A Pseudomonas koreensis BM06 and its applications

Abstract

The present invention discloses a strain of Pseudomonas koreensis BM06 and its application, which relates to the technical field of microorganisms. A strain of Pseudomonas koreensis BM06 disclosed by the present invention is preserved in the China Center for Type Culture Collection, with the preservation number: CCTCC NO: M 20241926, and the preservation date: September 5, 2024. The results of the examples of the present invention show that Pseudomonas koreensis BM06 significantly promotes the increase in the seedling height, the number of lateral roots, the ground diameter, the fresh weight, and the dry weight of Masson pine seedlings, and significantly promotes the growth of Masson pine seedlings. Compared with CK, the seedling height of Masson pine increased by 31.64%, the ground diameter increased by 51.13%, the number of lateral roots increased by 83.78%, the fresh weights of roots, stems, and leaves increased by 53.57%, 118.66%, and 104.17% respectively, and the dry weights of roots, stems, and leaves increased by 48.21%, 95.53%, and 77.07% respectively.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and more specifically, relates to a strain of Pseudomonas koreensis BM06 and its application. Background Art

[0002] Pine wilt disease ( Bursaphelenchus xylophilus ) is a major forest disease that has caused significant economic losses and environmental damage globally. The use of chemical pesticides can lead to environmental pollution, and nematodes are prone to developing drug resistance. The pathogenic mechanism of pine wilt disease is not yet clear, and its prevention and control are extremely difficult. Once the disease occurs, the entire pine forest will suffer devastating damage. For many years, domestic and foreign research on this disease has mainly focused on exploring pathogen-related scientific issues, such as pathogen species, pathogenic mechanisms of pathogens, ecological characteristics of pathogenic nematodes, pathogen vectors, disease diagnosis and prevention, etc. Due to the complexity of the occurrence and development of this disease, the pathogenic mechanism of the pathogen is not very clear, and there is still a lack of simple and effective prevention and control measures.

[0003] In recent years, highly effective and low-toxic biological control has become a research hotspot. Endophytic bacteria in plants have the characteristics of rapid reproduction and strong adaptability, can promote plant growth and development, and can be used as potential biological control carrier bacteria. Currently, some bacteria have been isolated from Masson pine and used for nematode killing, but whether these bacteria have the ability to promote growth and their colonization situation in Masson pine are still unknown. The colonization effect of bacteria in Masson pine has a certain impact on the biological control effect of the strain.

[0004] Therefore, developing an endophyte with high colonization and growth-promoting ability in Masson pine lays a foundation for the construction of a nematode-killing engineering bacterium, which is of great significance for the prevention and control of pine wilt disease. Summary of the Invention

[0005] Aiming at the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a strain of Pseudomonas koreensis BM06. Another technical problem to be solved by the present invention is to provide the application of a strain of Pseudomonas koreensis BM06 for promoting the growth of Masson pine.

[0006] To solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0007] A strain of Pseudomonas koreensis ( Pseudomonas koreensis ) BM06, which is deposited in the China Center for Type Culture Collection, with the deposit number: CCTCC NO: M 20241926, the deposit date: September 5, 2024, and the deposit address: Wuhan University, Bayi Road, Wuchang District, Wuhan City.

[0008] The application of Pseudomonas koreensis BM06 in promoting the growth of Masson pine.

[0009] The promotion of the growth of Pinus massoniana refers to the increase in the seedling height, the number of lateral roots, the ground diameter, the fresh weight, and the dry weight of Pinus massoniana seedlings.

[0010] The fresh weight includes the root fresh weight, the stem fresh weight, and the leaf fresh weight.

[0011] The dry weight includes the root dry weight, the stem dry weight, and the leaf dry weight.

[0012] Application of the bacterial suspension of Pseudomonas koreensis BM06 in promoting the growth of Pinus massoniana.

[0013] The preparation method of the bacterial suspension is as follows: Streak the strain Pseudomonas koreensis BM06 on an LB solid medium, and culture it in an inverted position at 28°C to obtain the activated strain; Inoculate the single colony obtained in the previous step of activation into an LB solid medium, and culture it at 28°C and 200 rpm for 8 h to obtain the seed liquid; Inoculate the seed liquid into an LB liquid medium, with an inoculation amount of 10%, a temperature of 28°C, and a rotation speed of 200 rpm, and culture it on a shaker for 24 h; After the culture is completed, centrifuge at 4°C and 10000 rpm to collect the bacterial cells, and resuspend them with sterile water to 10 8 CFU / mL.

[0014] The 16S rDNA sequence of Pseudomonas koreensis BM06 is shown in SEQ ID NO.1.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] Pseudomonas koreensis ( Pseudomonas koreensis ) BM06 disclosed in the present invention is preserved in the China Center for Type Culture Collection, with the preservation number: CCTCC NO: M 20241926, the preservation date: September 5, 2024, and the preservation address: Wuhan University, Bayi Road, Wuchang District, Wuhan. The results of the examples of the present invention show that: Pseudomonas koreensis BM06 significantly promoted the increase in the seedling height, the number of lateral roots, the ground diameter, the fresh weight, and the dry weight of Pinus massoniana seedlings. Compared with CK, the seedling height of Pinus massoniana increased by 31.64%, the ground diameter increased by 51.13%, the number of lateral roots increased by 83.78%, the fresh weights of the roots, stems, and leaves increased by 53.57%, 118.66%, and 104.17% respectively, and the dry weights of the roots, stems, and leaves increased by 48.21%, 95.53%, and 77.07% respectively. In summary, the results show that the strain BM06 significantly promoted the growth of Pinus massoniana seedlings (P < 0.05). Description of the Drawings

[0017] Figure 1 It is a phylogenetic tree diagram of the strain BM06 constructed based on the 16S rDNA gene sequence;

[0018] Figure 2 It is a diagram showing the effect of plasmid dosage on the transformation efficiency;

[0019] Figure 3 Colonization dynamics of the fluorescently labeled strain of Pseudomonas koreensis BM06 in the leaves, branches and roots of Pinus massoniana

[0020] Figure 4 Colonization sites of the fluorescently labeled strain of Pseudomonas koreensis BM06 in Pinus massoniana (A: Colonization of the fluorescently labeled strain BM06 in the branches and trunks of Pinus massoniana; B: Colonization of the fluorescently labeled strain BM06 in the leaves of Pinus massoniana; C: Colonization of the fluorescently labeled strain BM06 in the roots of Pinus massoniana; from left to right: GFP fluorescence field image, fluorescence and bright field composite image, bright field image).

[0021] Figure 5 Growth promotion effect of the fluorescently labeled strain of Pseudomonas koreensis BM06 on Pinus massoniana (A: Seedling height; B: Stem diameter; C: Lateral root number; D: Fresh weight; E: Dry weight; F: Phenotype of Pinus massoniana seedlings inoculated with strain BM06 for 90 d; Asterisks indicate significant differences according to Duncan's multiple range).

[0022] Figure 6 Nematicidal activity of the fermentation broth of Pseudomonas koreensis BM06 after dilution at different multiples for 24 h Detailed implementation mode

[0023] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described below in conjunction with specific embodiments. In the following embodiments, unless otherwise specified in detail, the technical means used are all conventional means well known to those skilled in the art.

[0024] The two-year-old Pinus massoniana seedlings used in this application are from Dianzhuang Flowering Trees in Baise City, Guangxi. Embodiment

[0025] 1. Isolation of the strain Pseudomonas koreensis BM06

[0026] Surface disinfection of the sample: The stem tissue sample of Pinus massoniana was first surface-disinfected, rinsed with sterile water on the surface, then soaked in 75% alcohol for 30 s, and finally rinsed with sterile water on the surface. The last rinse solution was collected and spread on an NA plate medium. If no colonies grew on the plate, it could prove that the surface disinfection was complete.

[0027] Strain isolation: After complete surface disinfection, the water was blotted dry with a sterile filter paper, cut into small pieces about 0.5 cm long with sterile surgical scissors, placed in an NA solid medium, and each treatment was repeated 3 times, and cultured in a bacterial incubator at 28 °C.

[0028] 2. Identification and preservation of the strain Pseudomonas koreensis BM06

[0029] 1) Morphological observation of BM06

[0030] The morphological characteristics of the endophytic bacterium BM06 were observed by culturing the strain on LB medium. The strain BM06 grew well on LB medium. The colonies were round in shape and of different sizes, smooth, moist, white, with characteristics such as neatness and semi-transparency. The results of Gram staining showed that the strain was a Gram-negative bacterium, and the cells were rod-shaped.

[0031] 2) Physiological and biochemical identification of BM06

[0032] The strain BM06 was able to utilize glucose, fructose, lactose, and xylose, but not sucrose. It was positive for oxidase and catalase, and was an aerobic bacterium. The nitrate reduction and nitrite reduction reactions were positive. It was unable to hydrolyze gelatin and starch.

[0033] Table 1 Physiological and biochemical characteristics of strain BM06

[0034]

[0035] 3) Molecular biological identification of BM06

[0036] The phylogenetic tree of strain BM06 was constructed based on the 16S rDNA gene sequence as Figure 1 shown. The 16S rDNA sequence of Pseudomonas koreensis BM06 is shown in SEQ ID NO.1.

[0037] 4) Preservation of strain BM06

[0038] Pseudomonas koreensis ( Pseudomonas koreensis ), BM06, was preserved in the China Center for Type Culture Collection, with the preservation number: CCTCC NO: M 20241926, the preservation date: September 5, 2024, and the preservation address: Wuhan University, Bayi Road, Wuchang District, Wuhan.

[0039] 3. P.koreensis BM06 competent cells

[0040] The BM06 competent cells were prepared using the kit Competent Cell Preparation Kit (TaKaRa, Beijing, China). The specific steps were as follows: P.koreensis 1) Purification of the bacterial strain

[0041] 1) Using an LB plate medium, pick the BM06 strain (preserved in glycerol at -80°C) with an inoculation needle and streak it on the plate medium in a hierarchical manner until single colonies appear. 2) Invert the above streaked plate medium and incubate it overnight at 30°C in a constant temperature incubator.

[0042] ① Using an LB plate medium, pick the BM06 strain (the glycerol-preserved bacteria at -80°C) with an inoculation needle and streak it on the plate medium in a hierarchical manner until single colonies appear. ② Invert the above streaked plate medium and incubate it overnight at 30°C in a constant temperature incubator.

[0043] 2) Bacterial culture

[0044] ① Transfer 20 mL of φb×broth to a 100 mL Erlenmeyer flask, plug with cotton plug and sterilize by high temperature and high pressure, then cool to room temperature. ② Pick a single colony on the streaked plate medium and inoculate it into the medium in ①. ③ Incubate at 30°C with shaking (about 120 rpm). ④ Measure the OD 600 value. When the OD 600 value reaches 0.35 - 0.5 (about 5 hours of incubation), place it on ice to stop the culture (if the OD 600 value exceeds this range, the transformation efficiency of competent cells cannot be guaranteed). ⑤ Proceed to the next step.

[0045] 3) Preparation of competent cells

[0046] ① Take 1 mL of the above bacterial culture solution into a 1.5 mL Microtube (determine the number of Microtubes according to the required amount). ② Centrifuge at 1,500×g (about 4,000 rpm for a general microcentrifuge) at 4°C for 5 minutes, discard the supernatant (pay attention to removing as much supernatant as possible). ③ Add 100 μL of Solution A pre-cooled on ice to each Microtube, gently flick the Microtube to suspend the precipitate, and avoid vigorous shaking. ④ Centrifuge at 1,500×g (about 4,000 rpm for a general microcentrifuge) at 4°C for 5 minutes, discard the supernatant (pay attention to removing as much supernatant as possible). ⑤ Add 100 μL of Solution B pre-cooled on ice to each Microtube, gently flick the Microtube to suspend the precipitate, and avoid vigorous shaking. ⑥ The preparation of competent cells is completed. These competent cells can be directly used for DNA transformation experiments or stored at -80°C for future use. When stored at -80°C, they can be effectively stored for more than one year, but cannot be repeatedly frozen and thawed. Once thawed, they cannot be stored at -80°C again.

[0047] 4. Fluorescent labeling of Pseudomonas koreensis BM06 strain

[0048] Transform plasmid pBBR1MCS2-Tac-EGFP into Pseudomonas koreensis BM06 by the freeze-thaw method. The specific steps are as follows:

[0049] ① Take a P.koreensis BM06 competent cell and thaw it on ice.

[0050] ②Respectively take the pBBR1MCS2-Tac-EGFP plasmids of corresponding masses (10 ng, 20 ng, 30 ng, 40 ng, 50 ng, 60 ng, 70 ng, 80 ng, 90 ng, 100 ng, 250 ng, 500 ng, 750 ng, 1000 ng respectively) and gently mix them with 100 μL of competent cells in a 1.5 mL centrifuge tube, and incubate on ice for 30 min.

[0051] ③Freeze in liquid nitrogen for 1 min and immediately place in a water bath at 37 °C for 2 min.

[0052] ④Incubate in the water bath for 5 min.

[0053] ⑤Add 800 μL of LB medium, shake culture at 200 rpm and 30 °C for 1 h.

[0054] ⑥Add 50 μL of 100 mg / mL Km antibiotic to the melted LB medium.

[0055] ⑦After incubating the bacterial solution for 1.0 h, centrifuge and concentrate the bacterial solution. Discard 500 μL of the supernatant, spread it on a plate, and culture at 28 °C for 36 h and observe the number of colonies.

[0056] ⑧Perform three biological replicates for each experiment.

[0057] The results are as Figure 2 shown. When the plasmid dosage is 20 ng - 70 ng, the plasmid dosage is relatively small and the transformation effect is not obvious. When the plasmid dosages are 500 ng and 750 ng, the transformation effect is the best. Example

[0058] 1. Preparation of Pseudomonas koreensis BM06 suspension

[0059] Take the fluorescently labeled strain Pseudomonas koreensis BM06 and streak it on an LB solid medium (containing 100 mg / mL Km), and culture it in an inverted position at 28 °C for activation to obtain the activated strain; pick a single colony from the further activated colonies and inoculate it into an LB solid medium (containing 100 mg / mL Km), culture it at 28 °C and 200 rpm on a shaker for 8 h to obtain a seed solution; inoculate the seed solution into an LB liquid medium (containing 100 mg / mL Km) with an inoculation amount of 10%, at a temperature of 28 °C and a rotation speed of 200 rpm, and culture it on a shaker for 24 h; after the culture is completed, centrifuge and collect the bacteria at 4 °C and 10000 rpm, and resuspend them with sterile water to 10 8 CFU / mL.

[0060] 2. Screening of specific genes

[0061] Use Mauve software to analyze nine Pseudomonas strains ( P. koreensis BM06 CP155621, P.abietaniphila ATCC 700689, P. abietaniphila KF701, P. abietaniphila KF717, P. aeruginosa NC_002516.2, P. fluorescens NZ_LT907842.1, P. kribbensis NZ_CP029608.1, P. putida A collinearity analysis was performed on the whole genomes of Pseudomonas koreensis BM06 (CP155621), ATCC 700689, KF701, KF717, NC_002516.2, NZ_LT907842.1, and NZ_CP029608.1), and the specific gene of Pseudomonas koreensis BM06 was screened out as BM06_00253 (the whole genome ID of BM06: CP155621, and the sequence of the specific gene BM06_00253 is shown in SEQ ID NO.2). The specificity was determined by NCBI alignment. Then, Topo cloning was performed on BM06_00253, and the plasmid was extracted. The standard curve equation was established as y = -3.213x + 20.582, R 2 = 0.994, Eff% = 104.

[0062] 3. Colonization dynamics and colonization sites of Pseudomonas koreensis BM06

[0063] Two-year-old Masson pine seedlings with consistent growth were taken, and the bacterial suspension and sterile water were sprayed respectively, 15 - 20 mL per plant, with 3 replicates for each treatment. At 0, 15, 30, 45, 60, and 90 d after inoculation, the leaves, stems, and roots of the Masson pine seedlings were collected. The total genomic DNA of each sample was extracted using the Plant Genome Kit, and the expression levels of specific genes in the Masson pine tissues at different times were detected by fluorescence quantitative qPCR, and the colonization dynamics of strain BM06 in the roots, stems, and leaves of Masson pine were calculated. When the BM06-labeled strain colonized for 30 d, the roots, stems, and leaves of Masson pine were sectioned, and the fluorescence of the strain in the leaf, stem, and root tissues of Masson pine was observed using a laser scanning confocal microscope (LSCM), with the excitation light at 488 nm.

[0064] The primer sequences for qPCR detection were:

[0065] BM06-F: 5’-CGCAATACAGGCAAATGGT-3’,

[0066] BM06-R: 5’TTAGACAGAAAGGCGAAGG-3’.

[0067] The results are as Figure 3 shown. When the BM06-labeled strain colonized in the roots, stems, and leaves of Masson pine for 15 d, the colonization number of the strain was the highest. At this time, the colonization numbers of the fluorescence-labeled strain BM06 in the roots, stems, and leaves of Masson pine were 7.96×10 4fu / g, 1.02×10 5 fu / g, 2.09×10 5 fu / g. The colonization state of the fluorescently labeled strain BM06 on the branches of Pinus massoniana is the most stable, and it is more suitable for the survival and reproduction of Pseudomonas koreensis BM06.

[0068] The results are as Figure 4 shown. The fluorescently labeled strain BM06 colonizes in the intercellular spaces of the plant tissues of Pinus massoniana, emitting strong fluorescent signals in the branches, leaves and roots of Pinus massoniana.

[0069] 4. Growth promotion effect of Pseudomonas koreensis BM06

[0070] Select two-year-old potted seedlings of Pinus massoniana with consistent growth (container specifications: 14.7 cm × 11.5 cm × 13.5 cm), spray 15 - 20 mL of the BM06 bacterial suspension (concentration 10 8 CFU / mL), inoculate each treatment separately, and use sterile water as the control. At 90 days after inoculation, measure the seedling height, ground diameter, number of lateral roots, fresh root weight, fresh stem weight, fresh leaf weight, dry root weight, dry stem weight, and dry leaf weight respectively.

[0071] The results are as Figure 5 shown. Pseudomonas koreensis BM06 significantly promoted the increase in the seedling height, number of lateral roots, ground diameter, fresh weight, and dry weight of Pinus massoniana seedlings. Compared with CK, the seedling height of Pinus massoniana increased by 31.64% ( Figure 5 A), the ground diameter increased by 51.13% ( Figure 5 B), the number of lateral roots increased by 83.78% ( Figure 5 C), the fresh weights of roots, stems and leaves increased by 53.57%, 118.66% and 104.17% respectively ( Figure 5 D), and the dry weights of roots, stems and leaves increased by 48.21%, 95.53% and 77.07% respectively ( Figure 5 E). In summary, the results show that the strain BM06 significantly promoted the growth of Pinus massoniana seedlings (P < 0.05) ( Figure 5 F). Example

[0072] 1. Culture of Bursaphelenchus xylophilus

[0073] AMA3 is a highly virulent strain of Bursaphelenchus xylophilus preserved in the Forest Pathology Laboratory of Nanjing Forestry University. The tested nematodes AMA3 were inoculated on a PDA medium plate covered with Botrytis cinerea, and cultured in a constant temperature incubator at 25°C until the hyphae of Botrytis cinerea were completely consumed. The nematodes were collected by the Berman funnel method, washed 3 times with sterile water, and prepared into a nematode suspension with a concentration of about 5000 nematodes / mL for standby.

[0074] 2. Preparation of BM06 Fermentation Broth

[0075] Take out the glycerol bacteria of strain BM06 from -80°C, activate it on an LB plate, and culture it overnight at 30°C. Pick a single colony with an inoculation loop and inoculate it into a 100 mL Erlenmeyer flask containing 20 mL of NB liquid medium, and culture it at 30°C and 200 r / min for 12 h as the original bacterial liquid. Inoculate the original bacterial liquid into a 500 mL Erlenmeyer flask containing 200 mL of NB liquid medium at a concentration of 1:100, culture it at 30°C and 200 r / min for 72 h, and add sterile water to adjust the supernatant of the fermentation broth to the concentration required for the experiment. Test the nematocidal activity of the fermentation broth of the strain against nematodes for 24 h.

[0076] 3. Corrected Mortality of BM06 Fermentation Broth against Nematodes for 24 h

[0077] Take 100 μL of the fermentation broth of strain BM06 at different dilution multiples respectively, add it into a 1.5 mL centrifuge tube, then add 100 μL of nematode suspension (about 500 nematodes), and mix well. After treating the nematodes for 24 h, suck 20 μL of the mixed solution (about 50 nematodes) and observe the number of dead pine wood nematodes (the nematode body is rigid, in a "J" shape or "C" shape, and the body surface of the nematode has no luster, which is regarded as dead) and the total number of pine wood nematodes under a microscope, calculate the mortality and corrected mortality, use sterile NB culture solution treatment as a control, and repeat each treatment 3 times.

[0078] Mortality (%) = Number of dead nematodes / Total number of nematodes × 100;

[0079] Corrected Mortality (%) = (Treatment Mortality - Blank Control Mortality) / (100 - Blank Control Mortality) × 100

[0080] The results are as Figure 6 shown. When the concentration of the fermentation broth of strain BM06 is 2 times and 5 times, the corrected mortality of pine wood nematodes is 100%. When diluted 10 times, the corrected mortality of pine wood nematodes is 35%. Strain BM06 has good nematocidal activity, which provides favorable conditions for the prevention and control of pine wood nematode disease.

[0081] The above description is only illustrative rather than restrictive for the present invention. Those of ordinary skill in the art understand that many modifications, changes or equivalents can be made without departing from the spirit and scope defined by the appended claims, but all will fall within the protection scope of the present invention.

Claims

1. A Korean strain of Pseudomonas ( Pseudomonas koreensis )BM06, which is deposited in the China Center for Type Culture Collection, with the deposit number: CCTCC NO: M 20241926, the deposit date: September 5, 2024, and the deposit address: Wuhan University, Bayi Road, Wuchang District, Wuhan City.

2. Use of the Korean Pseudomonas BM06 described in claim 1 in promoting the growth of Masson pine.

3. The use according to claim 2, characterized in that: The method for promoting the growth of Masson pine is to promote the increase of the height, number of lateral roots, ground diameter, fresh weight and dry weight of Masson pine seedlings.

4. The use according to claim 3, characterized in that: The fresh weight includes root fresh weight, stem fresh weight and leaf fresh weight.

5. The use according to claim 3, characterized in that: The dry weight includes root dry weight, stem dry weight and leaf dry weight.

6. Use of the bacterial suspension of Korean Pseudomonas BM06 according to claim 1 in promoting the growth of Masson pine.

7. The use according to claim 6, characterized in that: The preparation method of the bacterial suspension is as follows: streak the Korean Pseudomonas BM06 strain on an LB solid culture medium, invert and activate the culture at 28° C. to obtain an activated strain; take the activated single colony and inoculate it on an LB solid culture medium, shake and cultivate it at 28° C. and 200 rpm for 8 hours to obtain a seed solution; The seed liquid was inoculated into LB liquid culture medium with an inoculation volume of 10%, the temperature was 28°C, the rotation speed was 200 rpm, and the shaking culture was carried out for 24 hours; after the culture, the bacteria were collected by centrifugation at 4°C and 10000 rpm, and resuspended in sterile water to 10 8 CFU / mL.

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