Korean pseudomonas BM06 and application thereof

Abstract

The invention discloses Korean pseudomonas BM06 and application thereof, and relates to the technical field of microorganisms. The Korean pseudomonas BM06 disclosed by the invention is preserved in the China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC NO: M 20241926, the preservation date is September 5, 2024, and the preservation address is Wuhan University on eight road of Wuchang District, Wuhan City. The results of the embodiment of the invention show that the Korean pseudomonas BM06 obviously promotes the increase of the seedling height, the lateral root number, the ground diameter, the fresh weight and the dry weight of the pinus massoniana seedlings, and obviously promotes the growth of the pinus massoniana seedlings. Compared with CK, the seedling height of the masson pine is increased by 31.64%, the ground diameter is increased by 51.13%, the number of lateral roots is increased by 83.78%, the fresh weights of roots, stems and leaves are increased by 53.57%, 118.66% and 104.17% respectively, and the dry weights of the roots, the stems and the leaves are increased by 48.21%, 95.53% and 77.07% respectively.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and more specifically, relates to a Korean Pseudomonas BM06 and an application thereof. Background Art

[0002] Pine wilt disease (Bursaphelenchus xylophilus) is a major forest disease that has caused great economic losses and environmental damage worldwide. The use of chemical pesticides can lead to environmental pollution, and nematodes are prone to develop drug resistance. The pathogenic mechanism of pine wilt disease has not yet been clarified, and prevention and control are very difficult. Once the disease occurs, the entire pine forest will suffer devastating damage. The disease has been studied for many years at home and abroad, and the main focus has been on the exploration of scientific issues related to pathogens, such as pathogen types, pathogenic mechanisms, ecological characteristics of pathogenic nematodes, pathogen transmission vectors, disease diagnosis and prevention, etc. Due to the complexity of the occurrence and development of the disease, the pathogenic mechanism of the pathogen is not very clear, and there is still a lack of simple and effective prevention and control methods.

[0003] In recent years, high-efficiency 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 carriers. At present, bacteria have been isolated from Masson pine and used to kill nematodes, but whether these bacteria have the ability to promote growth and their colonization in Masson pine is 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 efficiency of colonization and growth promotion in Masson pine lays the foundation for the construction of nematicidal engineered bacteria, which is of great significance for the prevention and control of pine wood nematode disease. Summary of the invention

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

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A Korean Pseudomonas BM06, which is deposited in the China Center for Type Culture Collection, with the deposit number: CCTCCNO: M 20241926, the deposit date: September 5, 2024, and the deposit address: Wuhan University, Bayi Road, Wuchang District, Wuhan City.

[0008] Application of Korean Pseudomonas BM06 in promoting the growth of Masson pine.

[0009] 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.

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

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

[0012] Application of bacterial suspension of Korean Pseudomonas BM06 in promoting the growth of Masson pine.

[0013] The preparation method of the bacterial suspension is as follows: streak the Korean Pseudomonas BM06 strain on an LB solid medium, invert and activate the culture at 28°C to obtain an activated strain; inoculate the activated single colony on the LB solid medium, shake and culture at 28°C and 200rpm for 8h to obtain a seed liquid; inoculate the seed liquid into an LB liquid medium with an inoculation amount of 10%, a temperature of 28°C, a rotation speed of 200rpm, and shake culture for 24h; after the culture is completed, collect the bacterial cells by centrifugation at 4°C and 10000rpm, and resuspend them in sterile water to 10 8 CFU / mL.

[0014] The 16SrDNA sequence of Korean Pseudomonas BM06 is shown in SEQ ID NO.1.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The Korean Pseudomonas (Psedudomonas koreensis) BM06 disclosed in the present invention is deposited in the China Center for Type Culture Collection, with a deposit number of: CCTCC NO: M 20241926, a deposit date of September 5, 2024, and a deposit address of: Wuhan University, Bayi Road, Wuchang District, Wuhan City. The results of the embodiments of the present invention show that Korean Pseudomonas BM06 significantly promotes the increase in seedling height, lateral root number, ground diameter, fresh weight, and dry weight of Masson pine seedlings. Compared with CK, the height of Masson pine seedlings increased by 31.64%, the ground diameter increased by 51.13%, the number of lateral roots increased by 83.78%, the fresh weight of roots, stems, and leaves increased by 53.57%, 118.66%, and 104.17%, respectively, and the dry weight of roots, stems, and leaves increased by 48.21%, 95.53%, and 77.07%, respectively. In summary, the results showed that strain BM06 significantly promoted the growth of Masson pine seedlings (P < 0.05). BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the phylogenetic tree of strain BM06 constructed based on the 16SrDNA gene sequence;

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

[0019] Figure 3 This is a dynamic diagram of the colonization of the fluorescent-labeled Korean Pseudomonas BM06 strain in the leaves, branches, and roots of Pinus massoniana;

[0020] Figure 4 The colonization sites of the fluorescently labeled strain BM06 of Korean Pseudomonas in Masson pine (A is the colonization map of the fluorescently labeled strain BM06 in the branches and trunks of Masson pine; B is the colonization map of the fluorescently labeled strain BM06 in the leaves of Masson pine; C is the colonization map of the fluorescently labeled strain BM06 in the roots of Masson pine; from left to right: GFP fluorescence field image, fluorescence and bright field image, bright field image);

[0021] Figure 5 This is a graph showing the growth-promoting effect of the fluorescently labeled Korean Pseudomonas BM06 strain on Masson pine (A is seedling height; B is stem diameter; C is the number of lateral roots; D is fresh weight; E is dry weight; F is the phenotype of Masson pine seedlings inoculated with strain BM06 for 90 days; asterisks indicate significant differences based on the Duncan's multiple range).

[0022] Figure 6 This is a graph showing the nematicidal activity of Korean Pseudomonas BM06 fermentation broth after dilution at different times 24 hours later. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0024] The two-year-old Masson pine seedlings used in this application were from Dianzhuang Flowers and Trees in Baise City, Guangxi.

[0025] Example 1

[0026] 1. Isolation of Korean Pseudomonas BM06 strain

[0027] Surface disinfection of samples: First, the stem tissue samples of Masson pine were surface disinfected by washing the surface with sterile water, then soaking it in 75% alcohol for 30 seconds, and finally washing the surface with sterile water. The last washing liquid was collected and spread on the NA plate culture medium. If there was no colony growth on the plate, it proved that the surface disinfection was complete.

[0028] Strain isolation: After the surface is completely disinfected, use sterile filter paper to absorb the moisture, cut it into small sections of about 0.5 cm with sterile surgical scissors, and place it in NA solid culture medium. Repeat each treatment 3 times and culture it in a bacterial incubator at 28°C.

[0029] 2. Identification and preservation of Korean Pseudomonas BM06 strain

[0030] 1) BM06 morphological observation

[0031] The morphological characteristics of BM06 endogenous bacteria were observed by culturing the strain on LB medium. The strain BM06 grew well on LB medium, and the colonies were round in shape and of varying sizes. The colonies were smooth, moist, white, neat, and translucent. The Gram staining results showed that the strain was a Gram-negative bacterium with a rod-shaped body.

[0032] 2) Physiological and biochemical identification of BM06

[0033] Strain BM06 can utilize glucose, fructose, lactose and xylose, but cannot utilize sucrose. It is positive for oxidase and catalase, and is an aerobic bacterium. It is positive for nitrate reduction and nitrite reduction reactions, but cannot hydrolyze gelatin or starch.

[0034] Table 1 Physiological and biochemical characteristics of strain BM06

[0035] Physiological and biochemical experiments result Catalase + Oxidase + Methyl red test - VP(Vitamin Ethyl Methyl Methanol) - Starch hydrolysis - Nitrate reduction + Nitrite reduction + Gelatin hydrolysis - Indole - glucose +

[0036] 3) BM06 molecular biological identification

[0037] The phylogenetic tree of strain BM06 was constructed based on the 16S rDNA gene sequence. Figure 1 As shown, the 16SrDNA sequence of Korean Pseudomonas BM06 is shown as SEQ ID NO.1.

[0038] 4) BM06 strain preservation

[0039] Psedudomonas koreensis BM06 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.

[0040] 3. P.koreensis BM06 competent cells

[0041] P.koreensis BM06 competent cells were prepared using the Competent Cell Preparation Kit (TaKaRa, Beijing, China) as follows:

[0042] 1) Bacterial strain purification

[0043] ①Use LB plate culture medium, pick up BM06 strain (-80℃ glycerol preservation bacteria) with an inoculation needle, and streak on the plate culture medium in grades to allow single colonies to appear. ②Put the streaked plate culture medium upside down in a constant temperature incubator at 30℃ and culture overnight.

[0044] 2) Bacterial culture

[0045] ①Take 20mL Transfer to a 100mL Erlenmeyer flask, plug with cotton, sterilize under high temperature and high pressure, and then cool to room temperature. ② Pick a single colony on the streaked plate medium and inoculate it into the medium in ①. ③ Cultivate at 30℃ with shaking (about 120rpm). ④ Determine OD 600 Value, when OD 600 When the OD value reaches 0.35-0.5 (about 5 hours of cultivation), place it on ice and stop the cultivation (if the OD 600 If the value exceeds this range, the transformation efficiency of competent cells cannot be guaranteed). ⑤Proceed to the next step.

[0046] 3) Preparation of competent cells

[0047] ①Put 1mL of the above bacterial culture solution in a 1.5mL Microtube (determine the number of Microtubes as needed). ②Centrifuge at 1,500×g (about 4,000rpm in a general microcentrifuge) at 4℃ for 5 minutes and discard the supernatant (make sure to remove as much supernatant as possible). ③Add 100μL of Solution A pre-cooled in ice to each Microtube, gently flick the Microtube to suspend the precipitate, and avoid violent shaking. ④Centrifuge at 1,500×g (about 4,000rpm in a general microcentrifuge) at 4℃ for 5 minutes and discard the supernatant (make sure to remove as much supernatant as possible). ⑤Add 100μL of Solution B pre-cooled in ice to each Microtube, gently flick the Microtube to suspend the precipitate, and avoid violent shaking. ⑥The competent cells are ready. These competent cells can be used directly in DNA transformation experiments or stored at -80℃ for later use. When stored at -80℃, they can be effectively stored for more than one year, but they cannot be repeatedly frozen and thawed. Once thawed, they cannot be stored at -80℃ again.

[0048] 4. Fluorescence labeling of Korean Pseudomonas BM06 strain

[0049] The plasmid pBBR1MCS2-Tac-EGFP was transformed into Korean Pseudomonas BM06 by freeze-thaw method. The specific steps are:

[0050] ①Take one P.koreensis BM06 competent cell and melt it in an ice bath.

[0051] ② Take the corresponding mass of pBBR1MCS2-Tac-EGFP plasmid (10ng, 20ng, 30ng, 40ng, 50ng, 60ng, 70ng, 80ng, 90ng, 100ng, 250ng, 500ng, 750ng, 1000ng respectively) and 100μL competent cells in a 1.5mL centrifuge tube, gently mix and incubate on ice for 30min.

[0052] ③Freeze in liquid nitrogen for 1 min and immediately place in a 37°C water bath for 2 min.

[0053] ④Water bath for 5 minutes.

[0054] ⑤ Add 800 μL of LB medium and culture at 200 rpm and 30°C for 1 h.

[0055] ⑥ Add 50 μL of 100 mg / mL Km antibiotic into the melted LB medium.

[0056] ⑦ After incubating the bacterial solution for 1.0 h, centrifuge and concentrate the bacterial solution. Discard 500 μL of the supernatant and spread on the plate. Incubate at 28°C for 36 h and observe the number of colonies.

[0057] ⑧ Each experiment was repeated three times.

[0058] The results are as follows Figure 2 As shown in the figure, when the amount of plasmid is 20ng-70ng, the amount of plasmid is small and the transformation effect is not obvious. When the amount of plasmid is 500ng and 750ng, the transformation effect is the best.

[0059] Example 2

[0060] 1. Preparation of Korean Pseudomonas BM06 suspension

[0061] The fluorescently labeled strain Korean Pseudomonas BM06 line was placed in LB solid medium (containing 100 mg / mL of Km), inverted and activated at 28°C to obtain an activated strain; a single colony after activation was inoculated in LB solid medium (containing 100 mg / mL of Km), shaken at 28°C and 200 rpm for 8 hours to obtain a seed solution; the seed solution was inoculated in LB liquid medium (containing 100 mg / mL of Km), the inoculation amount was 10%, the temperature was 28°C, the speed was 200 rpm, and the shaking table was cultured for 24 hours; after the culture was completed, the bacteria were collected by centrifugation at 4°C and 10000 rpm, and resuspended to 10 8 CFU / mL.

[0062] 2. Specific gene screening

[0063] Mauve software was used to perform colinearity analysis on the whole genomes of 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, and P.putida NC_021505.1), and the specific gene of Korean Pseudomonas BM06 was screened out as BM06_00253 (BM06 whole genome ID: CP155621, and the sequence of the specific gene BM06_00253 is shown in SEQ ID NO.2), and the specificity was determined by NCBI alignment. Then, BM06_00253 was Topo cloned, plasmid was extracted, and the standard curve equation was established as y = -3.213x + 20.582, R 2 =0.994, Eff%=104.

[0064] 3. Colonization dynamics and colonization sites of Korean Pseudomonas BM06

[0065] Two-year-old Masson pine seedlings with consistent growth were sprayed with bacterial suspension and sterile water, 15-20 mL per plant, and 3 replicates per treatment. Leaves, stems and roots of Masson pine seedlings were collected at 0, 15, 30, 45, 60 and 90 days after inoculation, and the total genomic DNA of each sample was extracted using the Plant Genome Kit. Fluorescence quantitative qPCR was used to detect the expression of specific genes in Masson pine tissues at different periods, and the colonization dynamics of strain BM06 in roots, stems and leaves of Masson pine were calculated. When the BM06-labeled strain was colonized for 30 days, the roots, stems and leaves of Masson pine were sliced, and the fluorescence of the strain in the leaves, stems and root tissues of Masson pine was observed using a laser scanning confocal microscope (LSCM), with an excitation light of 488 nm.

[0066] The qPCR detection primer sequences are:

[0067] BM06-F:5'-CGCAATACAGGCAAATGGT-3',

[0068] BM06-R:5'TTAGACAGAAAGGCGAAGG-3'.

[0069] The results are as follows Figure 3As shown in the figure, the number of colonization of the BM06-labeled strain in the roots, stems, and leaves of Masson pine reached the highest level after 15 days of colonization. At this time, the number of colonization of the fluorescently labeled strain BM06 in the roots, stems, and leaves of Masson pine was 7.96×10 4 fu / g, 1.02×10 5 fu / g, 2.09×10 5 fu / g. The colonization state of fluorescent labeled strain BM06 in Masson pine branches was the most stable, which was more suitable for the survival and reproduction of Korean Pseudomonas BM06.

[0070] The results are as follows Figure 4 As shown, the fluorescently labeled strain BM06 colonized in the intercellular spaces of Masson pine plant tissues and emitted strong fluorescent signals in the branches, leaves, and roots of Masson pine.

[0071] 4. Growth-promoting effect of Korean Pseudomonas BM06

[0072] Select two-year-old Masson pine potted seedlings (container size: 14.7cm×11.5cm×13.5cm) with consistent growth, spray 15-20mL BM06 bacterial suspension (concentration 10 8 CFU / mL), each treatment was inoculated separately, and sterile water was used as a control. At 90 days after inoculation, seedling height, ground diameter, number of lateral roots, root fresh weight, stem fresh weight, leaf fresh weight, root dry weight, stem dry weight, and leaf dry weight were measured.

[0073] The results are as follows Figure 5 As shown in the results, Korean Pseudomonas BM06 significantly promoted the increase of seedling height, lateral root number, ground diameter, fresh weight and dry weight of Masson pine seedlings. Compared with CK, the height of Masson pine seedlings 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 weight of roots, stems and leaves increased by 53.57%, 118.66% and 104.17% respectively ( Figure 5 D), the dry weight of roots, stems and leaves increased by 48.21%, 95.53% and 77.07% respectively ( Figure 5 E). In summary, the results showed that strain BM06 significantly promoted the growth of Masson pine seedlings (P<0.05) ( Figure 5 F).

[0074] Example 3

[0075] 1. Cultivation of pine wood nematodes

[0076] AMA3 is a highly virulent pine wood nematode strain preserved in the Forest Pathology Laboratory of Nanjing Forestry University. The test nematode AMA3 was inoculated into a PDA medium plate covered with Botrytis cinerea and placed in a constant temperature incubator at 25°C until the Botrytis cinerea hyphae were completely eaten. The collected nematodes were collected using the Berman funnel method, washed three times with sterile water, and prepared into a nematode suspension with a concentration of about 5000 nematodes / mL for later use.

[0077] 2. Preparation of BM06 fermentation broth

[0078] Take out the BM06 glycerol culture at -80℃, activate it on the LB plate, and culture it at 30℃ overnight. Pick a single clone with an inoculation loop, inoculate it in a 100mL Erlenmeyer flask containing 20mL NB liquid culture medium, and culture it at 30℃ and 200r / min for 12h as the original bacterial solution. Inoculate the original bacterial solution in a 500mL Erlenmeyer flask containing 200mL NB liquid culture medium at a concentration of 1:100, culture it at 30℃ and 200r / min for 72h, and add sterile water to adjust the fermentation liquid supernatant to the fermentation liquid concentration required for the experiment. Test the nematode killing activity of the strain fermentation liquid for 24h.

[0079] 3. Corrected mortality of nematodes treated with BM06 fermentation liquid for 24 hours

[0080] Take 100 μL of fermentation liquid of strain BM06 at different dilution multiples, add it to a 1.5 mL centrifuge tube, then add 100 μL of nematode suspension (about 500 nematodes) and mix well. After 24 hours of nematode treatment, take 20 μL of the mixed solution (about 50 nematodes) and observe the number of dead pine wood nematodes under a microscope (nematode body stiffness, worm body in "J" or "C" shape, worm body surface dull, considered dead) and the total number of pine wood nematodes, calculate the mortality rate and corrected mortality rate, and use sterile NB culture solution treatment as control, and each treatment is repeated 3 times.

[0081] Mortality rate (%) = number of nematodes killed / total number of nematodes × 100;

[0082] Corrected mortality rate (%) = (treatment mortality rate - blank control mortality rate) / (100 - blank control mortality rate) × 100

[0083] The results are as follows Figure 6 As shown in the figure, when the concentration of the fermentation liquid of strain BM06 was 2 times and 5 times, the corrected mortality rate of pine wood nematodes was 100%, and when it was diluted 10 times, the corrected mortality rate of pine wood nematodes was 35%. Strain BM06 has good nematode killing activity, which provides favorable conditions for the prevention and control of pine wood nematode disease.

[0084] The above description is only illustrative rather than restrictive of the present invention. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all will fall within the scope of protection of the present invention.

Claims

1. A Korean Pseudomonas BM06, which is deposited in the China Center for Type Culture Collection, with the deposit number: CCTCCNO: 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, It is 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, It is characterized in that The fresh weight includes root fresh weight, stem fresh weight and leaf fresh weight.

5. The use according to claim 3, It is 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, It is 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 a single colony after activation 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 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 was completed, the bacteria were collected by centrifugation at 4°C and 10000 rpm, and resuspended in sterile water to 10 8 CFU / mL.

8. The 16S rDNA sequence of the Korean Pseudomonas BM06 according to claim 1 is shown in SEQ ID NO.1.

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