centrifugation and genomic DNA was extracted. The 16S rDNA fragment was amplified using universal primers 27F and 1492R. The amplified product was confirmed by electrophoresis (e.g., ...). Figure 3 (As shown) was then sent for sequencing. Its 16S rDNA sequence is shown in SEQ ID NO: 1, and the sequence length is 1441 bp. The sequenced 16S rDNA sequence was BLASTed in NCBI (https: / / www.ncbi.nlm.nih.gov / ), and a phylogenetic tree of the type strain and the tested strain was constructed using MEGA 6.0. The results showed that the new strain BP5 had high homology with Burkholderia, and... Burkholderia sp. strains 1-9 are in the same branch (e.g.) Figure 4 (As shown); Furthermore, by constructing a draft bacterial genome framework, a new strain BP5 was discovered to be related to... Burkholderia cepacia , Burkholderia cenocepacia , Burkholderia ambifaria The protein sequence similarity of the three strains was 30.43%, 13.82%, and 13.55%, respectively (e.g., ...). Figure 5 As shown in Table 1, the average nucleotide homogeneity (ANI) and DNA hybridization homology (dDDH) of the new strain BP5 were compared with those of the three strains mentioned above.

[0041] Table 1. Comparison of ANI and dDDH between the new strain BP5 and the three strains.

[0042] As shown in Table 1, the ANI of the new strain BP5 with the three strains mentioned above were 89.88%, 90.25%, and 92.42%, respectively; the dDDH of the new strain BP5 with the three strains mentioned above were 39.3%, 40.4%, and 47.4%, respectively. These values ​​are all below the threshold for identifying new strains (ANI below 95% and dDDH below 70% can identify the strain as a new strain).

[0043] In summary, based on morphological characteristics and molecular biological identification results, the new strain BP5 is named Burkholderia species (…). Burkholderia sp. BP5.

[0044] Example 2: Growth curve determination of the new strain BP5 Single colonies of the novel strain BP5 were inoculated into LB liquid medium and cultured with shaking at 30°C and 150 rpm. Medium uninoculated with the novel strain BP5 served as a blank control. OD values ​​were measured every 3 hours. 600 All experiments were repeated three times, and the results are as follows: Figure 6 As shown.

[0045] from Figure 6 As can be seen, the new strain BP5 was in the lag phase from 0 to 4 hours, entered the logarithmic phase from 4 to 16 hours, and then its growth tended to stabilize. The results indicate that the new strain BP5 has stable growth and good metabolic activity.

[0046] Example 3 Quantitative determination of the phosphorus solubilization ability of the new strain BP5 Take 5 μL of the new strain BP5 bacterial culture (OD) 600 The bacterial culture (with a value of 0.014) was inoculated onto a solid medium for phosphate-solubilizing bacteria using the spot staining method and cultured at 30°C. During the culture, the phosphate solubility index of the strain was observed and measured every two days. The results are as follows: Figure 7 As shown.

[0047] The solubility index (SI) is defined as the ratio of the diameter of the solubility zone to the diameter of the colony (D / d).

[0048] from Figure 7 As can be seen, the phosphorus solubility index (SI) of the new strain BP5 increases with the increase of culture time, indicating that the new strain BP5 has good phosphorus solubility.

[0049] Furthermore, the new strain BP5 was inoculated into LB liquid medium and cultured at 30℃ and 150 rpm for 24 h. 1 mL of the bacterial culture was added to 100 mL of phosphate-solubilizing bacteria isolation liquid medium (phosphate-solubilizing bacteria solid medium without agar is acceptable). Each treatment was performed in triplicate, with a control group not inoculated with the new strain BP5. The culture was continued at 30℃ and 150 rpm for 8 days. Samples were taken on day 8, and the pH and soluble phosphorus content of the samples were measured. The results are as follows: Figure 8 As shown.

[0050] from Figure 8 As can be seen, compared with the control group, the pH value of the experimental group decreased after the addition of the new strain BP5, while the soluble phosphorus content of the sample was 568.76 mg / L. The results indicate that the new strain BP5 has good phosphorus solubilization ability.

[0051] Example 4: Seed germination promotion test of new strain BP5 Preparation of bacterial agent: Activate the new strain BP5. After colonies grow, select colonies are inoculated into LB liquid medium and cultured at 30℃ and 150 rpm for 24 h on a shaker. After centrifugation, adjust the OD of the bacterial suspension with sterile water. 600 The value was 0.5, and a new strain of bacteria, BP5, was prepared.

[0052] First, disinfect the tobacco seeds, then wash them three times with sterile water. Soak them for 2 hours in the prepared BP5 bacterial agent. After drying with sterile filter paper, place them in petri dishes lined with moistened filter paper, with 50 seeds per dish. The strain was prepared in triplicate, with sterile water treatment as a control (CK). After incubation at 25℃ for 14 days, germination indicators such as germination energy, germination rate, germination numbers, and root length were measured. The results are as follows: Figure 9 As shown.

[0053] from Figure 9 As can be seen, treatment with the new strain BP5 significantly improved germination indicators such as germination potential, germination rate, number of germinations, and root length in flue-cured tobacco; the results indicate that treatment with the new strain BP5 inoculum has a general promoting effect on the early germination of tobacco seeds.

[0054] Example 5: Control of bacterial leaf streak in rice by the new strain BP5 This study investigated the inhibitory effect of a novel strain BP5 on bacterial leaf streak pathogen RH3 (which causes bacterial leaf streak in rice).

[0055] Specifically, it includes the following steps: Bacterial leaf streak pathogen RH3 was activated in NA solid medium, and then a single colony was picked and inoculated into NA liquid medium and cultured at 28°C with shaking at 200 rpm for 24 h. The bacterial cells were collected by centrifugation at 5000 rpm for 5 min, and then resuspended in sterile NA liquid medium (NA solid medium without agar is sufficient), and the OD was adjusted. 600 =0.2, as the seed culture. Add the seed culture to NA medium cooled to 45-50℃ at a 1% inoculum rate, mix well, and pour into plates. Add 5 μL of OD to the center of the medium. 600 A suspension of the novel strain BP5 with a pH of 0.8 was prepared, while the control group was prepared with an equal volume of sterile NA liquid medium. The culture dishes were then sealed and incubated upside down in a 28°C incubator for 2-6 days. The inhibitory effect of the novel strain BP5 on bacterial leaf streak causal agent RH3 was observed. The results are as follows: Figure 10 As shown.

[0056] from Figure 10 The results show that the new strain BP5 effectively inhibits the growth of bacterial leaf streak causal agent RH3, forming an inhibition zone. Furthermore, the diameter of the inhibition zone gradually increases over time, reaching its maximum at 6 days, with a diameter of 2.65 cm. These results indicate that the new strain BP5 can effectively inhibit bacterial leaf streak causal agent RH3, and therefore shows promising application prospects in the control of bacterial leaf streak and other plant pathogenic bacterial diseases in rice.

[0057] In summary, the new species of Burkholderia provided by this invention ( Burkholderia sp. BP5 has good phosphorus solubilization ability. It promotes seed germination by significantly improving seed germination rate, germination potential, number of germinations and root length. It can also significantly promote plant growth and has a good control effect on plant pathogenic bacterial diseases such as rice bacterial leaf streak.

[0058] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0059] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. 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 all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

[0060] The 16S rDNA sequence of the novel strain BP5 (SEQ ID NO: 1)

Claims

1. A new species of Burkholderia BP5, characterized in that, The preservation number of the new Burkholderia species BP5 is CCTCC NO:M 20252947.

2. A biological agent, characterized in that, Includes the new Burkholderia species BP5 as described in claim 1.

3. The biological agent according to claim 2, characterized in that, The biological agent is a liquid agent.

4. A method for preparing a biological agent as described in any one of claims 2-3, characterized in that, The process includes the following steps: inoculating the new Burkholderia species BP5 as described in claim 1 into a liquid culture medium for cultivation, separating the obtained culture and resuspending it in a solvent to obtain the biological agent.

5. The application of the new Burkholderia species BP5 as described in claim 1, the biological agent as described in any one of claims 2-3, or the biological agent prepared by the method in claim 4 in phosphorus solubilization.

6. The application of the new Burkholderia species BP5 as described in claim 1, the biological agent as described in any one of claims 2-3, or the biological agent prepared by the method in claim 4 in promoting seed germination.

7. The application according to claim 6, characterized in that, The seeds include flue-cured tobacco seeds.

8. The application of the new Burkholderia species BP5 as described in claim 1, the biological agent as described in any one of claims 2-3, or the biological agent prepared by the method in claim 4 in promoting plant growth.

9. The application according to claim 8, characterized in that, The plant mentioned includes tobacco.

10. The application of the new Burkholderia species BP5 as described in claim 1, the biological agent as described in any one of claims 2-3, or the biological agent prepared by the method in claim 4 in the prevention and control of plant diseases.

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