Application of Pseudomonas resistens thaxtoxin synthesis related genes pltB and pltC in biological control

Abstract

This invention belongs to the field of biotechnology, specifically disclosing the application of pltB and pltC, genes related to the synthesis of glucosamine from *Pseudomonas*, in biological control. This invention protects the glucosamine synthesis-related proteins pltB and pltC, or substances that promote the expression of the protein-coding genes, or substances that increase the activity or content of the proteins, in the biological control of bacterial plant diseases. This invention systematically reveals key genes involved in glucosamine synthesis. power plant and PltC The key role of the ZF509 strain in biocontrol was investigated, and its control effect on melon fruit spot disease, tomato canker and broccoli black rot was verified by pot experiment. This provides a theoretical basis and genetic resources for the further development and application of this biocontrol bacterium.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of pltB and pltC, genes related to the synthesis of gromwellin in Pseudomonas aeruginosa, in biological control. Background Technology

[0002] Watermelon acid-eating bacteria Acidovorax citrulli Bacterial fruit blotch (BFB) is a significant bacterial disease affecting watermelons and melons worldwide. It is characterized by rapid onset, reliance on a single control agent, and severe damage, seriously hindering the healthy development of the fruit industry. Currently, there are no commercially available resistant varieties for controlling BFB, and control primarily relies on copper-based chemical agents. However, the extensive use of these chemical agents has numerous negative impacts on the agricultural ecosystem, organisms, and human activities. Therefore, developing environmentally friendly and safe microbial agents for human health is of great importance for the control of BFB in cucurbits.

[0003] Defense against Pseudomonas ( Pseudomonas protegens This fungus can produce various antibiotics, including pyrrolnitrin (Prn), 2,4-diacetylphloroglucinol (2,4-DAPG), and pyoluteorin (Plt). Pyoluteorin is an aromatic polyketide Pseudomonas metabolite. The gene cluster responsible for the biosynthesis, transport, and regulation of pyoluteorin consists of a set of operons with opposite transcriptional expression directions: pltRM-pltLABCDEFG and pltZ-pltIJKNOP. Among them, pltB and pltC are key genes for pyoluteorin synthesis. Studies have shown that pyoluteorin has strong control effects on potato late blight, cucumber downy mildew, and damping-off of cotton and beet caused by Pythium oxysporum, but its control effect on bacterial diseases is rarely reported. This application constructs a gene to defend against Pseudomonas ZF509. pltB , pltC The deletion mutant strain was used in a greenhouse pot experiment to compare the control efficacy of wild-type and knockout strains against melon fruit spot disease, tomato bacterial canker, and broccoli black rot. The aim was to reveal whether pyoluteorin affects the biocontrol effect of ZF509 and to provide a theoretical basis for developing precision biological control strategies based on functional genes. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to improve the effectiveness of biological control of plant diseases.

[0005] To address the above technical problems, the present invention provides any one of the following applications of proteins, substances that promote the expression of said protein-coding genes, or substances that increase the activity or content of said proteins:

[0006] P1. Application in the biological control of bacterial plant diseases; P2. Application in the prevention and control of melon fruit spot disease; the pathogen of melon fruit spot disease is *Acidophilus hygrophila* (watermelon). Acidovorax citrulli ); P3, In reducing the acidophilic bacteria in watermelon ( Acidovorax citrulli Applications in pathogenicity; P4, In reducing watermelon acidophilus ( Acidovorax citrulli Application in the area of ​​lesions caused by ) P5, Inhibiting watermelon acidophilus ( Acidovorax citrulli Applications in growth and development; P6. Application in the prevention and control of tomato bacterial canker; the pathogen of tomato bacterial canker is *Corynebacterium miltanum* subsp. *miltanum* (… Clavibacter michiganensis subsp. michiganensis ); P7, In reducing the number of Corynebacterium micranthae subsp. micranthae ( Clavibacter michiganensis subsp. michi ganensis Applications in pathogenicity; P8. In reducing the number of Corynebacterium micranthae subspecies ( Clavibacter michiganensis subsp. michi ganensis Application in the area of ​​lesions; P9, Inhibition of Corynebacterium micranthae subsp. micranthae ( Clavibacter michiganensis subsp. michi ganensis Applications in growth and development; P10. Application in the prevention and control of black rot in broccoli; the pathogen causing black rot in broccoli is Xanthomonas spp. (Xanthomonas spp.) Xanthomonas campestris pv. campestris ); P11, In reducing the pathogenicity of Xanthomonas vegetans in rapeseed (a species of rapeseed) Xanthomonas campestris pv. campestris Applications in pathogenicity; P12, In reducing the pathogenic species of Xanthomonas spp. in rapeseed ( Xanthomonas campestris pv. campestris Application in the area of ​​lesions; P13, Inhibiting the pathogenicity of Xanthomonas laurentii in *Rhizopus spp.* (a strain of *Rhizopus spp.*) Xanthomonas campestris pv.campestris Applications in growth and development; The protein in question is a protein of type A1, A2, or A3: A1. The amino acid sequence is the protein that is the amino acid sequence shown in SEQ ID No. 2 or SEQ ID No. 4 in the sequence listing; A2. A protein that has more than 80% identity with the protein shown in A1 and is related to biocontrol function, obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 2 or SEQ ID No. 4 in the sequence listing. A3. A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1 or A2.

[0007] In the above applications, the protein tag refers to a polypeptide or protein fused with a target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0008] In the above applications, the protein can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically.

[0009] In the above applications, the identity of the protein refers to the identity of its amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambdaratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences to calculate the identity value (%), then the identity value can be obtained.

[0010] In the above applications, the 80% or more identity of the protein can be at least 81%, 85%, 90%, 91%, 92%, 95%, 96%, 98%, 99%, or 100% identity.

[0011] In the above applications, the protein is derived from *Pseudomonas aeruginosa* (a type of bacteria). Pseudomonas protegens Specifically, it originates from the defense against Pseudomonas ( Pseudomonas protegens) strain ZF509.

[0012] In the above applications, the substance that promotes the expression of the protein-coding gene or increases the activity or content of the protein is a biological material related to the protein, and the biological material is any one of B1 to B4 below: B1. The nucleic acid molecule encoding the protein; B2, an expression cassette containing the nucleic acid molecule described in B1; B3, a recombinant vector containing the nucleic acid molecule described in B1, or a recombinant vector containing the expression cassette described in B2; B4. Recombinant microorganisms containing the nucleic acid molecules described in B1, or recombinant microorganisms containing the expression cassette described in B2, or recombinant microorganisms containing the recombinant vector described in B3.

[0013] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0014] In the above applications, the nucleic acid molecule described in B1 may specifically be a DNA molecule whose coding sequence is SEQ ID No. 1 in the sequence listing or a DNA molecule whose coding sequence is SEQ ID No. 3 in the sequence listing; In the above applications, the expression cassette described in B2 refers to DNA capable of expressing the protein in a host cell. This DNA may include not only a promoter that initiates transcription of the protein's coding gene, but also a terminator that terminates transcription of the protein's coding gene.

[0015] In the above applications, the recombinant microorganism mentioned in B4 can specifically be bacteria.

[0016] In the above applications, the bacteria may specifically be Pseudomonas spp. ( Pseudomonas ) bacteria, the aforementioned *Pseudomonas* genus ( Pseudomonas The bacteria can specifically be Pseudomonas aeruginosa ( ) Pseudomonas protegens ).

[0017] In the above applications, the plant bacterial diseases can specifically be any one of melon fruit spot disease, tomato canker, and broccoli black rot. The pathogen causing melon fruit spot disease is *Acidophilus hygrophila* (watermelon). Acidovorax citrulli The pathogen causing tomato canker is *Corynebacterium miltanum* subsp. *miltanum* (…). Clavibacter michiganensis subsp. mich iganensis The pathogen causing the black rot of broccoli is *Xanthomonas spp.*, a pathogenic strain of *Xanthomonas spp.*. Xanthomonas campestris pv. campestris ).

[0018] The present invention also protects the protein.

[0019] The aforementioned biological materials also fall within the scope of protection of this invention.

[0020] To address the aforementioned technical problems, the present invention also provides an enhancement method for Pseudomonas spp. ( Pseudomonas Methods for the biological control of bacterial diseases in plants.

[0021] The enhanced Pseudomonas spp. provided by this invention ( Pseudomonas A method for the biological control of bacterial diseases in plants includes the step of increasing the expression level of the protein or its encoding gene in the recipient Pseudomonas spp. bacteria to obtain the target Pseudomonas spp. bacteria; wherein the control effect of the target Pseudomonas spp. bacteria on bacterial diseases in plants is stronger than that of the recipient Pseudomonas spp. bacteria on bacterial diseases in plants.

[0022] In the above method, the plant bacterial disease can specifically be any one of melon fruit spot disease, tomato canker, and broccoli black rot. The pathogen causing melon fruit spot disease is *Acidophilus hygrophila* (watermelon). Acidovorax citrulli The pathogen causing tomato canker is *Corynebacterium miltanum* subsp. *miltanum* (…). Clavibacter michiganensis subsp. mich iganensis The pathogen causing the black rot of broccoli is *Xanthomonas spp.*, a pathogenic strain of *Xanthomonas spp.*. Xanthomonas campestris pv. campestris ).

[0023] In the above method, increasing the expression level of the protein or its encoding gene in the recipient Pseudomonas bacteria can be achieved by introducing the encoding gene of the protein into the recipient Pseudomonas bacteria.

[0024] In the above method, the gene encoding the protein can be modified before being introduced into the recipient Pseudomonas bacteria to achieve better expression.

[0025] In the above method, the genome of the recipient Pseudomonas bacteria does not contain the gene encoding the protein paaC.

[0026] In the above method, the recipient Pseudomonas bacteria can be defensive Pseudomonas ( Pseudomonas protegens For example, in one embodiment of the present invention, the receptor Pseudomonas bacteria is a strain Δ that does not contain the gene encoding the protein pltB. pltB The Δ pltB For wild-type defense against Pseudomonas ( Pseudomonas protegens The ZF509 strain, which is knocked out, encodes the protein Δ.pltB Gene knockout strains obtained from the gene. For example, in one embodiment of the present invention, the recipient Pseudomonas bacteria may also be strains Δ that do not contain the gene encoding the protein pltC. pltC The Δ pltC For wild-type defense against Pseudomonas ( Pseudomonas protegens The ZF509 strain, which is knocked out, encodes the protein Δ. pltC The gene knockout strain ZF509 was obtained from the gene. This strain is registered in the China General Museum (CGMCC) with the number CGMCC No. 27638.

[0027] Defense against Pseudomonas ( Pseudomonas protegens ZF509 is a biocontrol bacterium with significant antagonistic effects against various plant pathogenic bacteria. To preliminarily elucidate the control mechanism of Pseudomonas ZF509 against bacterial fruit spot disease in melon, the effect of its secondary metabolite gambogein (Plt) on the growth of the fruit spot pathogen was tested, and a comparative analysis was conducted between ZF509 and its mutant strain Δ. pltB and Δ pltC The growth rate, motility, colonization, biofilm formation, and disease control effects of the mutant Δ were observed. The results showed that the mutant Δ pltB and Δ pltC The growth rate on KB medium was consistent with that of the wild-type strain ZF509, while the biofilm formation was reduced by 50.84% ​​and 54.97% respectively compared to ZF509. The plate antagonistic effect of gambogeysin standard against *Acidophilus hygrophila* increased with increasing concentration. Pot experiments showed that after inoculation with the pathogen, Δ pltB and Δ pltC The treatment significantly reduced the control efficacy against melon fruit spot disease, tomato bacterial canker, and broccoli black rot. Comprehensive analysis indicates that gromwellin (Plt) plays an important role in the control of plant bacterial diseases.

[0028] Preservation Instructions Bacterial species name: *Pseudomonas aeruginosa* Latin name: Pseudomonas protegens Strain number: ZF509 Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Collection Institution Abbreviation: CGMCC Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Deposit date: June 15, 2023 Registration number at the Preservation Center: CGMCC No. 27638. Attached Figure Description

[0029] Figure 1 This is the plasmid map of p2P24.

[0030] Figure 2 This is a plasmid map of pBBR1MCS-2.

[0031] Figure 3 This invention demonstrates the antibacterial effect of different concentrations of gambogeysin on watermelon acidophilus in Example 1 of this invention.

[0032] Figure 4 This is a schematic diagram of the gambogeysin synthesis gene cluster in Example 1 of the present invention.

[0033] Figure 5 This is a secondary structure model of pltB and pltC proteins in Example 1 of the present invention. Wherein, Figure 5 A is pltB. Figure 5 B is pltC.

[0034] Figure 6 This is a tertiary structure model of pltB and pltC proteins in Example 1 of the present invention. Wherein, Figure 6 A is pltB. Figure 6 B is pltC.

[0035] Figure 7 In Embodiment 1 of the present invention pltB Gene knockout and identification results. Figure 7 A represents the knockout vector p2P24- PltB Double enzyme digestion pattern, lanes 1-4 are transformants, M is DNA Marker; Figure 7 B represents the result of a single homologous recombination PCR identification; lanes 2, 3, 5, and 6 represent Δ... pltB The deletion mutant strains, lanes 1 and 4 are wild-type ZF509 strains that were not successfully knocked out, CK is the wild-type ZF509 control, and M is the DNA Marker; Figure 7 C is pltB PCR validation results for the deletion mutant: lanes 2, 3, 5, 7, 9, 11, 13, and 14 were Δ. pltB The deletion mutant strains are represented in lanes 1, 4, 6, 8, 10, 12 and 15, which are wild-type ZF509 strains that were not successfully knocked out. CK is the wild-type ZF509 control, and M is the DNA marker.

[0036] Figure 8 In Embodiment 1 of the present invention pltC Gene knockout and identification results. Figure 8 A represents the knockout vector p2P24- PltC Double enzyme digestion pattern, lanes 1-4 are transformants, M is DNA Marker; Figure 8 B represents the result of a single homologous recombination PCR identification, and lanes 1-7 represent Δ pltCDeletion mutant strain, CK is ZF509 wild-type control, M is DNA Marker; Figure 8 C is pltC PCR validation results for the deletion mutant: lanes 1-5, 12, and 13 showed Δ. pltC The deletion mutant strains, lanes 6-11, 14 and 15 are wild-type ZF509 strains that were not successfully knocked out, CK is the wild-type ZF509 control, and M is the DNA Marker.

[0037] Figure 9 C-Δ in Embodiment 1 of the present invention pltB and C-Δ pltB Identification of reintroduced strains. Figure 9 A is pltB PCR identification results of strains after gene deletion and restoration: lanes 1-15 are C-Δ pltB CK is the wild-type control of ZF509, and M is the DNA Marker. Figure 9 B is pltC PCR identification results of strains after gene deletion and restoration: lanes 1-15 are C-Δ pltC CK is the wild-type control of ZF509, and M is the DNA Marker.

[0038] Figure 10 The growth curves of ZF509 and its derivative strains in Example 1 of this invention are shown.

[0039] Figure 11 This invention relates to the generation and quantitative detection of biofilms from ZF509 and its derivative strains in Example 1 of this invention. Figure 11 A represents the biofilm formed by different bacterial strains as observed by crystal violet staining; A is KB, B is ZF509, and C is Δ. s D is C-Δ pltB E is Δ pltB F is C-Δ pltC . pltC b represents the biofilm formation rate of different strains at a wavelength of 570 nm, and ns represents no significant difference. The results of the significance analysis are as follows P <0.05.

[0040] [[ID=!13]]Figure 11 The results of the motility test of ZF509 and its derivative strains in Example 1 of this invention are shown. Figure 12 A represents the detection results for mobility and swarming. Figure 12 B represents the quantitative analysis result of motility; Figure 12 C represents the results of the quantitative analysis of clustering.

[0041] Figure 12This invention demonstrates the control effect of ZF509 and its derived mutations on melon fruit spot disease in Example 1. Figure 13 A represents the pathogen control; Figure 13 B is Δ Figure 13 Group; pltB [[ID=1!2]]Figure 13 C represents strain Δ pltC Group; Figure 13 D is group ZF509; Figure 13 E is C-Δ pltB Group; Figure 13 F is C-Δ pltC Group; Figure 13 G represents the healthy control group.

[0042] Figure 14 This presents the statistical results of the control efficacy of ZF509 and its derived mutations against melon fruit spot disease in Example 1 of this invention. Different lowercase letters represent the results of significance analysis. P <0.05.

[0043] Figure 15 This invention demonstrates the control effect of ZF509 and its derived mutant strains on tomato bacterial canker in Example 1 of this invention. Figure 15 A represents the pathogen control; Figure 15 B is Δ pltB Group; Figure 15 C represents strain Δ pltC Group; Figure 15 D is group ZF509; Figure 15 E is C-Δ pltB Group; Figure 15 F is C-Δ pltC Group; Figure 15 G represents the healthy control group.

[0044] Figure 16 This presents the statistical results of the control efficacy of ZF509 and its derived mutant strains against tomato bacterial canker in Example 1 of this invention. Different lowercase letters represent the results of significance analysis. P <0.05.

[0045] Figure 17 This invention demonstrates the control effect of ZF509 and its derived mutant strains on black rot of broccoli in Example 1 of this invention. Figure 17 A represents the pathogen control; Figure 17 B is Δ pltB Group; Figure 17 C represents strain Δ pltC Group; Figure 17 D is group ZF509; Figure 17 E is C-Δ pltB Group; Figure 17 F is C-Δ pltC Group; Figure 17G represents the healthy control group.

[0046] Figure 18 This presents the statistical results of the control efficacy of ZF509 and its derived mutant strains against black rot in broccoli in Example 1 of this invention. Different lowercase letters represent the results of significance analysis. P <0.05. Detailed Implementation

[0047] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0049] In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence listing is the 5' terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA / RNA.

[0050] The following examples use the anti-pseudomonas strain ZF509 ( Pseudomonas protegens ZF509, isolated from the rhizosphere soil of potato plants in Heilongjiang Province, was deposited on June 15, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 27638. It is hereinafter referred to as *Pseudomonas aeruginosa* CGMCC No. 27638 or strain ZF509.

[0051] The pathogen in the following examples is *Acidophilus citrus* (watermelon). Acidovorax citrulli The results have been published in the literature “Isolation and Identification of the Defensive Pseudomonas ZF509 and Its Control Effect on Bacterial Fruit Spot Disease in Melon”, and the public can obtain the results from the applicant to replicate the experiments described in this application.

[0052] The pathogen *Corynebacterium micranthae* subsp. *micranthae* in the following examples ( Clavibacter michiganensis subsp. michiganensis The results have been published in the literature “Isolation, Identification and Biocontrol Effect Study of ZF516, an Antagonistic Bacterium for Tomato Ulcer Disease”, and the public can obtain the results from the applicant to replicate the experiments described in this application.

[0053] The pathogen Xanthomonas spp. in the following examples is *Xanthomonas spp.*, a pathogenic strain of *Xanthomonas spp.*. Xanthomonas campestris pv. campestrisThe results have been published in the literature “Identification of resistance to black rot in seedlings of broccoli and its closely related species and screening of resistance sources”, and the public can obtain the results from the applicant to replicate the experiments in this application.

[0054] The p2P24 plasmid (gene knockout vector, Km) in the following examples r ,See Figure 1 (This has been discussed in the literature "The outer membrane protein OprF and the sigma factor SigX regulate antibiotic production in...") Pseudomonas fluorescens As disclosed in “2P24”, the public can obtain the experiments to replicate this application from the applicant.

[0055] The pRK2013 plasmid (triparental mating helper plasmid, Km) in the following examples r (This has been discussed in the literature "The outer membrane protein OprF and the sigma factor SigX regulate antibiotic production in...") Pseudomonas fluorescens As disclosed in “2P24”, the public can obtain the experiments to replicate this application from the applicant.

[0056] The pBBR1MCS-2 plasmid (wide-host cloning plasmid, Km) in the following examples r ,See Figure 2 The literature "The outer membrane protein OprF and the sigma factor SigX regulate antibiotic production in..." has been published. Pseudomonas fluorescens As disclosed in “2P24”, the public can obtain the experiments to replicate this application from the applicant.

[0057] Escherichia coli in the following examples ( Escherichia coli DH5α is a product of Nanjing Novizan Biotechnology Co., Ltd.

[0058] The culture medium preparation method in the following examples is as follows: LB (Luria-Bertani) liquid medium: 10 g tryptone, 5 g yeast extract, and 10 g NaCl were added to a final volume of 1 L with deionized water and adjusted to pH 7.0-7.2.

[0059] LB solid medium: Add 15 g of agar powder to each liter of LB liquid medium to make LB solid medium.

[0060] KB (King's B medium) liquid culture medium: Combine 20 g peptone, 1.5 g K2HPO4·H2O, 1.5 g MgSO4·7H2O, and 10 g glycerol with deionized water to a final volume of 1 L and adjust the pH to 7.0-7.2.

[0061] KB solid medium: Add 15 g of agar powder to each liter of KB liquid medium to make KB solid medium.

[0062] Mobility assay medium: 10 g of Bacto Peptone, 5 g of NaCl, and 3 g of Bacto Agar powder were diluted to 1 L with deionized water and adjusted to pH 7.0-7.2.

[0063] In the examples described below, restriction endonucleases, DNA polymerases, plasmid extraction kits, bacterial whole genome extraction kits, DNA recovery and purification kits, DNA markers, and agarose were all purchased from TaKaRa Technology Co., Ltd., seamless cloning kits were purchased from CloneSmarter, and other reagents were domestically produced analytical grade. Primer synthesis and DNA sequencing were performed by Sangon Biotech (Shanghai) Co., Ltd.

[0064] Unless otherwise specified, the quantitative experiments in the following examples were all repeated three times, and the results were averaged.

[0065] The primer sequences in the following examples are shown in Table 1: Table 1 Primers used in the embodiments of the present invention

[0066] Note: The underlined sequences in the table are restriction endonuclease recognition sites.

[0067] Example 1 Different concentrations of gambogeysin standard samples all showed antibacterial effects against *Acidophilus hygrophilus*, and the antibacterial ability increased with increasing gambogeysin concentration, producing a larger inhibition zone. See details... Figure 3 .

[0068] See the schematic diagram of the gambogeysin synthesis gene cluster. Figure 4 ,Include PltB Genes and PltC Gene.

[0069] PltB The CDS sequence of the gene is shown in SEQ ID No. 1, and the coding sequence of the PltB protein is shown in SEQ ID No. 2.

[0070] SEQ ID No.1 SEQ ID No.2 PltC The CDS sequence of the gene is shown in SEQ ID No. 3, and the coding sequence of the PltC protein is shown in SEQ ID No. 4.

[0071] SEQ ID No. 3 SEQ ID No.4 The physicochemical properties of proteins were analyzed using the online software Expasy-ProtParam (http: / / web,expasy.org / protparam / ); the secondary structure of proteins was predicted using the online software SOPMA (https: / / npsa.lyon.inserm.fr / cgi-bin / secpred_sopma.pl); and the three-dimensional structures of proteins encoded by two genes were predicted using Phyre2. Physicochemical property analysis of pltB and pltC proteins: Analysis using the Protparam tool (see Table 2) showed that pltB has 2458 amino acids, a molecular weight of 263053.44 Da, and an isoelectric point of 5.40. A value less than 7 indicates an acidic protein. The total number of negatively charged residues (Asp + Glu) is 258, accounting for 10.50% of the total protein, while the total number of positively charged residues (Arg + Lys) is 190, accounting for 7.73% of the total protein. The molecular formula is C1. 11631 H 18370 N 3322 O 3462 S 91 With a total of 36,876 atoms and an instability coefficient of 42.79, it is an unstable protein. Its aliphatic coefficient is 93.65, and its average hydrophilicity is 0.001. pltC has 1744 amino acids, a molecular weight of 190,392.60 Da, and an isoelectric point of 5.39 (values ​​less than 7 indicate an acidic protein). It contains 200 negatively charged residues (Asp + Glu), accounting for 11.47% of the total protein, and 151 positively charged residues (Arg + Lys), accounting for 8.66% of the total protein. Its molecular formula is C. 8439 H 13364 N 2388 O 2526 S 53 It has a total of 26,770 atoms, an instability coefficient of 37.93, is a stable protein, an aliphatic coefficient of 96.98, and an average hydrophilicity of -0.030.

[0072] Table 2 Physicochemical properties analysis of pltB and pltC proteins

[0073] Secondary structure prediction of pltB and pltC proteins: SOPMA analysis showed that the proportions of α-helix, β-sheet, and random coil in pltB protein were 39.38%, 13.10%, and 47.52%, respectively. Figure 5In the pltC protein, the proportions of α-helix, β-sheet, and random coil are 40.98%, 15.11%, and 43.91%, respectively. Figure 5 B).

[0074] Tertiary structure prediction of pltB and pltC proteins: Tertiary structure models of pltB and pltC proteins were constructed using the Phyre2 online software. c7wvzA, with the highest homology, was selected as the template for pltB. This model was derived from the β-ketoacyl acyl carrier protein synthase I module. Figure 6 A). pltC selects c7m7jB, which has the highest homology, as the template. This model comes from the EryAI module ( Figure 6 (B). Numerous α-helices and random curls can be observed in the prediction model diagram, indicating that the prediction result is consistent with the secondary structure prediction result.

[0075] 1. Construction of gene knockout vector (1) Using SnapGegne, primers for upstream and downstream homologous arms of the target gene were designed. The homologous arms were approximately 300-700 bp segments upstream and downstream of the target gene, and included a dozen or so bases, including the start codon and stop codon of the target gene. Amplification of ZF509 PltB Primer pairs for upstream homologous arms of genes are PltB -up-F and PltB -up-R composition, amplifying ZF509 PltB Primer pairs for the downstream homologous arms of the gene are formed by PltB -down-F and PltB Composed of -down-R.

[0076] Amplification of ZF509 PltC Primer pairs for upstream homologous arms of genes are PltC -up-F and PltC -up-R composition, amplifying ZF509 PltC Primer pairs for the downstream homologous arms of the gene are formed by PltC -down-F and PltC Composed of -down-R.

[0077] (2) PCR amplification was used. The PCR system and procedure are shown in Tables 3 and 4: Table 3 PCR reaction system

[0078] Table 4 PCR reaction procedure

[0079] Electrophoresis and gel purification were performed to obtain the following results: PltB Upstream and downstream homologous arm products of genes and PltC Products of upstream and downstream homologous arms of a gene.

[0080] Linearized plasmids were obtained by amplifying plasmid p2P24 using reverse PCR.

[0081] Purified PltB The upstream and downstream homologous arm products of the gene and the linearized plasmid p2P24 were ligated into three fragments using a multi-fragment seamless cloning kit, and then transformed. E.coli DH5α was plated on resistant plates containing 50 μg / mL kanamycin and incubated at 37°C. After single colonies grew on the plates, plasmid extraction and double enzyme digestion were performed for verification. The double enzyme digestion reaction system was prepared according to Table 5, and the digestion was carried out in a water bath at 37°C for 2 h. The size of the digested fragments was consistent with the expected transformants (see Table 5). Figure 7 A) The sample was sent to the company for sequencing. The correct strain, showing no mutations after sequencing, is considered to contain the target... PltB Gene knockout vector p2P24- PltB The strain of Escherichia coli is called DH5α / p2P24- PltB .

[0082] Purified PltC The upstream and downstream homologous arm products of the gene and the linearized plasmid p2P24 were ligated into three fragments using a multi-fragment seamless cloning kit, and then transformed. E.coli DH5α was plated on resistant plates containing 50 μg / mL kanamycin and incubated at 37°C. After single colonies grew on the plates, plasmid extraction and double enzyme digestion were performed for verification. The double enzyme digestion reaction system was prepared according to Table 4, and the digestion was carried out in a water bath at 37°C for 2 h. The size of the digested fragments was consistent with the expected transformants (see Table 4). Figure 8 A) The sample was sent to the company for sequencing. The correct strain, showing no mutations after sequencing, is considered to contain the target... PltC Gene knockout vector p2P24- PltC The strain of Escherichia coli is called DH5α / p2P24- PltC .

[0083] Table 5 Double enzyme digestion system

[0084] 2.2 Bacterial conjugation experiment The knockout vector p2P24- was introduced using a bacterial conjugation method. PltB and p2P24- PltC Transformed separately into ZF509: E. coli containing the knockout vector (DH5α / p2P24- PltB or DH5α / p2P24- PltCThe donor bacteria were Escherichia coli pRK2013, the assistant bacteria were wild-type ZF509 strain, and the recipient bacteria were wild-type ZF509 strain.

[0085] (1) The recipient bacteria, donor bacteria, and helper bacteria were inoculated into LB liquid medium, respectively. Both the LB liquid medium used to culture the donor bacteria and the LB liquid medium used to culture the helper bacteria should contain kanamycin (50 μg / mL). ZF509 was cultured at 30℃ in *Escherichia coli* containing the knockout vector (DH5α / p2P24-). PltB or DH5α / p2P24- PltC ) and pRK2013 were cultured at 37℃ and 200 rpm for 12 h to obtain various bacterial solutions; (2) Take 1 mL of each of ZF509 bacterial suspension, Escherichia coli bacterial suspension containing knockout vector and pRK2013 bacterial suspension, centrifuge at 12000 rpm for 1 min, remove the supernatant, add 1 mL of sterile water to wash the bacterial cells, repeat 3 times, and finally resuspend the bacterial cells with 100 μl + ddH2O to obtain the corresponding resuspended bacterial suspensions. (3) Transfer the three resuspended bacterial solutions, namely ZF509, Escherichia coli containing the knockout vector and pRK2013, to the same centrifuge tube, vortex to mix, centrifuge at 12000 rpm for 1 min, remove the supernatant, and finally resuspend the bacterial cells in 100 μl ddH2O sterile water to obtain a resuspended mixed bacterial solution. (4) After the resuspended mixed bacterial solution is combined for 10 min, it is evenly spotted on LB medium plates and incubated at 28℃ for 6-8 h.

[0086] 2.3 Gene Knockout - Single and Double Crossover (1) Use a spreader to scrape off the bacterial cells cultured in step (4) of 2.2, serially dilute them 10 times, spread them on solid LB double antibody plates containing ampicillin (100 μg / mL) and kanamycin (50 μg / mL), and incubate them upside down at 30°C for about 24 hours. (2) Single colonies were picked from the double antibody plate, and colony PCR was performed using primer combinations consisting of the upstream homologous arm F primer, the downstream homologous arm R primer, and knockout detection primers around the homologous arm. For... PltB -Gene, upstream homologous arm F primer finger PltB -up-F, downstream homologous arm R primer finger PltB -down-R, the knockout detection primer for the periphery of the homologous arm. PltB -F and PltB -R. For PltC Gene, upstream homologous arm F primer finger PltC -up-F, downstream homologous arm R primer finger PltC-down-R, the knockout detection primer for the periphery of the homologous arm. PltC -F and PltC -R.

[0087] (3) The correctly identified single colonies (see Figure 7 B and Figure 8 B) was inoculated into LB containing ampicillin (100 μg / mL) and kanamycin (50 μg / mL) and cultured at 30 °C with shaking at 200 rpm for 12 h; (4) Transfer the bacterial culture that has been cultured for 12 h to 5 mL of fresh, non-resistant LB liquid medium at a transfer volume of 1%, i.e., 50 μL, and culture at 30℃ and 200 rpm for 12 h to promote double exchange. (5) The bacterial culture above was serially diluted with fresh LB liquid medium and spread on LB solid plates containing 15% sucrose. It was then incubated upside down in a 30°C incubator for about 12 h (the plasmid contains a sucrose-related lethal gene) to screen for recombinants that have undergone double crossover and do not carry plasmids. (6) Targeting PltB The effect of gene knockout is measured using knockout detection primer pairs (made by...). PltB -F and PltB -R composition) Colony PCR identification of single colonies on sucrose plates was performed, and a 6.40 kb band was amplified from the ZF509 genomic DNA. PltB Gene deletion mutant strain Δ PltB Only a 1.54kb band was amplified from the genomic DNA, consistent with expectations (see...). Figure 7 (C). Streak single colonies with correct bands two to three times until the colony PCR electrophoresis bands are correct and singular. Recover the banded gel and send it for sequencing; correctly sequenced colonies are ZF509. PltB Gene knockout strains are called Δ strains. PltB The bacteria were preserved in glycerol at a final concentration of 20% at -80°C.

[0088] against PltC The effect of gene knockout is measured using knockout detection primer pairs (made by...). PltC -F and PltC -R composition) Colony PCR identification of single colonies on sucrose plates was performed, and a 5.33kb band was amplified from the ZF509 genomic DNA. PltC Gene deletion mutant strain Δ PltC Only a 1.59kb band was amplified from the genomic DNA, consistent with expectations (see...). Figure 8(C). Streak single colonies with correct bands two to three times until the colony PCR electrophoresis bands are correct and singular. Recover the banded gel and send it for sequencing; correctly sequenced colonies are ZF509. PltC Gene knockout strains are called Δ strains. PltC The bacteria were preserved in glycerol at a final concentration of 20% at -80°C.

[0089] 3. Obtaining the replacement strain In the knockout strain Δ constructed above PltB Based on this, using the ZF509 genome as a template, amplification was performed. PltB The gene's reading frame and its upstream and downstream promoter fragments were amplified and inserted into the multiple cloning site of the shuttle plasmid pBRR1MCS-2 to construct the gene complementation vector DH5α / pBRR1MCS-2. PltB And switch to Δ PltB strain, successfully obtained PltB The complement strain C-Δ of the deletion mutant PltB (See Figure 9 A).

[0090] In the knockout strain Δ constructed above PltC Based on this, using the ZF509 genome as a template, Δ PltC The gene's reading frame and its upstream and downstream promoter fragments were amplified and inserted into the multiple cloning site of the shuttle plasmid pBRR1MCS-2 to construct the gene complementation vector DH5α / pBRR1MCS-2. PltC And switch to Δ PltC strain, successfully obtained PltC The complement strain C-Δ of the deletion mutant PltC (See Figure 9 (B), specifically as follows: 3.1 Construction of the replenishment carrier (1) Download the target gene from the NCBI database ( PltB Primers were designed using SnapGene software based on the gene and its upstream and downstream 200 bp DNA sequences. PltB -com-F and PltB -com-R. Using the ZF509 genome as a template, PltB -com-F and PltB Using -com-R as primers, the target fragment was amplified by high-fidelity enzyme PCR, followed by electrophoresis, gel extraction, and recovery. PltB Amplified fragments of gene complementation.

[0091] Download the target gene from the NCBI database. PltC Primers were designed using SnapGene software based on the gene and its upstream and downstream 200 bp DNA sequences. PltC-com-F and PltC -com-R. Using the ZF509 genome as a template, PltC -com-F and PltC Using -com-R as primers, the target fragment was amplified by high-fidelity enzyme PCR, followed by electrophoresis, gel extraction, and recovery. PltC Amplified fragments of gene complementation.

[0092] (2) Plasmid pBBR1MCS-2 and the complemented amplification fragment ( PltB Gene complement fragments and PltC The gene complement fragment was digested with the same restriction endonuclease (XbaI and Hind III, the double digestion system is shown in Table 5), purified, and ligated (the ligation system is shown in Table 6) to obtain the fragment containing the target gene. PltB The plasmid vector pBBR1MCS-2- fragment PltB and carrying the target gene PltC The plasmid vector pBBR1MCS-2- fragment PltC .

[0093] Table 6 Connection System

[0094] Using heat shock, the target gene is... PltB The plasmid vector pBBR1MCS-2- fragment PltB Transformed into E. coli DH5α, it will carry the target gene PltC The plasmid vector pBBR1MCS-2- fragment PltC Transformed into Escherichia coli DH5α. Transformants grown on plates were identified by PCR using the universal M13 primer pair (consisting of M13-F and M13-R).

[0095] The target gene is included in the sequence that has the correct band size, is correctly sequenced, and has no mutations. PltB Fragment transformants are the constructed strains containing the complementation vector, named DH5α / pBBR1MCS-2- PltB The replenishment carrier is called pBBR1MCS-2- PltB The target gene is identified by the correct band size, accurate sequencing, and absence of mutations. PltC Fragment transformants are the constructed strains containing the complementation vector, named DH5α / pBBR1MCS-2- PltC The replenishment carrier is called pBBR1MCS-2- PltC .

[0096] 3.2 Construction of complement strains (1) Referring to the bacterial conjugation method in section 2.2 above, the complement vector pBBR1MCS-2-PltB Transplanting knockout strain Δ PltB In the process, the replenishment vector pBBR1MCS-2- will be used. PltC Transplanting knockout strain Δ PltC The bacteria were plated on plates containing 50 μg / mL kanamycin and 100 μg / mL ampicillin. Single colonies were detected by colony PCR using universal primers M13-F and M13-R. Transformants with the correct band size (see...) Figure 9 (2) Draw lines on the double-antibody plate two to three times consecutively.

[0097] (3) Identify the correct transformants by band size (see...) Figure 9 C-Δ pltB A 7.78 kb band was amplified, C-Δ pltC A 5.89 kb band was amplified (consistent with expectations). The sample was sent for sequencing. Recombinants with correct sequencing results were the successfully constructed complement strains. pltB The replacement strain was named C-Δ PltB , will sequence correctly pltC The replacement strain was named C-Δ PltC The bacteria were preserved in glycerol at a final concentration of 20% at -80°C.

[0098] 4. Determination of growth curves of *Pseudomonas ZF509* and its derivative strains To verify pltB , pltC Does gene deletion correction affect the growth of *Pseudomonas ZF509*? This was investigated on the effects of Δ... pltB and Δ pltC Deletion mutant and complement strain C-Δ pltB and C-Δ pltC To determine growth capacity.

[0099] Take 100 μL of bacterial culture (specifically, wild-type ZF509) from a glycerol tube stored at -80 ℃. PltB Gene knockout strain Δ PltB and the replenishment strain C-Δ PltB , PltC Gene knockout strain Δ PltC and the replenishment strain C-Δ PltC The culture was spread onto KB agar plates and incubated overnight at 28 °C. A single colony was picked and inoculated into a test tube containing 5 mL of KB liquid medium, and cultured at 28 °C with shaking at 200 rpm for 12 h to obtain the seed culture. The seed culture was then transferred at a 2% (v / v) inoculation rate to a 250 mL shake flask containing 30 mL of KB medium and incubated at 28 °C. Odionation (OD) was measured every 4 h. 600 .

[0100] Wild-type strain ZF509, knockout strain Δ PltB and the replacement strain C-Δ PltB Δ Knockout PltC and the replacement strain C-Δ PltC The growth curve is as follows Figure 10 As shown, throughout the entire growth cycle, the replenished strain C-Δ pltB The growth rate of this strain was slightly lower than that of the other four strains, but all five strains reached their maximum value at 20 hours, and their growth curves largely overlapped after entering the stationary phase. This indicates that the gene... pltB , pltC The absence / replenishment of ZF509 cells has no effect on their growth.

[0101] 5. Biofilm assay Wild-type strain ZF509 and knockout strain Δ PltB and the replacement strain C-Δ PltB Δ Knockout PltC and the replacement strain C-Δ PltC OD strains 600 20 µL of a 1.0 μL culture medium was transferred to a small glass vial containing 2 mL of KB liquid medium. After incubation at 28°C for 48 h, the culture medium was discarded, and the vials were rinsed thoroughly with sterile water. 2.2 mL of 0.01% crystal violet staining solution was added, and after 15 min, excess dye was rinsed off with sterile water and the vials were air-dried. Each vial was then thoroughly washed with 2 mL of 95% ethanol to remove any remaining purple substance from the inner wall. A blank vial was used as a negative control. OD was then measured. 595 Record the absorbance values, and repeat the experiment three times.

[0102] Biofilm formation assays showed that, compared to wild-type ZF509, the mutant Δ PltB and Δ PltC Biofilm formation ability was significantly reduced in all strains, but recovered in the replenished strains, indicating that the genes... pltB and pltC It can affect the biofilm formation of ZF509 (see Figure 11 ).

[0103] 6. Swimming ability test For analysis pltB Genes and pltC The versatility of the gene in defending against Pseudomonas ZF509 was further investigated. pltB Genes and pltC The influence of genes on bacterial motility. The swarming experiment is as follows: Each strain was cultured to OD using KB liquid medium. 600 =1.0, take 5 µL of wild-type strain ZF509 and mutant Δ respectively. pltB Δ pltCand the replenishment strain C-Δ pltB C-Δ pltC The bacterial suspension was dropped onto a plate for testing motility (using motility testing medium to prepare the plate), and incubated at 28°C for 10 h. The migration diameter of different strains was measured using the cross-cross method. The experiment was repeated 3 times.

[0104] Mobility and gregariousness enable bacteria to seek advantages and avoid disadvantages in environmental competition, and are important traits affecting the colonization and biocontrol ability of biocontrol mycorrhizal fungi. The swimming ability of ZF509 and its derivatives was detected in KB medium containing 0.2% agar. Figure 12 (A and 12B), the results show that, pltB , pltC Gene deletion and restoration had no effect on the motility of ZF509. The swimming ability of ZF509 and its derived strains was tested in KB medium containing 0.7% agar. Figure 12 (A and C of 12), the results show that Δ pltB The deletion mutant migrates faster, while the complemented strain C-Δ pltB The swimming behavior was not significantly different from that of the wild-type strain ZF509; Δ pltC and C-Δ pltC The swimming motion of the strain was significantly faster than that of the wild-type strain, Δ pltC The deletion mutant strain migrated the fastest. (Explanation) pltB Gene deletion promotes the swarming ability of ZF509 strain.

[0105] Example 2 pltB , pltC Effects on the biocontrol efficacy of ZF509 strain To investigate the key genes involved in chlorophyll synthesis pltB and pltC To investigate whether the biocontrol effect of strain ZF509 was affected, this application used a system of melon fruit spot disease, tomato bacterial canker, and cabbage black rot to test the biocontrol effect of strain ZF509 and its derived mutant strains.

[0106] 1. Determination of the protective efficacy of Pseudomonas ZF509 and its derivatives against melon fruit spot disease in potted plants Configure the following processing group: Pathogen control group: Inoculated with pathogenic bacterial suspension, and replaced with water to replace wild-type ZF509 or its derivatives.

[0107] Group ZF509: Inoculated with pathogenic bacterial solution, using wild-type ZF509 for control.

[0108] Δ pltB Group: Inoculated with pathogenic bacterial suspension, using mutant Δ pltB Prevention and control measures should be implemented.

[0109] C-Δ pltB Group: Inoculated with pathogenic bacterial suspension to replenish strain C-Δ pltB Prevention and control measures should be implemented.

[0110] Δ pltC Group: Inoculated with pathogenic bacterial suspension, using mutant Δ pltC Prevention and control measures should be implemented.

[0111] C-Δ pltC Group: Inoculated with pathogenic bacterial suspension to replenish strain C-Δ pltC Prevention and control measures should be implemented.

[0112] Healthy control group: The pathogenic bacterial solution was replaced with clean water, and wild-type ZF509 or its derivatives were replaced with clean water.

[0113] Inoculation method for pathogens: Inoculate the melon fruit spot disease pathogen, *Acidophilus hygrophila* (watermelon acidophilus). Acidovorax citrulli ) bacterial suspension (1.0×10 9 Spray the solution evenly (CFU / mL) onto both sides of the leaves of melon seedlings with 3-4 true leaves (variety name: Selected Yangjiaomi, purchased from Zhongshu Seed Industry Technology (Beijing) Co., Ltd., Agricultural Crop Seed Production and Operation License No.: D (Jinghai) Agricultural Seed Permit No. (2017) 0010). Ensure that the leaves are covered with the bacterial solution and about to drip. After inoculating once and letting it dry slightly, spray again. Each seedling should be inoculated with about 5mL of bacterial solution each time, for a total of three sprays.

[0114] Inoculation of test strains: ZF509 and its derivative strains in bacterial suspension (concentration approximately 1.0 × 10⁻⁶). 8 Dilute 100 times (CFU / mL) and spray to ensure that the bacterial solution adheres to both sides of the leaves. Inoculate an average of 5 mL per seedling.

[0115] Each treatment was first inoculated with the test strain, followed by inoculation with pathogenic bacterial solution after 48 hours, and then inoculated with the test strain again after another 48 hours. Each treatment had 3 replicates, with 15 seedlings in each replicate.

[0116] When water-soaked lesions appeared on the surface of half of the experimental seedlings in the pathogen control group, an investigation was conducted, followed by a second investigation 48 hours later.

[0117] Greenhouse pot experiment results showed that after inoculation with *Acidophilus hygrophilus*, the control effect of the ZF509 group (wild-type strain ZF509) was 71.43%. Compared with the wild-type strain, Δ pltB and Δ pltC The mutants showed control efficacy of only 36.94% and 40.88% against melon fruit spot disease, a significant difference, while the complement strain C-Δ... pltB and C-Δ pltC After treatment, the control efficacy against watermelon acidophilus reached 68.47% and 66.50% (see...).Figure 13 and Figure 14 ).

[0118] 2 Determination of the Pot Control Effect of Pseudomonas syringae pv. tomato DC3000 and Its Derivative Strains against Tomato Bacterial Canker The following treatment groups were set up: Pathogen control group: Inoculated with pathogen bacterial solution, and wild-type ZF509 or its derivative bacteria were replaced with clear water.

[0119] ZF509 group: Inoculated with pathogen bacterial solution, and wild-type ZF509 was used for control.

[0120] Δ pltB group: Inoculated with pathogen bacterial solution, and the mutant Δ pltB was used for control.

[0121] C-Δ pltB group: Inoculated with pathogen bacterial solution, and the complemented strain C-Δ pltB was used for control.

[0122] Δ pltC group: Inoculated with pathogen bacterial solution, and the mutant Δ pltC was used for control.

[0123] C-Δ pltC group: Inoculated with pathogen bacterial solution, and the complemented strain C-Δ pltC was used for control.

[0124] Health control group: The pathogen bacterial solution was replaced with clear water, and wild-type ZF509 or its derivative bacteria were replaced with clear water.

[0125] Pathogen inoculation method: The bacterial suspension (1.0×10 Clavibacter michiganensis subsp. michiganensis ) of Clavibacter michiganensis subsp. michiganensis, the pathogen of tomato bacterial canker, was evenly and suspendedly sprayed onto the front and back sides of the leaves of tomato seedlings (variety name: Zhongza 201, purchased from Zhongshu Seed Industry Technology (Beijing) Co., Ltd., crop seed business license: C (Beijing) Nongzhongjingxuzi (2017) No. 0010) at the stage of 3-4 true leaves, ensuring that the leaves were in a state where the bacterial solution was hanging and about to drip. After inoculation once and slightly drying, it was sprayed again. Each seedling was inoculated with about 5 mL of bacterial solution each time, and a total of three sprays were made. 9 CFU / mL) was evenly and suspendedly sprayed onto the front and back sides of the leaves of tomato seedlings (variety name: Zhongza 201, purchased from Zhongshu Seed Industry Technology (Beijing) Co., Ltd., crop seed business license: C (Beijing) Nongzhongjingxuzi (2017) No. 0010) at the stage of 3-4 true leaves, ensuring that the leaves were in a state where the bacterial solution was hanging and about to drip. After inoculation once and slightly drying, it was sprayed again. Each seedling was inoculated with about 5 mL of bacterial solution each time, and a total of three sprays were made.

[0126] Inoculation of the test strains: The bacterial solutions of ZF509 and its derivative strains (bacterial solution concentration was about 1.0×10 8 CFU / mL) were diluted 100 times and treated by spraying to ensure that the bacterial solution adhered to the front and back sides of the leaves, and 5 mL was inoculated per plant on average.

[0127] Each treatment was first inoculated with the test strain, followed by inoculation with pathogenic bacterial solution after 48 hours, and then inoculated with the test strain again after another 48 hours. Each treatment had 3 replicates, with 15 seedlings in each replicate.

[0128] When water-soaked lesions appeared on the surface of half of the experimental seedlings in the pathogen control group, an investigation was conducted, followed by a second investigation 48 hours later.

[0129] Greenhouse pot experiment results showed that after inoculation with *Corynebacterium micranthum* subsp. *micranthum*, the pathogen of tomato bacterial canker, Δ pltB and Δ pltC The mutant strain showed control efficacy against tomato bacterial canker of only 40.94% and 45.88%, significantly different from the wild-type strain (75.43%). The control efficacy against tomato bacterial canker was restored by the replacement strain (see...). Figure 15 and Figure 16 ).

[0130] 3. Determination of the protective efficacy of Pseudomonas ZF509 and its derivatives against black rot in potted broccoli Configure the following processing group: Pathogen control group: Inoculated with pathogenic bacterial suspension, and replaced with water to replace wild-type ZF509 or its derivatives.

[0131] Group ZF509: Inoculated with pathogenic bacterial solution, using wild-type ZF509 for control.

[0132] Δ pltB Group: Inoculated with pathogenic bacterial suspension, using mutant Δ pltB Prevention and control measures should be implemented.

[0133] C-Δ pltB Group: Inoculated with pathogenic bacterial suspension to replenish strain C-Δ pltB Prevention and control measures should be implemented.

[0134] Δ pltC Group: Inoculated with pathogenic bacterial suspension, using mutant Δ pltC Prevention and control measures should be implemented.

[0135] C-Δ pltC Group: Inoculated with pathogenic bacterial suspension to replenish strain C-Δ pltC Prevention and control measures should be implemented.

[0136] Healthy control group: The pathogenic bacterial solution was replaced with clean water, and wild-type ZF509 or its derivatives were replaced with clean water.

[0137] Inoculation method for pathogens: Inoculate with the pathogens causing black rot of broccoli, Xanthomonas oryzae, and the pathogenic strain of Xanthomonas oryzae (… Xanthomonas campestris pv. campestris ) bacterial suspension (1.0×10 9Evenly spray the bacterial solution (CFU / mL) onto both sides of the leaves of broccoli seedlings (variety name: Zhongqing 16, purchased from Zhongshu Seed Industry Technology (Beijing) Co., Ltd., Agricultural Crop Seed Production and Operation License No.: D (Jinghai) Nongzhongxuzi (2017) No. 0010) at the 3 true leaf stage, ensuring that the leaves are covered with bacterial solution and about to drip. After inoculating once and letting it dry slightly, spray again. Each seedling is inoculated with about 5mL of bacterial solution each time, for a total of three sprays.

[0138] Inoculation of test strains: ZF509 and its derivative strains in bacterial suspension (concentration approximately 1.0 × 10⁻⁶). 8 Dilute 100 times (CFU / mL) and spray to ensure that the bacterial solution adheres to both sides of the leaves. Inoculate an average of 5 mL per seedling.

[0139] Each treatment was first inoculated with the test strain, followed by inoculation with pathogenic bacterial solution after 48 hours, and then inoculated with the test strain again after another 48 hours. Each treatment had 3 replicates, with 15 seedlings in each replicate.

[0140] When water-soaked lesions appeared on the surface of half of the experimental seedlings in the pathogen control group, an investigation was conducted, followed by a second investigation 48 hours later.

[0141] The results of the greenhouse pot experiment showed that after inoculation with black rot fungus, Δ pltB and Δ pltC The control efficacy against black rot in broccoli was significantly reduced, with control efficacy of only 33.12% and 37.76%, significantly lower than that against the wild-type strain ZF509; while the supplementary strain C-Δ pltB and C-Δ pltC After treatment, the symptoms of black rot in broccoli were reduced, with control efficacy of 55.26% and 53.76% (see...). Figure 17 and Figure 18 ).

[0142] The above results indicate that, under greenhouse conditions, key genes involved in gromwellin synthesis... pltB and pltC The deletion of this gene affects the biocontrol effect of strain ZF509, indicating that the key gene for chlorophyll synthesis is missing. pltB and pltC It is the main biocontrol factor for bacterial diseases controlled by strain ZF509.

[0143] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The use of a protein, or a substance that promotes the expression of the protein-coding gene, or a substance that increases the activity or content of the protein, characterized in that: The application is any one of the following applications P1-P13: P1. Application in the biological control of bacterial plant diseases; P2. Application in the prevention and control of melon fruit spot disease; the pathogen of melon fruit spot disease is *Acidophilus hygrophila* (watermelon). Acidovorax citrulli ); P3, In reducing the acidophilic bacteria in watermelon ( Acidovorax citrulli Applications in pathogenicity; P4, In reducing watermelon acidophilus ( Acidovorax citrulli Application in the area of ​​lesions caused by ) P5, Inhibiting watermelon acidophilus ( Acidovorax citrulli Applications in growth and development; P6. Application in the prevention and control of tomato bacterial canker; the pathogen of tomato bacterial canker is *Corynebacterium miltanum* subsp. *miltanum* (… Clavibacter michiganensis subsp. michiganensis ); P7, In reducing the number of Corynebacterium micranthae subsp. micranthae ( Clavibacter michiganensis subsp. Michigan nsis Applications in pathogenicity; P8. In reducing the number of Corynebacterium micranthae subspecies ( Clavibacter michiganensis subsp. Michigan nsis Application in the area of ​​lesions; P9, Inhibition of Corynebacterium micranthae subsp. micranthae ( Clavibacter michiganensis subsp. Michigan nsis Applications in growth and development; P10. Application in the prevention and control of black rot in broccoli; the pathogen causing black rot in broccoli is Xanthomonas spp. (Xanthomonas spp.) Xanthomonas campestris pv. campestris ); P11, In reducing the pathogenicity of Xanthomonas vegetans in rapeseed (a species of rapeseed) Xanthomonas campestris pv. campestris Applications in pathogenicity; P12, In reducing the pathogenic species of Xanthomonas spp. in rapeseed ( Xanthomonas campestris pv. campestris Application in the area of ​​lesions; P13, Inhibiting the pathogenicity of Xanthomonas laurentii in *Rhizopus spp.* (a strain of *Rhizopus spp.*) Xanthomonas campestris pv. campestris Applications in growth and development; The protein in question is a protein of type A1, A2, or A3: A1. The amino acid sequence is the protein that is the amino acid sequence shown in SEQ ID No. 2 or SEQ ID No. 4 in the sequence listing; A2. A protein that has more than 80% identity with the protein shown in A1 and is related to biocontrol function, obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 2 or SEQ ID No. 4 in the sequence listing. A3. A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1 or A2.

2. The application according to claim 1, characterized in that: The substance that promotes the expression of the protein-coding gene or increases the activity or content of the protein is a biological material related to the protein of claim 1, wherein the biological material is any one of B1 to B4 below: B1. The nucleic acid molecule encoding the protein; B2, an expression cassette containing the nucleic acid molecule described in B1; B3, a recombinant vector containing the nucleic acid molecule described in B1, or a recombinant vector containing the expression cassette described in B2; B4. Recombinant microorganisms containing the nucleic acid molecules described in B1, or recombinant microorganisms containing the expression cassette described in B2, or recombinant microorganisms containing the recombinant vector described in B3.

3. The application according to claim 2, characterized in that: The nucleic acid molecule mentioned in B1 may specifically be a DNA molecule whose coding sequence is SEQ ID No. 1 in the sequence listing or a DNA molecule whose coding sequence is SEQ ID No. 3 in the sequence listing.

4. The protein as described in claim 1.

5. The biomaterial as described in claim 2 or 3.

6. A method for enhancing the biocontrol efficacy of Pseudomonas bacteria against bacterial diseases of plants, characterized in that: The method includes the step of increasing the expression level of the protein or its encoding gene as described in claim 1 in the recipient Pseudomonas bacteria to obtain the target Pseudomonas bacteria; the target Pseudomonas bacteria have a stronger control effect on plant bacterial diseases than the recipient Pseudomonas bacteria have on plant bacterial diseases.

7. The method according to claim 6, characterized in that: The plant disease mentioned is any one of the following: melon fruit spot disease, tomato canker, and broccoli black rot.

8. The method according to claim 7, characterized in that: The pathogen causing the melon fruit spot disease is *Acidophilus hygrophila* (watermelon acidophilus). Acidovorax citrulli The pathogen causing tomato canker is *Corynebacterium miltanum* subsp. *miltanum* (…). Clavibacter michiganensis subsp. Michiganensis The pathogen causing the black rot of broccoli is *Xanthomonas spp.*, a pathogenic strain of *Xanthomonas spp.*. Xanthomonas campestris pv. campestris ).

9. The method according to any one of claims 6-8, characterized in that: The receptor Pseudomonas bacteria are defensive Pseudomonas ( Pseudomonas protegens ).

10. The method according to claim 9, characterized in that: The genome of the receptor Pseudomonas bacteria does not contain a gene encoding the protein of claim 1.

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