Burkholderia gladioli and application of burkholderia gladioli in prevention and treatment of grape diseases

Abstract

The invention discloses burkholderia gladioli and application of the burkholderia gladioli in prevention and treatment of grape diseases, the classification name of the burkholderia gladioli is burkholderia gladioli, the burkholderia gladioli is preserved in China General Microbiological Culture Collection Center on April 1, 2024, the preservation number is CGMCC No.30228, and the address of the preservation unit is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The strain is wide in antibacterial spectrum, and has good antibacterial activity on common pathogenic bacteria in grape planting, especially on grape white rot and grape anthracnose. According to the research, through directional screening of grape rhizosphere soil microorganisms, biocontrol bacteria capable of efficiently antagonizing grape white rot bacteria are pre-obtained, and a basic material is provided for biological prevention and control of grape white rot.

Description

Technical Field

[0001] The invention belongs to the field of microbial strains, and particularly relates to Burkholderia gladiolus and application thereof in preventing and controlling grape diseases. Background Art

[0002] The information disclosed in this background technology section is only intended to increase some understanding of the overall background of the invention, and should not be necessarily regarded as an admission or any form of suggestion that the information constitutes the prior art already known to ordinary technicians in this field.

[0003] The genus Burkholderia is a Gram-negative, non-fermenting bacterium belonging to the phylum Proteobacteria. They are widely found in soil, water and a variety of hosts. In 1992, the genus was separated from the genus Pseudomonas. The interaction between Burkholderia and the host is complex and can be both pathogenic and beneficial. For example, the Burkholderia cepacia complex includes pathogenic and non-pathogenic bacteria, and melioidosis and glanders are also caused by it. Some Burkholderia species are plant or human pathogens, but many species have the functions of biological control, plant growth promotion and decomposition of toxic substances. Bacteria of this genus can fix nitrogen to promote plant growth, inhibit pests and degrade environmental pollutants. In addition, they have great potential in disease suppression and synthesis of antimicrobial substances, and can produce a variety of secondary metabolites with antifungal, antibacterial, herbicidal and insecticidal activities. Plant growth-promoting rhizobacteria (PGPR) are a type of bacteria that can promote plant growth. After colonizing the roots of plants, they can inhibit the invasion of harmful pathogens by promoting the absorption of plant nutrients, inducing the production of root surface hormones, or secreting antibacterial substances, thereby directly or indirectly affecting plant growth and development. The growth-promoting mechanisms of PGPR mainly include nitrogen fixation, phosphorus solubilization, potassium solubilization, secretion of siderophores, secretion of plant hormones and related regulatory substances, and release of volatile substances. The study of rhizosphere growth-promoting bacteria to enhance plant disease resistance has always been a hot topic of research. Growth-promoting strains that can enhance plant disease resistance have been isolated from the genus Bacillus. Studies have shown that inoculation of stress-tolerant PGPR strains under drought conditions can induce the production of plant hormones, promote the growth and development of lateral roots, and enhance the absorption and utilization rate of nutrients and water by plants.

[0004] So far, there are no reports on the application of Burkholderia gladiolus in preventing and controlling grape-related diseases. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a gladiolus Burkholderia and application thereof in preventing and controlling grape diseases.

[0006] The technical solution adopted by the present invention is as follows:

[0007] In the first aspect of the present invention, a gladiolus Burkholderia ZBSF BH07 is provided. The bacterium is classified and named as gladiolus Burkholderia (Burkholderia gladioli), which was deposited in the China General Microbiological Culture Collection Center on April 1, 2024, with a deposit number of: CGMCC No.30228, and the address of the deposit unit is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] In a second aspect of the present invention, a microbial mixture is provided, characterized in that it contains at least the strain of the present invention - Burkholderia gladiolus ZBSF BH07, and the microbial mixture may contain other strains that promote plant growth or prevent and control plant diseases and microorganisms that are symbiotic with the strain of the present invention.

[0009] In the third aspect of the present invention, a product comprising the strain of the present invention - Burkholderia gladioli ZBSF BH07 or the microbial mixture of the present invention is provided. The product may be a bacterial agent, a bacterial fertilizer or a composition.

[0010] The bacterial agent, bacterial fertilizer or composition contains the aforementioned Burkholderia gladioli ZBSF BH07 and / or contains a culture of the aforementioned Burkholderia gladioli ZBSF BH07.

[0011] The microbial agent may be a phosphorus-degrading microbial agent, a plant growth promoter, a plant pathogen inhibitor and / or a plant white rot control agent.

[0012] The term "culture" refers to a liquid or solid product (all materials in a culture container) containing a microbial community after artificial inoculation and culture. That is, the product obtained by growing and / or amplifying microorganisms, which may be a biologically pure culture of microorganisms, or may contain a certain amount of culture medium, metabolites or other components produced during the culture process.

[0013] In the present invention, the culture is a substance obtained by culturing the Burkholderia gladioli ZBSF BH07 in a microbial culture medium (all substances in a culture container).

[0014] In a fourth aspect of the present invention, a fermentation liquid, a fermentation product, a metabolite, etc. containing the Burkholderia gladioli ZBSF BH07 is provided.

[0015] In a fifth aspect of the present invention, a fermentation method of the gladiolus Burkholderia ZBSF BH07 is provided, which comprises inoculating the gladiolus Burkholderia ZBSF BH07 into a fermentation medium for fermentation culture.

[0016] In a sixth aspect of the present invention, any one of the following applications A1) to A8) is provided:

[0017] A1) Use of the above-mentioned Burkholderia gladiolus ZBSF BH07 in the preparation of a product for promoting plant growth;

[0018] A2) Use of the aforementioned Burkholderia gladioli ZBSF BH07 or the aforementioned microbial mixture or the aforementioned product in promoting plant growth;

[0019] A3) Use of the above-mentioned Burkholderia gladiolus ZBSF BH07 in the preparation of plant pathogen inhibitors;

[0020] A4) Use of the aforementioned Burkholderia gladioli ZBSF BH07 or the aforementioned microbial mixture or the aforementioned product in inhibiting plant pathogens;

[0021] A5) Use of the above-mentioned Burkholderia gladiolus ZBSF BH07 in the preparation of a product for preventing and / or treating plant white rot;

[0022] A6) Use of the aforementioned Burkholderia gladioli ZBSF BH07 or the aforementioned microbial mixture or the aforementioned product in preventing and / or treating plant white rot;

[0023] A7) Use of the aforementioned Burkholderia gladioli ZBSF BH07 or the aforementioned microbial mixture or the aforementioned product in degrading phosphorus;

[0024] A8) Use of the aforementioned Burkholderia gladioli ZBSF BH07 or the aforementioned microbial mixture or the aforementioned product in the preparation of siderophores.

[0025] Furthermore, in the above application, the plant is grape.

[0026] Furthermore, in the above application, the promoting plant growth may be promoting the vegetative growth of the plant.

[0027] Specifically, it can be to promote the growth of the aboveground part of the plant, promote the increase of the fresh weight of the plant and / or promote the increase of the dry weight of the plant.

[0028] Further, in the above application, the pathogen in A3) or A4) is a fungus;

[0029] The fungus is at least one of grape white rot pathogen - Coniella vitis, grape anthracnose pathogen - Colletotrichum aenigma, Fusarium oxysporum, grape pestalotiopsis clavispora, grape vine blight pathogen - Diaporthe eres, grape spike brown blight pathogen - Alternaria viticola, grape canker pathogen - Botryosphaeria dothidea, and grape gray mold pathogen - Botrytis cinerea.

[0030] In a seventh aspect of the present invention, a method for promoting plant growth is provided, the method comprising culturing the aforementioned Burkholderia gladioli in a microbial culture medium, collecting the culture, and treating plants with the culture.

[0031] In an eighth aspect of the present invention, a method for preventing and / or treating plant diseases is provided, the method comprising culturing the aforementioned Burkholderia gladiolus in a microbial culture medium, collecting the culture, and treating plants with the culture.

[0032] The plant disease is a fungus;

[0033] Further, the fungus can be at least one of grape white rot pathogen - Coniella vitis, grape anthracnose pathogen - Colletotrichum aenigma, Fusarium oxysporum, grape branch blight pathogen - Pestalotiopsis clavispora, grape vine blight pathogen - Diaporthe eres, grape canker pathogen - Botryosphaeria dothidea, grape gray mold pathogen - Botrytis cinerea, grape spike brown blight pathogen - Alternaria viticola, Fusarium graminearum, and Fusariumpseudograminearum.

[0034] More specifically, the plant diseases may be plant white rot, grape anthracnose, grape gray mold, grape cob brown blight and grape vine blight, and the pathogens may be grape white rot fungi - Coniella vitis, grape anthracnose fungi (Colletotrichum aenigma), grape gray mold fungi (Botrytis cinerea), grape cob brown blight fungi (Alternaria viticola), grape vine blight fungi (Diaporthe eres), and Fusarium oxysporum.

[0035] In the present invention, the microbial culture medium may be a solid culture medium or a liquid culture medium.

[0036] The solid culture medium of the present invention can be prepared by adding 15 g / L agar powder to a liquid culture medium.

[0037] In the present invention, the treatment of plants with the culture may specifically be root irrigation of plants with the fermentation liquid of Burkholderia gladioli, or contacting the surface of plants with the fermentation liquid of Burkholderia gladioli.

[0038] In the present invention, the method of treating pathogens with culture may specifically include spraying plants with fermentation liquid of Burkholderia gladioli before pathogens contact plants, or contacting plant surfaces with fermentation liquid of Burkholderia gladioli before / after pathogens contact plants.

[0039] Compared with the related art known to the inventor, one of the technical solutions of the present invention has the following beneficial effects:

[0040] The present invention isolates and screens a Burkholderia strain ZBSF BH07 with good antibacterial effect on grape white rot fungi from the rhizosphere soil of a grape plantation. The strain has a broad antibacterial spectrum and has good antibacterial activity against common pathogens in grape cultivation, especially grape white rot and grape anthracnose. This study obtains biocontrol bacteria with high efficiency antagonizing grape white rot fungi through targeted screening of grape rhizosphere soil microorganisms, providing basic materials for biological control of grape white rot. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute improper limitations on the present invention.

[0042] Figure 1 This is the colony morphology of strain ZBSF BH07.

[0043] Figure 2 This is the phylogenetic tree of strain ZBSF BH07 based on 16s rDNA sequence.

[0044] Figure 3 Growth rate and pH change of strain ZBSF BH07.

[0045] Figure 4 This is the inhibitory effect of strain ZBSF BH07 on different pathogens.

[0046] Figure 5 is the protease production ability of strain ZBSF BH07.

[0047] Figure 6 These are the siderophagic zone and phosphate-solubilizing zone of strain ZBSF BH07 on CAS and NBRIP medium, respectively.

[0048] Figure 7 This is the growth-promoting effect of strain ZBSF BH07 on grapes.

[0049] Figure 8 To test the effectiveness of strain ZBSF BH07 in controlling grape white rot. DETAILED DESCRIPTION

[0050] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0051] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations and / or combinations thereof.

[0052] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0053] The test culture medium in the following examples is as follows:

[0054] Beef extract peptone solid medium (NA) medium: 3g beef extract, 10g peptone, 5g NaCl, 15g agar, dilute to 1L with deionized water, pH 7.0-7.2.

[0055] Beef extract peptone liquid (NB) medium: 3g beef extract, 10g peptone, 5g NaCl, 1L distilled water.

[0056] Tryptone liquid medium (LB): 5g yeast powder, 10g tryptone, 10g NaCl, 1000mL distilled water. Tryptone solid medium (LB): 5g yeast powder, 10g tryptone, 10g NaCl, 15g agar, dilute to 1L with deionized water.

[0057] Potato dextrose medium (PDA): 200 g potatoes, 20 g glucose, 15 g agar, dilute to 1 L with deionized water.

[0058] NBRIP solid culture medium: glucose 10.0 g, Ca3(PO4)2 5.0 g, (NH4)2SO4 0.5 g, NaCl 0.2 g, KCl 0.2 g, MgSO4·7H2O 0.1 g, FeSO4·7H2O 0.03 g, MnSO4·4H2O 0.03 g, yeast powder 0.5 g, dilute to 1000 mL with deionized water, pH 6.8-7.2.

[0059] Protease detection medium: 2% skim milk powder, 2% agar powder, dilute to 1L with deionized water.

[0060] The above culture media were sterilized at 121°C for 20 min.

[0061] Example 1. Isolation, purification and identification of strain ZBSF BH07

[0062] 1.1. Isolation of strain ZBSF BH07

[0063] The strain was isolated from the rhizosphere soil of the grape plant. 10 g of soil was weighed and added to a conical flask containing 90 mL of sterile saline. The soil was shaken at 180 r / min for 1 hour, then placed in a water bath at 80°C for 20 minutes, and the strain was isolated by the dilution plate method. The strain was cultured at 28°C for 48 hours. After the colonies grew, a single colony was picked and streaked on NA solid culture medium for purification to obtain the strain ZBSF BH07. The strain was cultured at 28°C for 48 hours and stored in a refrigerator at 4°C for later use.

[0064] 1.2. Identification of strain ZBSF BH07

[0065] The strain ZBSF BH07 screened above was identified as follows:

[0066] 1) Morphological observation and physiological characteristics of strain ZBSF BH07

[0067] The isolated strain was streaked into LB medium and cultured at 28°C for 48 hours to observe the colony morphology of the strain. The physiological and biochemical characteristics were identified according to the Bergey's Manual of Bacterial Identification and the Manual of Common Bacterial Systematic Identification.

[0068] The strain ZBSF BH07 was cultured on LB medium for 4 days. The colonies were round, off-white, with neat edges, smooth and moist surfaces. Under the microscope, the bacteria were short rod-shaped and Gram-negative. No hemolysis zone was produced in the blood plate, indicating that the strain GSBZ09 was non-pathogenic and safe for the human body.

[0069] The culture morphology of the strains Figure 1 As shown, Figure 1 The first picture from the left in the middle shows the culture morphology of strain ZBSF BH07 on LB medium. Figure 1 The second picture from the left in the middle shows the culture morphology of strain ZBSF BH07 on blood plate (purchased from Beekman Biotechnology Co., Ltd.) culture medium.

[0070] 1.3 Molecular identification

[0071] DNA was extracted using the Omega genomic DNA miniprep kit, and the 16S rRNA sequence of the pathogen was amplified using primers 27F and 1492R. The PCR reaction conditions were: 95°C pre-denaturation for 10 min; 95°C denaturation for 30 s, 55°C renaturation for 30 s, 72°C extension for 45 s, 34 cycles; and finally 72°C extension for 8 min. The PCR product was detected by 1% agarose gel electrophoresis, and the product molecular weight was detected using DNA Marker BM5000 as a control. A 1387 bp 16S rDNA target band was obtained. After sequencing, the nucleotide sequence of 16S rDNA was SEQ ID No. 1, and the specific sequence was:

[0072] AGGGGAATGCCTTACCATGCAAGTCGAACGGCAGCACGGGTGCTTGCACCTGGTGGCGAGTGGCGA

[0073] ACGGTGAGTAATACATCGGAACATGTCCTGTAGTGGGGGATAGCCCGGCGAAAGCCGGATTAATA

[0074] CCGCATACGATCTACGGATGAAAGCGGGGGACCTTCGGGCCTCGCGCTATAGGGTTGGCCGATGGC

[0075] TGATTAGCTAGTTGGTGGGGTAAAGGCCCACCAAGGCGACGATCAGTAGCTGGTCTGAGAGGACGA

[0076] CCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGAC

[0077] AATGGGCGAAAGCCTGATCCAGCAATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTT

[0078] TTGTCCGGAAAGAAATCCTGAGGGCTAATATCCTTCGGGGATGACGGTACCGGAAGAATAAGCACC

[0079] GGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACTGGGCG

[0080] TAAAGCGTGCGCAGGCGGTTTGTTAAGACCGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATT

[0081] GGTGACTGGCAAGCTAGAGTATGGCAGAGGGGGGTAGAATTCCACGTGTAGCAGTGAAATGCGTAG

[0082] AGATGTGGAGGAATACCGATGGCGAAGGCAGCCCCCTGGGCCAATACTGACGCTCATGCACGAAAG

[0083] CGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTAGTTGTTGG

[0084] GGATTCATTTCCTTAGTAACGTAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGTCGCAAGAT

[0085] TAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGATTAATTCGATGCAAC

[0086] GCGAAAAACCTTACCTACCCTTGACATGGTCGGAACCTTGGAGAGATCTGAGGGTGCTCGAAAGAG

[0087] AACCGATACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCA

[0088] ACGAGCGCAACCCTTGTCCTTAGTTGCTACGCAAGAGCACTCTAGGGAGACTGCCGGTGACAAACC

[0089] GGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGGGTAGGGCTTCACACGTCATACAA

[0090] TGGTCGGAACAGAGGGTCGCCAACCCGCGAGGGGGAGCTAATCCCAGAAAACCGATCGTAGTCCGG

[0091] ATTGCACTCTGCAACTCGAGTGCATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGT

[0092] GAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTTTACCAGAAGTGGCTAGTCTAACCGCAAGGAGGACGGTCACCACCGTTTG.

[0093] The nucleotide sequences of the primers are as follows:

[0094] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0095] 1492R: 5'-CTACGGCTACCTTGTTACGA-3'.

[0096] The obtained sequence was compared with the data in the NCBI ribosomal RNA sequence database using the NCBI Blast program, and the maximum likelihood method in MEGA 6.0 software was used to construct a phylogenetic tree between strain ZBSF BH07 and other similar strains. It was found that the strain ZBSF BH07 had a 99% affinity with Burkholderia gladioli. Combined with morphological observations and physiological and biochemical characteristics, strain ZBSF BH07 was determined to be Burkholderia gladioli of the genus Burkholderia. Figure 2This is a phylogenetic tree based on 16S rDNA.

[0097] The strain ZBSF BH07 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 1, 2024, with the deposit number CGMCC No.30228 and the classification name Burkholderia gladiolus.

[0098] 1.4. Growth rate and pH value determination of Burkholderia gladiolus

[0099] The strain ZBSF BH07 was inoculated into liquid LB medium and cultured at 28°C with shaking for 52 hours. The bacterial suspension after 16 hours of culture was added to the LB medium. A total of 3 replicates were set up and cultured in a shaking incubator at 28°C. The OD and pH values ​​were measured every 4 hours, and a total of 14 points were measured. The growth curve and pH change curve of the strain ZBSF BH07 are shown in Figure 2. Figure 3 As shown, strain ZBSFBH07 was in an exponential growth phase before 12 hours, the absorbance reached 4.002 at 28 hours, and entered a stable growth phase after 28 hours. The pH value first decreased and then increased with time, reaching 6.69 at 52 hours and remained stable.

[0100] 1.5. Antibiotic determination of strain ZBSF BH07

[0101] 1) Determination of resistance to different antibiotics

[0102] Ampicillin, Vancomycin, Chloramphenicol, Gentamicin, Tetracycline, Spectinomycin, Kanamycin, Streptomycin, Rifampicin were inoculated into 5 mL of liquid LB medium at a ratio of 0.1%, and a single colony of strain ZBSF BH07 was picked, inoculated into 5 mL and added to liquid LB medium containing different antibiotics, and cultured at 28°C for 24 hours. Strain ZBSF BH07 LY7 can grow at the working concentrations of ampicillin, chloramphenicol, and vancomycin, but not at the other concentrations. LY7 can grow at twice the working concentration of ampicillin (100 mg / L), but the growth is slow, and it cannot grow at twice the working concentration of chloramphenicol.

[0103] Example 2: Functional study of strain ZBSF BH07

[0104] 2.1. Application of strain ZBSF BH07 in antibacterial activities

[0105] 2.1.1 Determination of fungal antibacterial spectrum of strain ZBSF BH07

[0106] Using the plate confrontation method, 7mm of the selected fungal cake was inoculated in the center of the 90mm PDA plate, cultured at 25℃ for 1d, and the strain ZBSF BH07 was inoculated in the liquid LB medium, cultured at 28℃ for 16h, and the ZBSF BH07 bacterial suspension was obtained. 2.5uL ZBSF BH07 bacterial suspension was inoculated at 4 points relative to the edge of the culture dish at 10mm. 2.5uL ZBSF BH07 bacterial suspension was inoculated at 4 points relative to the edge of the culture dish at 10mm, and the inoculation of liquid LB medium was used as the blank control. Cultured at 25℃ for 3d, when the blank control was about to grow all over the culture dish, the control growth (colony radius) and treatment growth (growth radius after inoculation of ZBSF BH07) of the target bacteria were measured, and expressed as the inhibition rate.

[0107] Inhibition rate (%) = (control growth - treatment growth) / control growth × 100%

[0108] The pathogenic fungi come from the following sources:

[0109] Grape white rot pathogen - Coniella vitis, grape anthracnose pathogen - Colletotrichum aenigma, Fusarium oxysporum, grape branch blight pathogen - Pestalotiopsis clavispora, grape vine blight pathogen - Diaporthe eres, grape canker pathogen - Botryosphaeria dothidea, grape gray mold pathogen - Botrytis cinerea, grape spike brown blight pathogen - Alternaria viticola, Fusarium graminearum, Fusarium pseudograminearum have been reported in the literature Yuan L, Jiang H, Jiang X, Li T, Lu P, Yin X and Wei Y (2022) Comparative genomic and functional analyses of Paenibacillus peoriae ZBSF16 with biocontrol potential against grapevine diseases, provide insights into its genes related to plant growth-promoting and biocontrol mechanisms. Front. Microbiol. 13: 975344. discloses that the public can obtain the above pathogens from Shandong Grape Research Institute or through other channels.

[0110] The results of the determination of the antibacterial spectrum of the strain ZBSF BH07 are as follows Figure 4 As shown in Table 1 below.

[0111] Table 1 Inhibitory effect of strain ZBSF BH07 on pathogenic fungi

[0112] Pathogens Antibacterial zone Grape White Rot (Coniella vitis) +++ Grape Anthracnose (Colletotrichum aenigma) +++ Grape gray mold (Botrytis cinerea) +++ Grape blight pathogen (Pestalotiopsis clavispora) ++ Grapevine cob brown blight pathogen (Alternaria viticola) +++ Diaporthe eres +++ Fusarium oxysporum +++ Grape canker pathogen (Botryosphaeria dothidea) ++ Fusarium graminearum ++ Fusarium pseudograminearum ++

[0113] Note: +++ means that the diameter of the bacterial inhibition zone is greater than or equal to 5.0 cm or the fungal inhibition rate is greater than or equal to 60%; ++ means that the diameter of the bacterial inhibition zone is greater than or equal to 4.0 cm and less than 5.0 cm or the fungal inhibition rate is greater than or equal to 40% and less than 60%.

[0114] 2.2. Determination of enzyme production capacity of strain ZBSF BH07

[0115] Protease detection: 2.5 μL of the above bacterial suspension was spotted in the protease detection medium, and the diameter of the transparent circle was observed after 3 days. Figure 5 After culturing Burkholderia gladioli ZBSF BH07 in protease medium for 3 days, the diameter of the transparent zone was 2.9 cm, indicating that the strain ZBSF BH07 had the ability to produce protease.

[0116] 2.3. Detection of growth-promoting effect of strain ZBSF BH07

[0117] 2.3.1 Qualitative detection of phosphate dissolution plate

[0118] The strain ZBSF BH07 was inoculated into a test tube containing 5 mL of liquid LB medium and cultured at 28°C and 170 r / min for 24 h to prepare 10 7 CFU / mL of bacterial suspension, spot 2.5uL of bacterial suspension on the center of NBRIP solid culture medium plate, culture for 7 days, observe the phosphate dissolution circle and measure the diameter, and repeat each treatment 3 times.

[0119] The results are as follows Figure 6 As shown, Figure 6 The second picture from the left is the result of qualitative detection of phosphorus solubilization plate. The results show that after culturing Burkholderia gladioli ZBSF BH07 in NBRIP medium for 7 days, the diameter of the phosphorus solubilization zone is 1.2 cm, indicating that Burkholderia gladioli ZBSF BH07 has the ability to degrade phosphorus.

[0120] 2.3.2 Siderophore detection

[0121] General CAS plate test, in which the CAS test solution is prepared as follows:

[0122] 6mL 10mmol·L -1 Hexadecyltrimethylammonium bromide (HDTMA) was placed in a 100 mL volumetric flask and diluted appropriately with deionized water. 1.5 mL of 1 mmol·L -1 FeCl3·6H2O and 7.5mL 2mmol·L -1 CAS solution was mixed and slowly added to the volumetric flask along the glass rod. 4.3 g of anhydrous piperazine was dissolved in water and 6.25 mL of 12 mol·L -1 Hydrochloric acid was added to obtain a buffer solution of pH 5.6, and then the solution was transferred into the aforementioned volumetric flask and made up to 100 mL with double distilled water. The solution was sterilized at 121°C for 15 min and then set aside.

[0123] Preparation of CAS test plate: Prepare 1 mol·L -1 CaCl2 solution, 1mmol·L -1MgSO4·6H2O solution and 10% acid hydrolyzed casein solution are sterilized separately at 121℃ for 15min and then used. Take 0.2mL of CaCl2 solution, 0.2mL of MgSO4·7H2O solution, and 6mL of 10% acid hydrolyzed casein solution, add biological buffer solution Pipes, and adjust the pH value to 6.8-7.0. Make up to 100mL with deionized water, add 2g of agar powder, sterilize at 121℃ for 15min, and when the temperature drops to 60℃, add 5mL of the prepared CAS blue detection solution and mix well. Be careful not to generate bubbles that will affect the plate detection experiment. Then pour 25mL into each culture dish. A positive reaction is recorded by the CAS color changing from blue to yellow or orange. Record the formation of a halo around the colony on the CAS agar plate.

[0124] The results are as follows Figure 6 As shown, Figure 6 The first figure on the left in the middle is the siderophore detection result, and the results show that after the strain ZBSF BH07 was cultured on the CAS agar plate for 7 days, a yellow halo was produced around the colony, indicating that siderophore was produced.

[0125] 2.3.3 Grape growth promotion experiment

[0126] After transplanting the one-year-old red grape seedlings, 50 seedlings with the same growth were selected for the test. 6 CFU / mL of fermentation liquid and fermentation liquid supernatant were evenly poured into the root area of ​​the seedlings. At the same time, a clear water control group was set up. There were 10 plants in each treatment, 50mL of bacterial liquid per plant, and the roots were irrigated once every 7 days, for a total of 5 times. The growth of the plants was observed regularly.

[0127] After 60 days, all plants were uprooted, washed and dried, and the plant height, root length, fresh weight and dry weight were counted respectively, and all values ​​were calculated as average values.

[0128] Wherein, the preparation method of fermentation broth and fermentation broth supernatant is:

[0129] (1) The preparation method of the fermentation broth of strain ZBSF BH07 is as follows:

[0130] A single colony of strain ZBSF BH07 was selected and incubated in 50 mL LB medium at 28°C and 180 r·min -1 Culture for 24 hours. Transfer to LB medium at a ratio of 0.1% at 28°C and 180 r / min -1 After 48 hours of cultivation, the fermentation liquid of strain ZBSF BH07 was obtained, and the content of strain ZBSF BH07 in the fermentation liquid of strain ZBSF BH07 was 1×10 8 cfu / mL.

[0131] The results are shown in Table 2 and Figure 7The results showed that the fermented liquid could significantly promote the growth of grape plants after root irrigation, and the fresh weight, dry weight and length of the aboveground parts and roots increased. The fresh weight and dry weight of the aboveground parts treated with the fermented liquid increased by 56.56% and 55.30%, respectively. The fresh weight and dry weight of the roots increased by 172.97% and 236.36%, respectively. The root length and branch length increased by 100% and 35.45%, respectively.

[0132] Table 2 Growth-promoting effect of Burkholderia gladioli ZBSF BH07 on grape

[0133]

[0134] Note: The lowercase letters after the numbers indicate significant differences. The same letters indicate no significant differences between the results, and different letters indicate significant differences (P < 0.05).

[0135] 2.3.4 Effects of strain ZBSF BH07 on the activities of grape defense enzymes

[0136] The leaves of each grape seedling (the second and third fully expanded leaves from the bottom) were taken at 60 days after the treatment in 2.3.3, and the superoxide dismutase (SOD) activity, polyphenol oxidase (PPO) activity, phenylalanine ammonia lyase (PAL) activity, total protein content, proline content and malondialdehyde content were determined using kits (Suzhou Keming Biotechnology Co., Ltd.).

[0137] 1) Determination of SOD activity

[0138] The superoxide dismutase (SOD) activity was determined using a superoxide dismutase (SOD) test kit (Suzhou Keming Biotechnology Co., Ltd., catalog number 5SOD-2-W).

[0139] The SOD activity was determined by the nitro blue tetrazolium method. The 3 mL reaction system contained 2.2 mL of 50 mmol / L pH 7.8 phosphate buffer, 0.2 mL of 60 μmol / L riboflavin, 0.2 mL of 195 mmol / L methionine, 0.1 mL of 3 μmol / LEDTA-Na2, 0.1 mL of SOD crude extract (the control was replaced by buffer) and 0.2 mL of 1.125 mmol / L NBT. The reaction system was placed under 4 000 lx sunlight for 20 min. After the reaction was completed, it was covered with a black cloth to terminate the reaction. The absorbance at a wavelength of 560 nm was measured, and 50% inhibition of NBT photoreduction was defined as 1 enzyme activity unit.

[0140] The preparation method of the crude SOD extract is as follows: weigh 0.1 g of tissue, add 1 mL of extract, homogenize in an ice bath, centrifuge at 8000 g and 4°C for 10 min, take the supernatant, and place on ice for testing.

[0141] The calculation formula of SOD activity: SOD activity (U / g fresh weight) = [inhibition percentage ÷ (1-inhibition percentage) × V total] ÷ (W × V sample ÷ V total);

[0142] Wherein, inhibition percentage = (A control tube - A assay tube) ÷ A control tube × 100%; Vreaction: total volume of reaction system; Vsample: volume of sample added to reaction system; Vsample: volume of extract added; Cpr: sample protein concentration; W: sample mass.

[0143] 2) PPO enzyme activity determination

[0144] The polyphenol oxidase (PPO) activity was determined using a polyphenol oxidase (PPO) test kit (Suzhou Keming Biotechnology Co., Ltd., catalog number PPO-2-Y).

[0145] Specifically, 1g of plant tissue is ground with 1mL of precooled pH7.8 (0.05M) phosphate buffer, and then 1mL of buffer is added, poured into a 5mL centrifuge tube, centrifuged at 4°C, 10000rpm for 20min, and the supernatant is collected. The reaction system is 3mL of 0.2M catechol (prepared with pH7.8 phosphate buffer), 1mL of enzyme solution, and the inactivated enzyme solution is used as a blank control. The reaction is water bathed at 30°C for 10min, and the reaction is immediately terminated with 20% trichloroacetic acid. Centrifuged at 5000rpm for 10min, and the absorbance is measured at 525nm.

[0146] PPO enzyme activity = (U / g fresh weight) = 60 × ΔA ÷ W

[0147] Wherein, ΔA=A assay tube-A control tube; OD525nm is the absorbance value of the test reaction solution measured at 525nm; W: sample mass.

[0148] 3) PAL enzyme activity assay

[0149] The PPO activity was determined using a phenylalanine ammonia lyase (PAL) test kit (Suzhou Keming Biotechnology Co., Ltd., catalog number PAL-2-Y).

[0150] Specifically, 0.2g of grape leaves were weighed, 6mL of 0.05mol / L pH 8.8 borax-hydrochloric acid buffer (containing 5mmol / L mercaptoethanol) and 0.2g of polyvinylpyrrolidone (PVP) were added, and the mixture was ground and homogenized in an ice bath. The homogenate was centrifuged at 4°C and 12,000r / min for 15min. 0.2mL of supernatant, 1mL of 0.02mol / L phenylalanine, and 3.8mL of 0.05mol / L borax-hydrochloric acid buffer were taken to detect PAL activity. The control group did not add phenylalanine but only added 0.2mL of supernatant and 4.8mL of borax-hydrochloric acid buffer. After mixing, the mixture was placed in a 30°C constant temperature water bath for 30min, and 0.5mL of 6mol / L hydrochloric acid solution was added to terminate the reaction. The absorbance was measured at a wavelength of 290 nm (A290 nm), and a change of 0.1 in the A290 nm value per hour was defined as one enzyme activity unit (U). The PAL activity was calculated according to the following formula.

[0151] PAL (U / g fresh weight) = ΔA × V total ÷ (W × V sample ÷ V total) ÷ ​​0.1 ÷ T = 17.3 × ΔA ÷ W

[0152] In the formula, Vreaction: total volume of reaction system, 1.04 mL; Vsample: added sample volume, 0.02 mL; Vsample: added extract volume, 1 mL; T: reaction time, 30 min; Cpr: sample protein concentration, mg / mL; W: sample mass.

[0153] 4) Total protein determination

[0154] The total protein content was determined using the Coomassie Brilliant Blue protein content test kit (Suzhou Keming Biotechnology Co., Ltd., catalog number KMSP-2-W). The total protein determination includes:

[0155] (1) Preparation of standard curve: Take 100 μl of BSA protein standard solution (0, 25, 125, 250, 500, 750, 1000, 1500, 2000 μg / mL) in a 5 mL centrifuge tube, add 2 mL of BCA working solution, mix thoroughly and place at 37°C for 30 min, and measure the OD value at a wavelength of 562 nm. Plot the OD value against the BSA protein mass concentration to obtain the BSA protein standard curve.

[0156] (2) Determination of protein content in grape leaves: Weigh 100 mg of grape leaves and dissolve them in 50 mL of distilled water to prepare a 1 mg / mL protein test solution. Take 100 μL of the test solution in a 5 mL centrifuge tube, add 2 mL of BCA working solution, mix thoroughly and place at 37°C for 30 min, measure the OD value at a wavelength of 562 nm, and substitute it into the standard curve equation to calculate the protein mass concentration.

[0157] Cpr (mg / g) = C standard × (A measuring tube - A blank tube) ÷ (A standard tube - A blank tube) ÷ sample mass = 0.5 × (A measuring tube - A blank tube) ÷ (A standard tube - A blank tube) ÷ sample mass.

[0158] 5) Proline determination

[0159] The proline content was determined using a proline (PRO) content test kit (Suzhou Keming Biotechnology Co., Ltd., product number PRO-2-Y).

[0160] Proline determination includes: (1) Take 0.5mL sample + 0.5mL glacial acetic acid + 0.5mL reagent 2 in a covered test tube, place in a boiling water bath for 30 minutes (cover tightly to prevent water loss), and shake once every 10 minutes. (2) After cooling, add 1mL reagent 3 to the test tube, shake for 30 seconds, let it stand for a while to allow the pigment to transfer to toluene; aspirate 0.8mL -1 mL of upper solution was placed in a 1 mL glass cuvette, and the colorimetry was performed at a wavelength of 520 nm, and the absorbance A was recorded.

[0161] Pro content (μg / g fresh weight) = [(A520nm+0.0021)÷0.0521×V1]÷(W×V1÷V2)

[0162] =19.2×(A520nm+0.0021)÷W.

[0163] The sample preparation method is as follows: weigh about 0.1g of tissue, add 1mL of extracting solution, and homogenize in an ice bath; then extract at 90℃ with oscillation for 10min; centrifuge at 10000g, 25℃ for 10min, take the supernatant, and cool it for testing.

[0164] 6) Malondialdehyde determination

[0165] The proline content was determined using a malondialdehyde (MDA) test kit (Suzhou Keming Biotechnology Co., Ltd., product number MDA-2-Y).

[0166] The malondialdehyde determination includes: taking 0.5g of leaves, adding 2ml of pre-cooled 0.05mol / l pH7.8 phosphate buffer, adding a small amount of quartz sand, grinding into a homogenate in a mortar after an ice bath, transferring to a 5ml centrifuge tube, making the volume up to 5ml with buffer, centrifuging at 4500r / min for 10min, and the supernatant is the malondialdehyde extract. Pipette 2ml of the extract into a graduated test tube, add 0.5, 3ml of 5% trichloroacetic acid solution of thiobarbituric acid, boil in water for 10min, 4500r / min for 10min, and measure the absorbance of the supernatant at 532 and 600nm wavelengths, using distilled water as a blank and control.

[0167] MDA content (nmol / g fresh weight) = [ΔA×V total ÷ (ε×d)×109] ÷ (W×V sample ÷ V total)

[0168] =25.8×ΔA÷W.

[0169] Wherein, ΔA=A532nm-A600nm, W represents the sample mass.

[0170] The test results are shown in the table. The results show that compared with the control, the activities / contents of the three leaf protective enzymes measured after being treated with the fermentation liquid of strain ZBSF BH07 were all increased.

[0171] Table 3 Effects of strain ZBSF BH07 on protective enzymes in grape leaves

[0172] deal with SOD(U / g) PPO (IU / g) PAL(U / g) Total protein (mg / mL) PRO (ng / g) MDA (nmol / g) C 1009.25d 23.75a 12.22b 4.08c 599.69b 1.55a ZBSF BH07 fermentation broth 1442.35a 35.16a 10.09b 4.93a 655.21a 1.09c

[0173] Note: The lowercase letters after the numbers indicate significant differences, and the same letters indicate no significant differences between the results.

[0174] Different letters indicate significant differences (P<0.05).

[0175] Example 3: Detection of the effect of strain ZBSF BH07 on controlling white rot

[0176] 4.1. Test Drugs

[0177] 4.1.1 Spore suspension of grape white rot pathogen

[0178] Preparation of spore suspension of grape white rot fungus - Coniella vitis: Grape white rot fungus was cultured on a PDA plate at 28°C. After sufficient spore production, 10 mL of sterile 1% glucose solution was added, and the spores were washed off with a sterile cotton swab. Then, a suspension was prepared with sterile 1% glucose solution, and the spore content was adjusted to 1×10 7 cfu / mL

[0179] 4.1.2 Preparation of fermentation broth of strain ZBSF BH07

[0180] After the strain ZBSF BH07 was activated on the LB plate, a single colony was picked and cultured in LB liquid medium at 28°C and 180r / min for 12h to obtain seed solution. The seed solution was transferred to LB medium at a ratio of 0.1%, and cultured at 28°C and 180r / min for 48h. The fermentation liquid of the strain ZBSF BH07 was obtained, and the content of the strain ZBSF BH07 in the fermentation liquid of the strain ZBSF BH07 was 1×10 8 cfu / mL.

[0181] 4.2. Prevention and treatment of grape white rot

[0182] The experiment set up four treatments: grape white rot bacteria treatment, fermentation liquid treatment, fermentation liquid + white rot (prevention) treatment, and fermentation liquid + white rot (treatment) treatment. Select red grape fruits with neat appearance, no pests and diseases, and no external injuries, first rinse them with tap water, then spray them with 75% alcohol for disinfection, and wipe them dry with sterile filter paper after 2 minutes. Put the fruits in a culture dish with a diameter of 100mm covered with sterile filter paper, add sterile water to keep them moist, and use a sterilized inoculation needle to pierce a wound of about 1mm on each fruit. 20 fruits for each treatment, repeated 3 times.

[0183] The specific experimental methods are as follows:

[0184] 1) Treatment with grape white rot fungus: 40 μL of grape white rot fungus spore suspension was inoculated at the wound.

[0185] 2) Fermentation broth treatment: 40 μL of fermentation broth of strain ZBSF BH07 was inoculated on the wound

[0186] 3) Prevention of grape white rot by fermentation liquid + white rot fungus treatment: 20 μL of the fermentation liquid of strain ZBSF BH07 was inoculated on the wound for 24 hours, and then 20 μL of grape white rot fungus spore suspension was inoculated.

[0187] 4) Treatment of grape white rot fermentation liquid + white rot fungus treatment: 20 μL of grape white rot fungus spore suspension was inoculated on the wound for 24 hours, and then 20 μL of strain ZBSF BH07 fermentation liquid was inoculated. The above treatments were cultured at 28℃ for 7 days, and the disease statistics were calculated and the control effect was calculated.

[0188] White rot classification standards

[0189] Grade 0: No disease;

[0190] Level 1: The diseased area accounts for ≤5% of the fruit area;

[0191] Level 3: The diseased area accounts for 5%-20% of the fruit area;

[0192] Level 5: The diseased area accounts for 21%-50% of the fruit area;

[0193] Level 7: The diseased area accounts for 51%-75% of the fruit area;

[0194] Level 9: The diseased area accounts for 76%-100% of the fruit area.

[0195] Disease index = 100 × ∑ (number of diseased leaves / fruits at each level × corresponding level) / (total number of leaves / fruits investigated × highest representative value)

[0196] Preventive effect = [(disease index of sterile water control - disease index of treatment) / disease index of sterile water control] × 100% The test data were analyzed for significant differences using DPS software, and graphed using Microsoft Excel software.

[0197] The results are shown in Table 4 and Figure 8 The results showed that compared with the single inoculation of white rot fungus, the fermentation liquid of strain ZBSF BH07 had a better control effect on white rot of red grape fruit. The incidence rate and disease index were significantly reduced. And the preventive effect was greater than the therapeutic effect.

[0198] Table 4 Control effect of gladiolus Burkholderia ZBSF BH07 on grape white rot

[0199] Serial number deal with Incidence / % Disease index Control effect / % 1) Grape white rot 100.00a 87.8a / 2) ZBSF BH07 fermentation broth 0.00f 0.00f / 3) ZBSF BH07 fermentation liquid + white rot (prevention) 15.00e 5.00e 94.31a 4) ZBSF BH07 fermentation liquid + white rot (treatment) 20.00e 7.78d 91.14b

[0200] Note: The lowercase letters after the numbers in the table indicate significant differences. There is no significant difference between treatments with at least one identical letter at the 0.05 level, and there is a significant difference between treatments without the same letter at the 0.05 level.

[0201] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A gladiolus Burkholderia ZBSF BH07, characterized in that: The fungus was classified and named Burkholderia gladioli. It was deposited in the China General Microbiological Culture Collection on April 1, 2024 with the deposit number: CGMCC No.30228. The address of the deposit unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. A microbial mixture, characterized in that: The microbial mixture contains at least the Burkholderia gladiolus ZBSFBH07 described in claim 1, and further contains other strains that promote plant growth or prevent and control plant diseases and are symbiotic microorganisms with the Burkholderia gladiolus.

3. A product comprising the Burkholderia gladioli ZBSF BH07 of claim 1 or the microbial mixture of claim 2, characterized in that: The product is a bacterial agent, a bacterial fertilizer or a combination.

4. The product according to claim 3, characterized in that: The bacterial agent, bacterial fertilizer or composition contains the Burkholderia gladioli ZBSF BH07 and / or contains the culture of the Burkholderia gladioli ZBSF BH07; The microbial agent is a phosphorus-degrading microbial agent, a plant growth promoter, a plant pathogen inhibitor and / or a plant white rot control agent. 5 . A fermentation liquid, fermentation product or metabolite comprising the Burkholderia gladioli ZBSF BH07 of claim 1 .

6. A fermentation method of the gladiolus Burkholderia ZBSF BH07 according to claim 1, comprising inoculating the gladiolus Burkholderia ZBSF BH07 into a fermentation medium for fermentation culture.

7. An application according to any one of A1) to A8), characterized in that: A1) Use of the gladiolus Burkholderia ZBSF BH07 according to claim 1 in the preparation of a product for promoting plant growth; A2) Use of the gladiolus Burkholderia ZBSF BH07 according to claim 1 or the microbial mixture according to claim 2 or the product according to claim 3 in promoting plant growth; A3) Use of the gladiolus Burkholderia ZBSF BH07 according to claim 1 in the preparation of plant pathogen inhibitors; A4) Use of the gladiolus Burkholderia ZBSF BH07 according to claim 1, the microbial mixture according to claim 2, or the product according to claim 3 in inhibiting plant pathogens; A5) Use of the gladiolus Burkholderia ZBSF BH07 according to claim 1 in the preparation of a product for preventing and / or treating plant white rot; A6) Use of the gladiolus Burkholderia ZBSF BH07 according to claim 1 or the microbial mixture according to claim 2 or the product according to claim 3 in preventing and / or treating plant white rot; A7) Use of the gladiolus Burkholderia ZBSF BH07 according to claim 1 or the microbial mixture according to claim 2 or the product according to claim 3 in degrading phosphorus; A8) Use of the Burkholderia gladiolus ZBSF BH07 according to claim 1, the microbial mixture according to claim 2, or the product according to claim 3 in the preparation of siderophores.

8. The use according to claim 7, characterized in that: The plant is grape; The promoting of plant growth may be promoting the nutritional growth of plants; specifically, promoting the growth of the aboveground parts of plants, promoting the increase of plant fresh weight and / or promoting the increase of plant dry weight; the plant pathogenic bacteria is at least one of grape white rot fungus - Coniella vitis, grape anthracnose fungus - Colletotrichum aenigma, Fusarium oxysporum, grape club-shaped pseudo-disk hairy spore (Pestalotiopsis clavispora), grape vine blight fungus - Diaporthe eres, grape spike brown blight fungus - Alternaria viticola, grape ulcer fungus - Botryosphaeria dothidea, and grape gray mold fungus - Botrytis cinerea.

9. A method for promoting plant growth, characterized in that: The method comprises culturing the Burkholderia gladioli ZBSF BH07 of claim 1 in a microbial culture medium, collecting the culture, and treating plants with the culture.

10. A method for preventing and / or treating plant diseases, characterized in that: The method comprises culturing the gladiolus Burkholderia ZBSF BH07 of claim 1 in a microbial culture medium, collecting the culture, and treating a plant with the culture; the plant is grape; The plant disease is at least one of grape white rot fungus - Coniella vitis, grape anthracnose fungus - Colletotrichum aenigma, Fusarium oxysporum, grape Pestalotiopsis clavispora, grape vine blight fungus - Diaporthe eres, grape spike brown blight fungus - Alternaria viticola, grape canker fungus - Botryosphaeria dothidea, and grape gray mold fungus - Botrytis cinerea.

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