An engineered strain for controlling Bursaphelenchus xylophilus, its construction method and application

Abstract

The present invention discloses a genetically engineered strain for controlling Bursaphelenchus xylophilus, its construction method and application, which relates to the technical field of microorganisms. The construction method of a genetically engineered strain for controlling Bursaphelenchus xylophilus disclosed by the present invention is to construct the oprL promoter, the SD sequence and the Peptidase03 gene sequence into the modified vector pBBR1MCS2-Tac-EGFP to obtain the recombinant plasmid pBBR1MCS2-P03; transform the plasmid pBBR1MCS2-P03 into the recipient bacterium BM06 to obtain the genetically engineered bacterium BM06-P03; the Peptidase03 gene sequence is as shown in SEQ ID NO.1; the sequence of the oprL promoter is as shown in SEQ ID NO.4; the SD sequence is as shown in SEQ ID NO.5. The genetically engineered strain BM06-P03 constructed by the present invention delays the disease onset speed of Pinus massoniana and has an effect on controlling Bursaphelenchus xylophilus.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and more specifically, relates to an engineering strain for controlling Bursaphelenchus xylophilus, a construction method thereof, and an application thereof. Background Art

[0002] Bursaphelenchus xylophilus is a major forest disease that has caused great 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 Bursaphelenchus xylophilus disease has not been clearly defined, and its prevention and control are very 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 scientific issues related to the pathogen, such as pathogen species, pathogenic mechanism of the pathogen, ecological characteristics of the pathogenic nematode, pathogen transmission vector, 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. By genetically introducing exogenous resistance genes into plant endophytic bacteria, not only can the biological control potential of endophytic bacteria be enhanced, but also the host plant can obtain certain stress resistance. A large number of studies on genetic engineering transformation of endophytic bacteria have been carried out at home and abroad. For example, after introducing the Bt gene into endophytic bacteria of sugarcane, corn, cotton, rice, etc., the host plants have obtained strong insecticidal ability, and the transformed endophytic bacteria still maintain their inherent nitrogen fixation, growth promotion and disease resistance abilities.

[0004] Bursaphelenchus xylophilus is the pathogen of Bursaphelenchus xylophilus disease and is the direct pathogenic factor of the disease. The cuticle is the barrier between Bursaphelenchus xylophilus and the external environment and plays a crucial role in maintaining the body shape and integrity of the nematode. The epidermis of Bursaphelenchus xylophilus is composed of various proteins, which are connected by peptide bonds. How to break through the nematode body wall barrier is of great significance for finding nematicidal factors.

[0005] Peptidase 03 (NlpC / P60) is from Pseudomonas syringae MB03 and contains the protease catalytic triad Cys-His-His, which has the activity of catalyzing peptide bonds. The hydrolysis of peptide bonds leads to the decomposition of proteins, which can seriously interfere with the normal physiological activities of nematodes and cause their death. The NlpC / P60 domain is structurally similar to papain and can exist alone or bind to other domains. Its catalytic function changes with the structural change, and it can form proteins with diverse functions. For example, the SH3 domain in the endopeptidase domain of NlpC / P60 participates in the process of anchoring hyaluronidase to the cell wall, regulates the concentration and anchoring position of hyaluronic acid, and forms an effective enzyme-substrate complex to play a role. Peptidoglycan (PG) is the main component of the bacterial cell wall and is crucial for maintaining the structural integrity, internal osmotic pressure, and shaping the morphology of bacteria. However, peptidoglycan is also highly dynamic, and its glycosidic bonds and amide bonds can be broken or formed by different enzymes. NlpC / P60 endopeptidase is one of them. The degradation products generated by the catalytic activity of NlpC / P60 endopeptidase can be recycled for the resynthesis of peptidoglycan, or can act as signaling molecules to trigger antibiotic resistance or the regrowth of dormant cells, or as effector molecules in immune responses. NlpC / P60 endopeptidase can also be secreted into the environment or enter the periplasm of other bacteria through the type VI secretion system to affect their survival, giving the bacteria secreting NlpC / P60 endopeptidase a competitive advantage.

[0006] Therefore, developing a strain that can efficiently colonize in Masson pine and has strong nematocidal effects indoors and biocontrol potential outdoors is of great significance for the control of pine wilt disease. Summary of the Invention

[0007] Aiming at the above problems existing in the prior art, the technical problems to be solved by the present invention are to provide an engineered strain for controlling pine wood nematodes. Another technical problem to be solved by the present invention is to provide a construction method of the engineered strain for controlling pine wood nematodes. The technical problem to be solved by the present invention is also to provide the application of the engineered strain for controlling pine wood nematodes for controlling pine wood nematodes.

[0008] To solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0009] A method for constructing an engineered strain for controlling Bursaphelenchus xylophilus, which constructs the oprL promoter, SD sequence and Peptidase03 gene sequence into the modified vector pBBR1MCS2-Tac-EGFP to obtain the recombinant plasmid pBBR1MCS2-P03; transforms the plasmid pBBR1MCS2-P03 into the recipient bacterium Pseudomonas koreensis BM06 to obtain the engineered strain BM06-P03;

[0010] The Peptidase03 gene sequence is shown in SEQ ID NO.1;

[0011] The sequence of the oprL promoter is shown in SEQ ID NO.4;

[0012] The SD sequence is shown in SEQ ID NO.5.

[0013] The amplification primers of the oprL promoter are as follows:

[0014] oprL-F: 5’-ACTACAACGCCAACCCGAAGCTGTC-3’,

[0015] oprL-R: 5’-TGAAACTCCTAATGAACCCCAGTGT-3’.

[0016] The specific steps of the construction method are as follows:

[0017] 1) Refer to the oprL gene sequence GenBank AAN66847 of Pseudomonas putida KT2440 strain to design primers oprL-F: 5’-ACTACAACGCCAACCCGAAGCTGTC-3’, oprL-R: 5’-TGAAACTCCTAATGAACCCCAGTGT-3’, extract the genomic DNA of Pseudomonas koreensis, and obtain the oprL gene promoter by PCR method;

[0018] The sequence of the oprL promoter is shown in SEQ ID NO.4;

[0019] 2) Insert the oprL promoter, SD sequence and Peptidase03 gene sequence into the EcoRI and HindIII restriction sites of the modified vector pBBR1MCS2-Tac-EGFP to obtain the vector pBBR1MCS2-P03, and extract the plasmid pBBR1MCS2-P03;

[0020] The SD sequence is shown in SEQ ID NO.5;

[0021] The Peptidase03 gene sequence is as shown in SEQ ID NO.1;

[0022] 3) Transform the plasmid pBBR1MCS2-P03 into DH5α, pick positive clones and extract the pBBR1MCS2-P03 plasmid again, then transform it into the recipient bacterium Pseudomonas koreensis BM06 to obtain the recombinant engineering strain BM06-P03.

[0023] The recombinant engineering strain BM06-P03 for controlling Bursaphelenchus xylophilus.

[0024] The application of the recombinant engineering strain BM06-P03 in controlling Bursaphelenchus xylophilus.

[0025] The control of Bursaphelenchus xylophilus is to delay the disease onset speed of Pinus massoniana.

[0026] The application of the fermentation filtrate of the recombinant engineering strain BM06-P03 in killing Bursaphelenchus xylophilus.

[0027] The preparation method of the fermentation filtrate is to activate the recombinant engineering strain BM06-P03 on an LB plate containing knna and culture it overnight at 30°C; pick monoclonal colonies with an inoculation loop and inoculate them into 20 mL of NB liquid medium containing 50 μg / mL kanamycin antibiotic, and culture at 30°C and 200 r / min for 12 h to obtain the fermentation filtrate of the recombinant engineering strain BM06-P03.

[0028] The protein expressed by the recombinant engineering strain BM06-P03 has an amino acid sequence as shown in SEQ ID NO.3.

[0029] The application of the protein expressed by the recombinant engineering strain BM06-P03 in controlling Bursaphelenchus xylophilus disease.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] 1) The construction method of an engineering strain for controlling Bursaphelenchus xylophilus disclosed in the present invention constructs the oprL promoter, SD sequence and Peptidase03 gene sequence into the modified vector pBBR1MCS2-Tac-EGFP to obtain the recombinant plasmid pBBR1MCS2-P03; transform the plasmid pBBR1MCS2-P03 into the recipient bacterium Pseudomonas koreensis BM06 to obtain the engineering strain BM06-P03; the Peptidase03 gene sequence is as shown in SEQ ID NO.1; the sequence of the oprL promoter is as shown in SEQ ID NO.4; the SD sequence is as shown in SEQ ID NO.5.

[0032] 2) The results of the embodiments of the present invention show that when the purified Peptidase03 (120.00 μg / mL) protein was used to treat nematodes for 24 h, the mortality rate of nematodes was 100%. The nematode intestines were damaged, vesicles appeared, and the worm bodies broke. As the concentration of Peptidase03 increased, the mortality rate of Bursaphelenchus xylophilus also increased. The EC 50 value of the purified Peptidase03 was 25.73 μg / mL. Peptidase03 has high toxicity to Bursaphelenchus xylophilus and is an important factor for killing Bursaphelenchus xylophilus.

[0033] 3) The results of the embodiments of the present invention show that when the fermentation filtrate of strain BM06-P03 was uniformly diluted 5-fold, 10-fold or 20-fold, it all had the effect of killing nematodes and had higher application potential, providing an effective strategy for the control of Bursaphelenchus xylophilus disease.

[0034] 4) The results of the embodiments of the present invention show that the engineered strain BM06-P03 delayed the disease onset rate of Pinus massoniana and achieved the effect of controlling Bursaphelenchus xylophilus. Description of the Drawings

[0035] Figure 1 It is the structural diagram of the recombinant vector pET-32a(+)-P03;

[0036] Figure 2 It is the detection result diagram of the purified protein Peptidase 03 expression by SDS and Western blot;

[0037] Figure 3 It is the diagram of the influence of Peptidase03 treatment on the body surface of Bursaphelenchus xylophilus observed by scanning electron microscope;

[0038] Figure 4 It is the diagram of the influence of Peptidase03 treatment on the intestine of Bursaphelenchus xylophilus observed by transmission electron microscope;

[0039] Figure 5 It is the lethal rate diagram of the purified Peptidase03 protein at different concentrations on Bursaphelenchus xylophilus;

[0040] Figure 6 It is the structural diagram of the vector pBBR1MCS2-P03;

[0041] Figure 7 It is the detection result diagram of the expression of the protein Peptidase 03 in strain BM06 by SDS and Western blot;

[0042] Figure 8The fermentation filtrates of the wild-type strain BM06 and the engineered strain BM06-P03 were cultured for 12 h, 24 h, 36 h, and 48 h respectively, and diluted 5-fold, 10-fold, and 20-fold respectively. After treatment for 24 h, the corrected mortality rate of Bursaphelenchus xylophilus was shown in the figure (A is the corrected mortality rate of nematodes when the fermentation filtrate was diluted 5-fold; B is the corrected mortality rate of nematodes when the fermentation filtrate was diluted 10-fold; C is the corrected mortality rate of nematodes when the fermentation filtrate was diluted 20-fold);

[0043] Figure 9 It is the colonization dynamics diagram of the engineered strain BM06-P03 colonized on the leaves, branches, and roots of Pinus massoniana for 15 d, 30 d, 45 d, and 60 d;

[0044] Figure 10 It is the control effect diagram of the engineered strain BM06-P03 on Bursaphelenchus xylophilus disease of Pinus massoniana seedlings. Specific implementation manners

[0045] 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, if not otherwise specified in detail, the technical means used are all conventional means well known to those skilled in the art.

[0046] The receptor bacterium Pseudomonas koreensis BM06 used in this application 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 host bacterium Pseudomonas koreensis BM06 has a type VI secretion system, which provides favorable conditions for the extracellular secretion of Peptidase 03 (NlpC / P60).

[0047] Example 1

[0048] 1. Construct the vector

[0049] The pET-32a(+) vector used in this example has a full length of 5887 bp and contains multiple restriction enzyme sites such as EcoRI, HindIII, NotI, and XhoI. There is an Amp resistance gene on the vector, which plays a screening role when constructing the plasmid.

[0050] The target gene sequence Peptidase 03 (SEQ ID NO.1) was inserted into the EcoRI and HindIII restriction sites of the vector pET-32a(+), and the target gene sequence was fused with the vector sequence for expression (synthesized by GenScript). The vector pET-32a(+)-P03 was constructed. The plasmid was extracted using a plasmid extraction kit (Aidlab Biotechnologies Co., Ltd., Nanjing), and the target gene was subjected to PCR and sequencing verification (Sepgen, Nanjing). The sequence of the vector pET-32a(+)-P03 is shown in SEQ ID NO.2, and the vector structure is as Figure 1 shown.

[0051] 2. Protein expression and purification

[0052] The plasmid pET-32a(+)-P03 was transformed into E.coil BL21(DE3) cells, and positive clones were picked for PCR and sequencing verification of the recombinant plasmid (Sepgen, Nanjing).

[0053] The recombinant plasmid glycerol bacteria pET-32a(+)-P03 were inoculated into LB liquid medium (Amp, 50 μg / mL) and shaken at 37 °C for 12 h to obtain a seed culture solution. Then, the seed culture solution was inoculated into LB liquid medium (Amp, 50 μg / mL) at a ratio of 1:100 and cultured until OD 600 = 0.6, IPTG (0.5 mM) was added to induce growth, and the cells were shaken at 200 r / min at 28 °C and incubated for 8 h. Finally, the induced cells were collected by centrifugation, resuspended in phosphate buffered saline (1×PBS) buffer, lysed with a cell disruptor, centrifuged at 10000 r / min at 4 °C, and the supernatant was collected. The Peptidase03 protein (SEQ ID NO.3) was purified from the supernatant using a His-Bind column chromatography (Sangon Biotech) according to the instructions. The purified product was placed in a dialysis bag containing 0.01 M phosphate buffered saline and dialyzed overnight at 4 °C. Using bovine serum albumin (BSA) as a standard, the protein concentration was determined by the Bradford method.

[0054] SDS-PAGE molecular weight analysis was performed using a 10% polyacrylamide gel. Western blot analysis was performed using an anti-his-tag primary antibody and a goat anti-mouse IgG horseradish peroxidase (HRP)-conjugated secondary antibody (Abmart Shanghai, China). First, the purified protein sample was separated on an SDS-PAGE gel and then physically transferred to a nitrocellulose membrane. The membrane was immersed in a 1:5000 diluted anti-his-tag primary antibody overnight. After washing, the membrane was incubated with a 1:6000 diluted enzyme-labeled secondary antibody. Then, the signal was observed using an enhanced chemiluminescence (ECL) substrate (BioRAD).

[0055] The results are as follows Figure 2 shown. Both the SDS and Western blot results indicate that the purified protein Peptidase 03 was successfully expressed.

[0056] 3. Effect of Protein Peptidase 03 on Bursaphelenchus xylophilus

[0057] The impregnation method was used to test the effect of the pure protein Peptidase 03 on nematodes. The specific steps are as follows: 200 μL of pure protein extract was added to 200 μL of nematode solution as the experimental group; 200 μL of BSA was added to 200 μL of nematode solution as the control group. The changes in nematodes were observed 24 h after protein treatment of the nematodes. The pure protein extract of Peptidase03 was diluted to 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, and 80 μg / mL respectively, and the EC 50 value of Peptidase03 was measured.

[0058] The results are as follows Figure 3 shown. When the nematodes were treated with the purified Peptidase03 (120.00 μg / mL) protein for 24 h, the nematode mortality rate was 100%. The nematode epidermis was observed by scanning electron microscopy. After treatment with the purified Pepti dase03, the nematode body was swollen, broken, and the body surface was decomposed. The body surface of the control CK nematodes was intact and smooth.

[0059] The results are as follows Figure 4 shown. The nematode intestine was observed by transmission electron microscopy. After treatment with the purified Peptidase03, vacuolization and cell disorder occurred in the nematode body, the muscle fibers were damaged, and inclusions exuded, and the intestinal structure was incomplete. The internal structure of the control CK was dense, the muscle fibers were arranged neatly, and the intestinal structure was complete.

[0060] The results are as follows Figure 5 shown. As the concentration of Peptidase03 increased, the mortality rate of Bursaphelenchus xylophilus also increased. The EC 50 value of the purified Peptidase03 was 25.73 μg / mL. Peptid ase03 has high toxicity to Bursaphelenchus xylophilus and is an important factor for killing Bursaphelenchus xylophilus.

[0061] Example 2

[0062] 1. Construction of BM06-P03 Engineering Strain

[0063] The reconstructed vector pBBR1MCS2-Tac-EGFP used in this example is 5134 bp in full length. The original vector is pBBR1MCS2-Tac-EGFP. The fluorescence tag fragment of the original vector pBBR1MCS2-Tac-EGFP was removed from the reconstructed vector pBBR1MCS2-Tac-EGFP. The reconstructed vector pBBR1MCS2-Tac-EGFP contains restriction enzyme sites such as EcoRI, HindIII, SalI, and KpnI, and the Kana resistance gene on the vector, which plays a screening role during plasmid construction.

[0064] Primers oprL-F: 5’-ACTACAACGCCAACCCGAAGCTGTC-3’ and oprL-R: 5’-TGAAACTCCTAATGAACCCCAGTGT-3’ were designed referring to the oprL gene sequence (GenBank AAN66847) of Pseudomonas putida KT2440 strain. The genomic DNA of Pseudomonas koreensis was extracted, and the oprL gene promoter (SEQ ID NO.4) was obtained by PCR method.

[0065] The oprL promoter, SD sequence (SEQ ID NO.5) and Peptidase03 gene sequence were inserted into the EcoRI and HindIII restriction enzyme sites of the reconstructed vector pBBR1MCS2-Tac-EGFP, named pBBR1MCS2-P03, and synthesized by GenScript. The plasmid was extracted using a plasmid extraction kit (Aikerui Co., Ltd., Nanjing), and the target gene was subjected to PCR and sequencing verification (Suprogen, Nanjing). The sequence of the vector pBBR1MCS2-P03 is shown in SEQ ID NO.6, and the vector structure is as Figure 6 shown.

[0066] The plasmid pBBR1MCS2-P03 was transformed into DH5α, and positive clones were picked for verification. The pBBR1MCS2-P03 plasmid was extracted using a plasmid extraction kit (Aikerui Co., Ltd., Nanjing) and transformed into the recipient bacterium Pseudomonas koreensis BM06 to obtain the recombinant engineering strain BM06-P03.

[0067] 2. Protein expression and purification

[0068] The glycerol bacteria BM06-P03 were inoculated into LB liquid medium (kana, 50 μg / mL) and shaken at 30 °C for 12 h to obtain a seed culture solution. Then, the seed culture solution was inoculated into LB liquid medium (Amp, 50 μg / mL) at a ratio of 1:100 and cultured until OD 600When IPTG (0.5 mM) was added at 0.6 for induction of growth, it was shaken at 200 r / min at 28 °C and incubated for 8 h. Finally, the induced cells were collected by centrifugation, resuspended with phosphate buffered saline (1×PBS) buffer, broken by a cell disruptor, and centrifuged at 10,000 r / min at 4 °C, and the supernatant was collected. Peptidase03 protein was purified from the supernatant using His-Bind column chromatography (Sangon Biotech) according to the instructions. The protein concentration was determined by the Bradford method using bovine serum albumin (BSA) as a standard.

[0069] SDS-PAGE molecular weight analysis was performed using 10% polyacrylamide gel. Western blot analysis was performed using anti-his-tag primary antibody and goat anti-mouse IgG horseradish peroxidase (HRP)-conjugated secondary antibody (Abmart Shanghai, China). First, the purified protein samples were separated on SDS-PAGE gels and then physically transferred to nitrocellulose membranes. The membranes were immersed in a 1:5000 diluted anti-his-tag primary antibody overnight. After washing, the membranes were incubated with a 1:6000 diluted enzyme-labeled secondary antibody. Then, the signals were observed using enhanced chemiluminescence (ECL) substrate (BioRAD).

[0070] The results were as Figure 7 shown. SDS and Western blot analyses detected the correct secretion and expression of Peptidase03 in Pseudomonas koreensis BM06.

[0071] 3. Nematicidal activity of the engineered strain against Bursaphelenchus xylophilus

[0072] 1) Culture of Bursaphelenchus xylophilus

[0073] The test nematodes AMA3 were inoculated onto 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. They were collected using the Berman funnel method, and the collected nematodes were washed 3 times with sterile water and prepared into a nematode suspension with a concentration of 3000 nematodes / mL for standby.

[0074] 2) Preparation of bacterial fermentation filtrate

[0075] The wild-type strain Pseudomonas koreensis BM06 and the engineered strain BM06-P03 were taken out at -80°C and activated on LB (antibiotic-free) plates and LB (containing knna) plates respectively, and cultured overnight at 30°C. A single colony was picked with an inoculation loop and inoculated into 100 mL Erlenmeyer flasks containing 20 mL NB liquid medium and 20 mL NB liquid medium containing kanamycin antibiotic (50 μg / mL) respectively, and cultured at 30°C and 200 r / min for 12 h as the original bacterial solution. The original bacterial solution was inoculated into 500 mL Erlenmeyer flasks containing 200 mL NB liquid medium at a concentration of 1:100, and cultured at 30°C and 200 r / min for 12 h, 24 h, 36 h, and 48 h respectively. After centrifugation at 10000 r / min for 10 min, the supernatant was taken and filtered through a 0.22 μm filter membrane. The fermentation filtrate was diluted 5 times, 10 times, and 20 times with sterile water respectively and kept for use.

[0076] 3) Nematicidal activity of the engineered strain against Bursaphelenchus xylophilus

[0077] 100 μL of the fermentation filtrates of the wild-type strain Pseudomonas skoreensis BM06 and the engineered strain BM06-P03 cultured for different times and diluted by different multiples were added into 1.5 mL centrifuge tubes respectively, and then 100 μL of nematode suspension (about 500 nematodes) was added and mixed evenly. After treating the nematodes for 24 h, 20 μL of the mixed solution (about 50 nematodes) was taken and observed under a microscope for the number of dead Bursaphelenchus xylophilus (when the nematode body was rigid, in a "J" shape or "C" shape, and the body surface of the nematode had no luster, it was regarded as dead. The total number and the number of dead Bursaphelenchus xylophilus were counted, and the mortality rate and corrected mortality rate of Bursaphelenchus xylophilus were calculated. The treatment with sterile NB culture solution was used as a control, and each treatment was repeated 3 times. The nematicidal activity of the fermentation filtrate of the strain was measured at the 24th h when cultured for different times and diluted by different multiples.

[0078] Mortality rate (%) = number of dead nematodes / total number of nematodes × 100,

[0079] Corrected mortality rate (%) = (treatment mortality rate - blank control mortality rate) / (100 - blank control mortality rate) × 100.

[0080] The results are as Figure 8As shown, when the fermentation filtrate was uniformly diluted 5-fold, there were significant differences in the nematicidal activities of the fermentation filtrates of strains BM06 and BM06-P03 at 12 h, 24 h, 36 h, and 48 h (P < 0.05). When the fermentation filtrate was uniformly diluted 10-fold, there were significant differences in the corrected mortality rates of Bursaphelenchus xylophilus when the fermentation filtrates of strains BM06 and BM06-P03 were cultured for 24 h, 36 h, and 48 h (P < 0.05). When the fermentation filtrate was uniformly diluted 20-fold, there were significant differences at 36 h of the fermentation filtrates of strains BM06 and BM06-P03 (P < 0.05).

[0081] In summary, the insertion of the exogenous insecticidal gene enhanced the nematicidal effect of the wild-type strain, had higher application potential, and provided an effective strategy for the control of Bursaphelenchus xylophilus disease.

[0082] 4. Colonization of the engineered strain in Pinus massoniana seedlings

[0083] The engineered strain BM06-P03 was inoculated into LB medium (kanamycin 50 μg / mL) and shaken at 30 °C for 12 h to obtain the seed culture solution. Then, the seed culture solution was inoculated into LB liquid medium (kanamycin 50 μg / mL) at a ratio of 1:100, incubated at 30 °C, 200 rpm for 24 h, centrifuged at 10,000 r / min for 10 min, and suspended in sterile water to generate an inoculum with a concentration of approximately 10 8 cfu / mL. 20 mL of the inoculum was sprayed onto Pinus massoniana and placed in the greenhouse. Subsequently, the leaves, stems, and roots of the pine seedlings in the treatment group and the control group were collected at 0 d, 15 d, 30 d, 45 d, 60 d, and 90 d after inoculation and stored at -80 °C. The total genomic DNA of each sample was extracted using the Plant Genome Kit, and the concentration and quality of the extracted genomic DNA were measured using Nanodrop. The DNA of each sample was diluted to a concentration of less than 100 ng / μL and stored at -20 °C.

[0084] The copy number of the specific target gene of the engineering strain BM06-P03 was determined by the absolute quantification method. First, Mauve software was used to perform collinearity analysis on the whole genomes of 9 Pseudomonas species (P. koreensis BM06 CP155621, P. abietaniphila BHJ04 CP155619, 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 NC_021505.1), and the BM06-specific gene was selected for comparison on NCBI to ensure its specificity. Bacterial DNA was extracted by the freeze-thaw method. The PCR amplification products of the target gene were collected and detected at a concentration of 80 ng / μL. Then, the 10-fold diluted products were used as templates for fluorescence quantitative PCR amplification. The effectiveness of the established fluorescence quantitative PCR method was tested by the R 2 , slope, and amplification efficiency (E) of the standard curve.

[0085] Using the extracted total genomic DNA as a template, real-time fluorescence quantitative PCR was performed. Four technical replicates were established for each sample, and the amplification reaction was carried out in two steps.

[0086] The reaction conditions were 95°C for 30 s; 95°C for 5 s, 60°C for 30 s, for 40 cycles.

[0087] The conditions for melt curve analysis were 95°C for 15 s; 60°C for 60 s; 95°C for 30 s.

[0088] The results were as Figure 9 shown. The colonization number of the engineering strain BM06-P03 was the highest in the branches of Pinus massoniana. At 0 d, 15 d, 30 d, 45 d, and 60 d of colonization, the colonization numbers were 4.82×10 2 fu / g, 5.88×10 4 fu / g, 3.49×10 4 fu / g, 1.02×10 4 fu / g, 1.67×10 3 fu / g, respectively. The colonization number of the engineering strain BM06-P03 was the highest at 15 d of colonization in the roots, stems, and leaves of Pinus massoniana.

[0089] 5. Outdoor biocontrol experiment of the engineering strain

[0090] 1) Prepare the engineering strain suspension (1×10 8 cells / mL)

[0091] Inoculate the engineered strain BM06-P03 into LB medium (kana, 50 μg / mL) and shake it at 30 °C for 12 h. Add the inoculum culture solution at a ratio of 1:100 to inoculate LB broth (kana, 50 μg / mL), incubate at 30 °C, 200 rpm for 24 h, and then centrifuge at 4 °C, 10,000 rpm for 10 min. Discard the supernatant and suspend the precipitate with sterile water to obtain an inoculant with a concentration of approximately 10 8 cfu / mL.

[0092] 2) Inoculate the two-year-old seedlings of Pinus massoniana with the engineered strain

[0093] Use the spraying method to inoculate the two-year-old seedlings of Pinus massoniana with the bacterial suspension of the engineered strain, and inoculate 15 - 20 mL for each Pinus massoniana seedling. After 3 d, inject 3000 nematodes of Bursaphelenchus xylophilus AMA3 strain into the middle part of the stem of the Pinus massoniana seedlings. Set the following treatment groups respectively:

[0094] ① No strain + no nematode; ② Inoculate sterile water + inoculate nematodes; ③ Inoculate wild-type strain BM06 + nematodes; ④ Inoculate engineered strain BM06-P03 + nematodes.

[0095] Observe the disease symptoms of the inoculated hosts every 7 d, and count the disease incidence rate and disease index. Calculate the disease index according to the disease grading standard. The disease severity of Pinus massoniana is divided into 5 levels. Level Ⅰ: The needles of the plant are green and grow healthily, with a representative value of 0; Level Ⅱ: 0 - 25% of the needles turn chlorotic and yellow, with a representative value of 1; Level Ⅲ: 25% - 50% of the needles turn chlorotic and yellow, and the branch tips are bent, with a representative value of 2; Level Ⅳ: 50% - 70% of the needles turn chlorotic and yellow, and the branch tips droop, with a representative value of 3; Level Ⅴ: 75% - 100% of the needles turn chlorotic and brown, and the plant withers and dies, with a representative value of 4.

[0096] The results are as Figure 10 shown. When inoculating the AMA3 strain for 14 d, the disease incidence rates of the wild-type strain and the engineered strain are the same, the number of diseased plants is 2, and the grades are both 1; all the controls are diseased; when inoculating for 30 d, all the wild-type strains die, and the number of dead plants of the engineered strain is 1.

[0097] In summary, the engineered strain BM06-P03 delays the disease incidence rate of Pinus massoniana and has an effect on controlling Bursaphelenchus xylophilus.

[0098] The above description is illustrative rather than restrictive to the present invention. Those of ordinary skill in the art understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all of them will fall within the protection scope of the present invention.

Claims

1. A method for constructing an engineered strain for controlling Bursaphelenchus xylophilus, characterized in that, The specific steps are as follows: 1) Based on the gene sequence GenBank AAN66847 of Pseudomonas putida KT2440 strain oprL Design primers oprL -F: 5’-ACTACAACGCCAACCCGAAGCTGTC-3’, oprL -R: 5’-TGAAACTCCTAATGAACCCCAGTGT-3’, Extract the genomic DNA of Pseudomonas koreensis, and obtain the oprL gene promoter by PCR method; The oprL sequence of the promoter is shown in SEQ ID NO.4; 2) Insert the oprL promoter, SD sequence and Peptidase03 gene sequence into the EcoRI and HindIII restriction sites of the modified vector pBBR1MCS2-Tac-EGFP to obtain the vector pBBR1MCS2-P03, and extract the plasmid pBBR1MCS2-P03; The said SD sequence is as shown in SEQ ID NO.5; The said Peptidase03 gene sequence is as shown in SEQ ID NO.1; 3) Transform plasmid pBBR1MCS2-P03 into DH5α, pick positive clones and extract plasmid pBBR1MCS2-P03 again, then transform it into the recipient bacterium Pseudomonas koreensis Pseudomonas koreensis BM06 to obtain the recombinant engineering strain BM06-P03; The said Pseudomonas koreensis Pseudomonas koreensis The preservation number of BM06 is: CCTCC NO: M20241926.

2. The recombinant engineering strain BM06-P03 for controlling Bursaphelenchus xylophilus constructed by the method described in claim 1.

3. The application of the recombinant engineering strain BM06-P03 described in claim 2 in controlling Bursaphelenchus xylophilus.

4. The application according to claim 3, characterized in that, The control of Bursaphelenchus xylophilus is to delay the disease onset speed of Pinus massoniana.

5. Use of the fermentation filtrate of the recombinant engineered strain BM06-P03 according to claim 2 in causing the death of Bursaphelenchus xylophilus, characterized in that, The preparation method of the said fermentation filtrate is to activate the recombinant engineering strain BM06-P03 on an LB plate containing kana and culture it overnight at 30°C; pick a monoclonal with an inoculation loop and inoculate it into 20 mL of NB liquid medium containing 50 μg / mL kanamycin antibiotic, 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, and culture it at 30°C and 200 r / min for 12 h, 24 h, 36 h, 48 h, centrifuge at 10000 r / min for 10 min, take the supernatant, and filter it with a 0.22 μm filter membrane to prepare the fermentation filtrate of the recombinant engineering strain BM06-P03.

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