Berkhoutia calomelanos and application thereof in prevention and treatment of grape diseases

Abstract

The application discloses a Burkholderia gladioli and application thereof in prevention and treatment of grape diseases, the classification and naming of the bacteria is Burkholderia gladioli, which is preserved in the China General Microbiological Culture Collection Center on April 1, 2024, and the preservation number is CGMCC No.30228, and the address of the preservation unit is No.3, Xili, Beichen West Road, Chaoyang District, Beijing City. The strain has a wide antibacterial spectrum, and has good antibacterial activity on common pathogenic bacteria in grape planting, especially on grape white rot and grape anthracnose. Through directional screening of grape rhizosphere soil microorganisms, a biocontrol bacterium with high efficiency of antagonizing grape white rot bacteria is obtained, and basic materials are provided for biological control of grape white rot.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial strains, and particularly relates to a Burkholderia gladioli and application thereof in prevention and treatment of grape diseases. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing an understanding of the general background of the application and does not necessarily constitute an admission or a recognition that the information forms part of the prior art already known to a person of ordinary skill in the art.

[0003] Burkholderia is a genus of Gram-negative non-fermenting bacteria belonging to the phylum Proteobacteria. They are widely present in soil, water and various hosts. In 1992, the genus was separated from Pseudomonas. Burkholderia interacts with hosts in a complex way, which can be pathogenic or beneficial. For example, the onion Burkholderia complex includes pathogenic and non-pathogenic bacteria, and melioidosis and glanders are also caused by it. Some Burkholderia are plant or human pathogenic bacteria, but many species have the functions of biological control, plant growth promotion and decomposition of toxic substances. The bacteria of the genus can fix nitrogen to promote plant growth, inhibit pests and degrade environmental pollutants. In addition, they have great potential in disease inhibition and synthesis of antibacterial substances, and can produce a variety of secondary metabolites with antifungal, antibacterial, herbicidal and insecticidal activities. Plant growth-promoting rhizobacteria (PGPR) is a kind of bacteria that can promote plant growth, and can inhibit the invasion of harmful pathogenic bacteria by promoting the absorption of plant nutrients, inducing the production of root surface hormones or secreting antibacterial substances, etc. after planting in the plant roots, so as to directly or indirectly affect the growth and development of plants. The growth-promoting mechanism of PGPR mainly includes nitrogen fixation, phosphorus dissolution, potassium dissolution, secretion of siderophores, secretion of plant hormones and related regulatory substances and release of volatile substances, etc. The research on the enhancement of plant disease resistance by PGPR has always been a hot topic. In the genus Bacillus, strains of PGPR that can enhance plant disease resistance have been isolated. 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] At present, there is no report on the application of Burkholderia gladioli in prevention and treatment of grape diseases. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a Burkholderia gladioli and application thereof in prevention and treatment of grape diseases.

[0006] The technical scheme adopted by the present application is as follows:

[0007] In a first aspect of the present application, there is provided a strain of Burkholderia gladioli, designated as ZBSF BH07, which was deposited at China General Microbiological Culture Collection Center on April 1, 2024, and assigned accession number CGMCC No. 30228, and address of the depositary is No. 3, Beichen West Road, Chaoyang District, Beijing.

[0008] In a second aspect of the present application, there is provided a microbial mixture comprising at least the strain of the present application, i.e., ZBSF BH07. The microbial mixture can comprise other strains that promote plant growth or control plant diseases, etc., and coexist with the strain of the present application.

[0009] In a third aspect of the present application, there is provided a product comprising the strain of the present application, i.e., ZBSF BH07, or the microbial mixture of the present application. The product can be a microbial agent, a microbial fertilizer, or a composition, etc.

[0010] The microbial agent, the microbial fertilizer, or the composition contain the above-mentioned ZBSF BH07 and / or the culture of the above-mentioned ZBSF BH07.

[0011] The microbial agent can be a phosphorus-degrading microbial agent, a plant growth promoter, a plant pathogenic bacteria inhibitor, and / or a plant white rot disease control agent.

[0012] The term "culture" refers to a general term for a liquid or solid product (all substances in the culture vessel) that has been inoculated and cultured by humans and has grown into a microbial population. That is, a product obtained by growing and / or amplifying microorganisms, which can be a biologically pure culture of microorganisms, or can contain a certain amount of culture medium, metabolites, or other components produced during the culture process.

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

[0014] In a fourth aspect of the present application, there is provided a fermentation broth or a fermentation product or a metabolite, etc., comprising the ZBSF BH07.

[0015] In a fifth aspect of the present application, there is provided a fermentation method of the ZBSF BH07, which comprises inoculating the ZBSF BH07 into a fermentation medium and then performing fermentation culture.

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

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

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

[0019] A3) Use of the above mentioned Burkholderia gladioli ZBSF BH07 for the preparation of a plant pathogen inhibitor;

[0020] A4) Use of the above mentioned Burkholderia gladioli ZBSF BH07 or the above mentioned microbial mixture or the above mentioned product for inhibiting plant pathogen;

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

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

[0023] A7) Use of the above mentioned Burkholderia gladioli ZBSF BH07 or the above mentioned microbial mixture or the above mentioned product for degrading phosphor;

[0024] A8) Use of the above mentioned Burkholderia gladioli ZBSF BH07 or the above mentioned microbial mixture or the above mentioned product for the preparation of siderophores.

[0025] Further, in the above mentioned uses, the plant is grape.

[0026] Further, in the above mentioned uses, the promoting plant growth can be promoting vegetative growth of the plant.

[0027] In particular, it can be promoting growth of the above ground parts of the plant, promoting fresh weight increase of the plant and / or promoting dry weight increase of the plant.

[0028] Further, in the above mentioned uses, the pathogen of A3) or A4) is a fungus.

[0029] The plant disease is at least one of Coniella vitis, Colletotrichum aenigma, Fusarium oxysporum, Pestalotiopsis clavispora, Diaporthe eres, Botryosphaeria dothidea, Botrytis cinerea, Alternaria viticola, Fusarium graminearum, and Fusarium pseudograminearum.

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

[0031] In an eighth aspect of the present application, there is provided a method for preventing and / or treating a plant disease, the method comprising culturing the Burkholderia gladioli described above in a microbial culture medium, collecting the culture, and treating a plant with the culture.

[0032] The plant disease is a fungus;

[0033] Further, the fungus can be at least one of Coniella vitis, Colletotrichum aenigma, Fusarium oxysporum, Pestalotiopsis clavispora, Diaporthe eres, Botryosphaeria dothidea, Botrytis cinerea, Alternaria viticola, Fusarium graminearum, and Fusarium pseudograminearum.

[0034] More specifically, the plant disease can be grape white rot, grape anthracnose, grape botrytis blight, grape cordon brown blight and grape vine blight, and the pathogenic bacteria can be grape white rot pathogen Coniella vitis, grape anthracnose pathogen (Colletotrichum aenigma), grape botrytis blight pathogen (Botrytis cinerea), grape cordon brown blight pathogen (Alternaria viticola), grape vine blight pathogen (Diaporthe eres), and fusarium oxysporum.

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

[0036] In the present application, the solid culture medium can be prepared by adding 15 g / L agar powder to the liquid culture medium.

[0037] In the present application, the plant treated with the culture can be specifically treated with the fermentation broth of Burkholderia gladioli by root irrigation, or the plant surface is contacted with the fermentation broth of Burkholderia gladioli.

[0038] In the present application, the pathogenic bacteria treated with the culture can be specifically sprayed with the fermentation broth of Burkholderia gladioli before the pathogenic bacteria contact the plant, or the plant surface is contacted with the fermentation broth of Burkholderia gladioli before / after the pathogenic bacteria contact the plant.

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

[0040] In the present application, a Burkholderia ZBSF BH07 with good control effect on grape white rot pathogen is isolated and screened from the rhizosphere soil of grape plantation, which has a wide antibacterial spectrum and good antibacterial activity on common pathogenic bacteria in grape plantation, especially on grape white rot and grape anthracnose. Through directional screening of grape rhizosphere soil microorganisms, the present application pre-obtains biocontrol bacteria with high efficiency against grape white rot pathogen, and provides basic materials for biological control of grape white rot. BRIEF DESCRIPTION OF DRAWINGS

[0041] The drawings accompanying the specification of the present application form a part thereof and serve to provide further understanding of the present application, the illustrative embodiments of the present application and its description serve to explain the present application, and do not constitute an improper limitation on the present application.

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

[0043] Figure 2 It 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 Antibacterial effect of strain ZBSF BH07 on different pathogenic bacteria.

[0046] Figure 5 Protease production capacity of strain ZBSF BH07.

[0047] Figure 6 Siderophore halo and phosphorus solubilization halo of strain ZBSF BH07 on CAS and NBRIP media, respectively.

[0048] Figure 7 Growth-promoting effect of strain ZBSF BH07 on grape.

[0049] Figure 8 Effect detection of strain ZBSF BH07 in preventing and treating grape botrytis. DETAILED DESCRIPTION

[0050] It should be noted that the following detailed description is exemplary in nature and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0051] It is to be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments consistent with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

[0052] In order to enable persons skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in combination with specific examples.

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

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

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

[0056] Lysogeny Broth liquid medium (LB): Yeast extract 5 g, Tryptone 10 g, NaCl 10 g, distilled water 1000 mL. Lysogeny Broth solid medium (LB): Yeast extract 5 g, Tryptone 10 g, NaCl 10 g, agar 15 g, deionized water to 1 L.

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

[0058] NBRIP solid 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 extract 0.5 g, deionized water to 1000 mL, pH 6.8-7.2.

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

[0060] The above-mentioned media were sterilized at 121℃ 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 grape plants. 10 g of soil was weighed into a flask containing 90 mL of sterilized physiological saline, and cultured at 180 r / min for 1 h, then in a water bath at 80℃ for 20 min. The strain was isolated by dilution plating method, and cultured at 28℃ for 48 h. After the colonies grew, single colonies were picked and streaked on NA solid medium for purification, and strain ZBSF BH07 was obtained. The strain was cultured at 28℃ for 48 h and stored in a refrigerator at 4℃ for standby.

[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 determination of strain ZBSF BH07

[0067] The isolated strain was inoculated on LB medium and cultured at 28℃ for 48 h, and the colony morphology of the strain was observed. According to the "Berger's Bacterial Identification Manual" and "Common Bacterial System Identification Manual", the physiological and biochemical characteristics were identified.

[0068] The strain ZBSF BH07 was cultured on LB medium for 4 days, the colony was round, off-white, with neat edge and smooth surface. Under microscope, the bacterial body was short rod-shaped and gram-negative. No hemolytic ring was produced on blood plate, indicating that the strain GSBZ09 was non-pathogenic and safe to human body.

[0069] The culture morphology of the strain is shown in Figure 1 Figure 1 The left 1st figure in the middle is the culture morphology of the strain ZBSF BH07 on LB medium, Figure 1 The left 2nd figure in the middle is the culture morphology of the strain ZBSF BH07 on blood plate (purchased from Biomerieux) medium.

[0070] 1.3, Molecular identification

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

[0072] AGGGGAATGCCTTACCATGCAAGTCGAACGGCAGCACGGGTGCTTGCACCTGGTGGCGAGTGGCGA

[0073] ACGGGTGAGTAATACATCGGAACATGTCCTGTAGTGGGGGATAGCCCGGCGAAAGCCGGATTAATA

[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 by using the NCBI Blast program, and the phylogenetic tree between the strain ZBSF BH07 and other similar strains was constructed by using the maximum likelihood method in the MEGA 6.0 software. It was found that the strain ZBSF BH07 had a 99% genetic relationship with Burkholderia gladioli. Combined with the observation of morphological characteristics and the analysis of physiological and biochemical characteristics, it was determined that the strain ZBSF BH07 was Burkholderia gladioli. Figure 2A phylogenetic tree based on 16S rDNA.

[0097] Strain ZBSF BH07 was preserved in China General Microbiological Culture Collection Center on April 1, 2024, with a preservation number of CGMCC No. 30228 and a classification name of Burkholderia gladioli.

[0098] 1.4, Growth rate and pH value determination of Burkholderia gladioli

[0099] Strain ZBSF BH07 was inoculated in liquid LB medium and cultured at 28°C for 52h. A 16h cultured bacterial suspension was added to the LB medium. Three replicates were set up and placed in a 28°C shaker. The OD value and pH value were measured every 4h, and a total of 14 points were recorded. The growth curve and pH change curve of strain ZBSF BH07 are shown in Figure 3 The strain ZBSF BH07 was in the exponential growth phase before 12h, and the absorbance reached 4.002 at 28h. After 28h, it entered the stable growth phase. The pH value decreased first and then increased with time, and reached 6.69 at 52h and remained stable.

[0100] 1.5, Antibiotic determination of strain ZBSF BH07

[0101] 1) Determination of different antibiotic resistance

[0102] Nine antibiotics, ampicillin, vancomycin, chloramphenicol, gentamicin, tetracycline, spectinomycin, kanamycin, streptomycin, and rifampicin, were added to 5mL of liquid LB medium at a proportion of 0.1%. Strain ZBSF BH07 single colony was picked and inoculated into 5mL of liquid LB medium containing different antibiotics and cultured at 28°C for 24h. Strain ZBSF BH07 LY7 could grow at the working concentration of ampicillin, chloramphenicol, and vancomycin, but not at other concentrations. LY7 could grow at 2 times the working concentration (100mg / L) of ampicillin, but grew slowly. It could not grow at 2 times the working concentration of chloramphenicol.

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

[0104] 2.1, Application of strain ZBSF BH07 in bacteriostasis

[0105] 2.1.1, Measurement of the fungal inhibition spectrum of strain ZBSF BH07

[0106] The plate confrontation method was used. A 7-mm fungal cake was inoculated in the center of a 90-mm PDA plate, and the plate was incubated at 25°C for 1 day. Strain ZBSF BH07 was inoculated in liquid LB medium and incubated at 28°C for 16 h. The ZBSF BH07 bacterial suspension was obtained. 2.5 uL of the ZBSF BH07 bacterial suspension was inoculated at 4 points opposite each other at a distance of 10 mm from the edge of the plate. 2.5 uL of the ZBSF BH07 bacterial suspension was inoculated at 4 points opposite each other at a distance of 10 mm from the edge of the plate. Liquid LB medium was used as a blank control. The plates were incubated at 25°C for 3 days. When the blank control was about to cover the entire plate, the control growth (colony radius) and the treatment growth (growth radius after inoculation of ZBSF BH07) of the target fungus were measured, and the inhibition rate was used as an indicator.

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

[0108] The pathogenic fungi are as follows:

[0109] Coniella vitis, Colletotrichum aenigma, Fusarium oxysporum, Pestalotiopsis clavispora, Diaporthe eres, Botryosphaeria dothidea, Botrytis cinerea, Alternaria viticola, Fusarium graminearum, Fusarium pseudograminearum have been disclosed 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. The above pathogens are available to the public from Shandong Grape Research Institute or through other means.

[0110] The results of the determination of the fungal inhibition spectrum of strain ZBSF BH07 are shown in Table 1 below. Figure 4

[0111] Table 1 Inhibition of pathogenic fungi by strain ZBSF BH07

[0112] Pathogenic bacteria Inhibition zone Coniella vitis +++ Colletotrichum aenigma +++ Botrytis cinerea +++ Pestalotiopsis clavispora ++ Alternaria viticola +++ Diaporthe eres +++ Fusarium oxysporum +++ Botryosphaeria dothidea ++ Fusarium graminearum ++ Fusarium pseudograminearum ++

[0113] Note: +++ means the diameter of 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 the diameter of bacterial inhibition zone is greater than or equal to 4.0 cm but less than 5.0 cm or the fungal inhibition rate is greater than or equal to 40% but 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 on protease detection medium, and the diameter of transparent circle was observed after 3 d. The results are shown in Figure 5 , the transparent circle diameter of ZBSF BH07 was 2.9 cm, indicating that the strain ZBSF BH07 had the ability to produce protease.

[0116] 2.3, Promoting effect of strain ZBSF BH07 was detected

[0117] 2.3.1, qualitative detection of phosphorus-solubilizing plate

[0118] The strain ZBSF BH07 was inoculated in a test tube containing 5 mL of liquid LB medium, and after 24 h of shaking culture at 28°C and 170 r / min, a bacterial suspension of 10 7 CFU / mL was prepared, 2.5 uL of the bacterial suspension was spotted in the center of the NBRIP solid culture medium plate, and after 7 d of culture, the phosphorus-solubilizing circle was observed and the diameter was measured, and each treatment was repeated 3 times.

[0119] The results are shown in Figure 6 Figure 6 The second picture from the left is the result of qualitative detection of phosphorus-solubilizing plate. The results show that ZBSF BH07 was cultured in NBRIP medium for 7 d, and the diameter of the phosphorus-solubilizing circle was 1.2 cm, indicating that ZBSF BH07 had the ability to degrade phosphorus.

[0120] 2.3.2, siderophore detection

[0121] General CAS plate detection, wherein the preparation of CAS detection solution is as follows:

[0122] Put 6 mL of 10 mmol / L -1 hexadecyltrimethylammonium bromide (HDTMA) in a 100 mL volumetric flask, and dilute with double distilled water as appropriate, add 1.5 mL of 1 mmol / L -1 FeCl3·6H2O and 7.5 mL of 2 mmol / L -1 CAS solution, slowly add it to the volumetric flask along the glass rod. Dissolve 4.3 g of anhydrous piperazine in water and add 6.25 mL of 12 mol / L -1 hydrochloric acid to obtain a buffer solution with pH 5.6, then transfer this solution to the aforementioned volumetric flask, and make up to 100 mL with double distilled water, sterilize at 121°C for 15 min, and then use.

[0123] Preparation of CAS detection plate: prepare 1 mol / L -1 CaCl2solution, 1 mmol / L -1 ​MgSO4.6H2O solution, 10% acid hydrolysis casein solution 121℃ 15min sterilized alone for standby. Respectively take 0.2mL of CaCl2 solution, 0.2mL of MgSO4.7H2O solution, 6mL 10% acid hydrolysis casein solution, add biological buffer solution Pipes, adjust pH to 6.8~7.0. Deionized water to 100mL, add 2g agar powder, 121℃ 15min sterilization, when the temperature drops to 60℃, add 5mL of CAS blue detection solution has been configured, mix well. Note not to produce bubbles, affect the plate detection experiment. Then according to every dish 25mL pour into the culture dish. Positive reaction by CAS color from blue to yellow or orange to record. Record CAS agar plate halo around the colony.

[0124] Results as shown in Table 2 and Figure 6 Figure 6 The first left side of the first drawing is the result of siderophore detection, the results show that: strain ZBSF BH07 after 7d culture on CAS agar plate, halo around the colony produced yellow, indicating that there is siderophore production.

[0125] 2.3.3, grape growth promotion test

[0126] After transplanting annual red grape seedlings, 50 seedlings with consistent growth were selected for the test. The prepared 10 6 CFU / mL of fermentation broth and fermentation broth supernatant were evenly irrigated into the root zone of the seedlings, and a water control group was set up, 10 seedlings per treatment, 50mL of bacterial solution per seedling, irrigated once every 7d, a total of 5 times, and the growth status of the plants was observed regularly.

[0127] After 60d, all plants were pulled up, washed and dried, and the plant height, root length, fresh weight and dry weight were counted respectively, and all values were counted as average.

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

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

[0130] Pick strain ZBSF BH07 single colony in 50mL LB medium, 28℃, 180r·min -1 Cultured for 24h. According to 0.1% proportion, transfer to LB medium, 28℃, 180r·min -1 Cultured for 48h. Strain ZBSF BH07 fermentation broth was obtained, and the content of strain ZBSF BH07 in the strain ZBSF BH07 fermentation broth was 1×10 8 cfu / mL.

[0131] Results as shown in Table 2 and Figure 7 ​The results showed that the fermentation broth could significantly promote the growth of grape plants after root irrigation, and the fresh weight, dry weight, length of the aboveground part and root were increased. The fresh weight and dry weight of the aboveground part treated by fermentation broth increased by 56.56% and 55.30% respectively. The fresh weight and dry weight of the root increased by 172.97% and 236.36% respectively. The root length and branch length increased by 100% and 35.45% respectively.

[0132] Table 2 Promoting effect of Burkholderia ZBSF BH07 on grape

[0133]

[0134] Note: The small letters after the numbers indicate the significance of the difference, the same letters indicate that there is no significant difference between the results, and different letters indicate that there is a significant difference (P<0.05).

[0135] 2.3.4, Effect of strain ZBSF BH07 on the defense enzyme activity of grape

[0136] The leaves (the second and third fully expanded leaves from the bottom) of grape seedlings treated for 60 days in 2.3.3 were taken, and the activities of superoxide dismutase (SOD), polyphenol oxidase (PPO), phenylalanine ammonialyase (PAL), total protein content, proline content and malondialdehyde content were determined using a kit (Suzhou Keming Biotechnology Co., Ltd.).

[0137] 1) Determination of SOD activity

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

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

[0140] The preparation method of the SOD crude extract is as follows: 0.1 g of tissue is weighed, 1 mL of extraction solution is added, and homogenization is performed in an ice bath, 8000 g centrifugation is performed at 4°C for 10 min, the supernatant is taken, and the supernatant is placed on ice for detection.

[0141] The calculation formula of SOD activity is SOD activity (U / g fresh weight) = [inhibition percentage ÷ (1-inhibition percentage) × Vtotal] ÷ (W × Vsample ÷ Vtotal);

[0142] In the formula, inhibition percentage = (Acontrol tube-Adetermination tube) ÷ Acontrol tube × 100%; Vtotal: total volume of the reaction system; Vsample: sample volume added to the reaction system; Vtotal: volume of the extraction solution added; Cpr: sample protein concentration; and W: sample mass.

[0143] 2) PPO enzyme activity determination

[0144] The polyphenol oxidase (PPO) test kit (Suzhou Keming Biotechnology Co., Ltd., item number PPO-2-Y) is used to determine PPO activity.

[0145] Specifically, 1 g of plant tissue is ground with 1 mL of pre-cooled pH 7.8 (0.05 M) phosphate buffer, 1 mL of buffer is added, poured into a 5 mL centrifuge tube, centrifuged at 4°C and 10000 rpm for 20 min, and the supernatant is collected. The reaction system is 3 mL of 0.2M catechol (prepared with pH 7.8 phosphate buffer), 1 mL of enzyme solution, and the inactivated enzyme solution is used as a blank control. The reaction is stopped immediately after 10 min of water bath at 30°C with 20% trichloroacetic acid. Centrifugation is performed at 5000 rpm for 10 min, and the absorbance value is measured at 525 nm.

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

[0147] In the formula, ΔA = Adetermination tube-Acontrol tube OD525 nm is the absorbance value of the reaction solution to be measured at 525 nm; and W: sample mass.

[0148] 3) PAL enzyme activity determination

[0149] The phenylalanine ammonia lyase (PAL) test kit (Suzhou Keming Biotechnology Co., Ltd., item number PAL-2-Y) is used to determine PPO activity.

[0150] Specifically comprising: take 0.2 g of grape leaves, add 0.05 mol / L pH 8.8 borax-hydrochloric acid buffer (containing mercaptoethanol 5 mmol / L) 6 mL, polyvinylpyrrolidone (polyvinylpyrrolidone, PVP) 0.2 g, grind homogenate in ice bath, homogenate liquid at 4℃, 12 000 r / min under the condition of centrifugation for 15 min. Take 0.2 mL supernatant, 0.02 mol / L 1 mL phenylalanine, 0.05 mol / L 3.8 mL borax-hydrochloric acid buffer to detect PAL activity. The control group does not add phenylalanine, only adds 0.2 mL supernatant, 4.8 mL borax-hydrochloric acid buffer. Mix well and place in 30℃ constant temperature water bath for 30 min, add 6 mol / L 0.5 mL hydrochloric acid solution to terminate the reaction. Measure the absorbance (A290 nm) at 290 nm wavelength, and take 0.1 as an enzyme activity unit (U) per hour A290 nm value change. Calculate the PAL activity according to the following formula.

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

[0152] In the formula, V total reaction: total volume of reaction system, 1.04 mL; V sample: added sample volume, 0.02 mL; V total sample: 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 by using the Coomassie brilliant blue method protein content test box (Suzhou Keming Biotechnology Co., Ltd., product 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℃ for 30 min. Measure the OD value at a wavelength of 562 nm. Plot the OD value against the mass concentration of BSA protein to obtain the BSA protein standard curve.

[0156] (2) Determination of protein content of grape leaves: Weigh 100 mg of grape leaves into 50 mL of distilled water to prepare a 1 mg / mL protein 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℃ for 30 min. Measure the OD value at a wavelength of 562 nm and calculate the protein mass concentration by substituting the standard curve equation.

[0157] Cpr(mg / g) = Cstandard x (A test tube - A blank tube) ÷ (A standard tube - A blank tube) ÷ sample mass = 0.5 x (A test tube - A blank tube) ÷ (A standard tube - A blank tube) ÷ sample mass.

[0158] 5) Proline determination

[0159] The proline (PRO) content test kit (Suzhou Keming Biotechnology Co., Ltd., PRO-2-Y) was used to determine the proline content.

[0160] The proline determination included: (1) Take 0.5 mL sample + 0.5 mL glacial acetic acid + 0.5 mL reagent two in a covered test tube, place in a boiling water bath for 30 min (cover tightly to prevent water loss), shake every 10 min. (2) After cooling, add 1 mL reagent three in the test tube, shake for 30 s, stand for a while, let the pigment transfer to toluene; take 0.8 mL -1 mL upper solution in 1 mL glass cuvette, colorimetric at 520 nm wavelength, record the absorbance A.

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

[0162] = 19.2 x (A520nm+0.0021) ÷ W.

[0163] Wherein, the sample preparation method is: take about 0.1 g of tissue, add 1 mL of extraction solution, and homogenize in ice bath; then place in a 90℃ oscillation extraction for 10 min; 10000g, 25℃ centrifuge for 10 min, take the supernatant, cool and measure.

[0164] 6) Malondialdehyde determination

[0165] The malondialdehyde (MDA) test kit (Suzhou Keming Biotechnology Co., Ltd., MDA-2-Y) was used to determine the proline content.

[0166] The malondialdehyde determination included: take 0.5 g of leaf, add 2 ml of pre-cooled 0.05 mol / l pH 7.8 phosphate buffer, add a small amount of quartz sand, grind into homogenate in an ice bath mortar, transfer to a 5 ml centrifuge tube, dilute to 5 ml with buffer, centrifuge at 4500 r / min for 10 min, the supernatant is the malondialdehyde extract. Take 2 ml of extract in a calibrated test tube, add 3 ml of 5% thiobarbituric acid solution in 3% trichloroacetic acid, boil in a water bath for 10 min, centrifuge at 4500 r / min for 10 min, the supernatant is measured at 532, 600 nm wavelength with distilled water as blank control.

[0167] MDA content (nmol / g fresh weight) = [ΔA x Vtot / (ε x d) x 109] ÷ (W x Vsample ÷ Vtotal)

[0168] = 25.8 x ΔA ÷ W.

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

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

[0171] Table 3 Effect of strain ZBSF BH07 on grape leaf protective enzymes

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

[0173] Note: The small letters after the numbers indicate the significance of the difference, and the same letters indicate that there is no significant difference between the results,

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

[0175] Example 3, Detection of the effect of strain ZBSF BH07 in preventing and treating grape white rot

[0176] 4.1, Test agent

[0177] 4.1.1 Spore suspension of grape white rot fungus

[0178] Preparation of grape white rot fungus Coniella vitis spore suspension: Grape white rot fungus was cultured on PDA plates at 28°C, and after sufficient spore production, 10 mL of sterile 1% glucose solution was added, and the spores were washed down with a sterile cotton swab, and then a sterile 1% glucose solution was prepared to adjust the spore content to 1 x 10 7 cfu / mL

[0179] 4.1.2 Preparation of strain ZBSF BH07 fermentation broth

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

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

[0182] The experiment was treated with grape Botryosphaeria dothidea, fermentation broth, fermentation broth + Botryosphaeria dothidea (prevention) treatment, fermentation broth + Botryosphaeria dothidea (treatment) treatment. Select the appearance of neat, no disease and insect pests, no trauma red globe grape fruit, first washed with tap water, then use 75% alcohol disinfection, 2 min after the sterile filter paper dry. The fruit was placed in a sterile filter paper with a diameter of 100 mm in a petri dish, add sterile water, use a sterile inoculation needle to prick a 1 mm wound on each fruit, 20 fruits for each treatment, repeated 3 times.

[0183] The specific experimental method is as follows:

[0184] 1) Grape Botryosphaeria dothidea treatment: inoculate 40 μL grape Botryosphaeria dothidea spore suspension at the wound site.

[0185] 2) Fermentation broth treatment: inoculate 40 μL fermentation broth of strain ZBSF BH07 at the wound site

[0186] 3) Prevention of grape Botryosphaeria dothidea fermentation broth + Botryosphaeria dothidea treatment: inoculate 20 μL fermentation broth of strain ZBSF BH07 at the wound site 24 h, then inoculate 20 μL grape Botryosphaeria dothidea spore suspension.

[0187] 4) Treatment of grape Botryosphaeria dothidea fermentation broth + Botryosphaeria dothidea treatment: inoculate 20 μL grape Botryosphaeria dothidea spore suspension at the wound site 24 h, then inoculate 20 μL fermentation broth of strain ZBSF BH07. The above treatments were incubated at 28℃ for 7 days, and the disease was counted and the control effect was calculated.

[0188] Botryosphaeria dothidea grading standard

[0189] 0 level: no disease;

[0190] 1 level: the area of disease is ≤5% of the fruit area;

[0191] 3 level: the area of disease is 5%-20% of the fruit area;

[0192] 5 level: the area of disease is 21%-50% of the fruit area;

[0193] 7 level: the area of disease is 51%-75% of the fruit area;

[0194] 9 level: the area of disease is 76%-100% of the fruit area.

[0195] Disease index = 100 x ∑ (level of each disease leaf / fruit x corresponding level) / (total number of leaves / fruits surveyed x highest level representative value)

[0196] Preventive effect = [(disease index of sterile water control - disease index of treatment) / disease index of sterile water control] x 100% The experimental data were analyzed for significant difference by DPS software and plotted by Microsoft Excel software.

[0197] The results are shown in Table 4 and Figure 8 The results show that the fermentation liquor of strain ZBSF BH07 has better control effect on the white rot of red globe grape fruit than inoculation of white rot fungus alone. The incidence and disease index are significantly reduced. And the preventive effect is greater than the treatment effect.

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

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

[0200] Note: The small letters after the numbers in the table indicate significant difference. Treatments with at least one same letter are not significantly different at the 0.05 level, and treatments without same letter are significantly different at the 0.05 level.

[0201] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. A Burkholderia caryophila ZBSF BH07, characterized in that, The classification and naming of the bacteria is Burkholderia gladioli, which was preserved in the China General Microbiological Culture Collection Center on April 1, 2024, with the preservation number CGMCC No. 30228 and the address of the preservation unit being No. 3, Beichen West Road, Chaoyang District, Beijing.

2. A product comprising the B. taraxacum ZBSF BH07 of claim 1, characterized by, The product is a bacterial agent or a bacterial fertilizer.

3. The product of claim 2, wherein The bacterial agent or the bacterial fertilizer contains the Burkholderia gladioli ZBSF BH07 and / or contains the culture of the Burkholderia gladioli ZBSF BH07.

4. A fermentation method of the Burkholderia gladioli ZBSF BH07 according to claim 1, wherein the Burkholderia gladioli ZBSF BH07 is inoculated into a fermentation medium to carry out fermentation culture.

5. Any one of the following A1) to A8), characterized in that: A1) the Burkholderia gladioli ZBSF BH07 according to claim 1 is used in the preparation of a product for promoting the growth of plants; A2) the Burkholderia gladioli ZBSF BH07 according to claim 1 or the product according to claim 2 is used in promoting the growth of plants; A3) the Burkholderia gladioli ZBSF BH07 according to claim 1 is used in the preparation of a plant pathogenic bacteria inhibitor; A4) the Burkholderia gladioli ZBSF BH07 according to claim 1 or the product according to claim 2 is used in inhibiting plant pathogenic bacteria; A5) the Burkholderia gladioli ZBSF BH07 according to claim 1 is used in the preparation of a product for preventing and / or treating plant white rot; A6) the Burkholderia gladioli ZBSF BH07 according to claim 1 or the product according to claim 2 is used in preventing and / or treating plant white rot; A7) the Burkholderia gladioli ZBSF BH07 according to claim 1 or the product according to claim 2 is used in the preparation of siderophores; the plant is grape; the promotion of the growth of the plant is the promotion of the vegetative growth of the plant; the plant pathogenic bacteria are at least one of Coniella vitis, Colletotrichum aenigma, Fusarium oxysporum, Pestalotiopsis clavispora, Diaporthe eres, Alternaria viticola, Botryosphaeria dothidea, and Botrytis cinerea.

6. The use according to claim 5, characterized in that the promotion of the vegetative growth of the plant is the promotion of the growth of the aboveground part of the plant, the promotion of the increase of the fresh weight of the plant, and / or the promotion of the increase of the dry weight of the plant.

7. A method of promoting plant growth, characterized by, The method comprises culturing the Burkholderia gladioli ZBSF BH07 of claim 1 in a microbial culture medium, collecting the culture, and treating a plant with the culture; the plant being a grape.

8. A method for preventing and / or treating plant diseases, characterized by, The method comprises culturing the Burkholderia gladioli ZBSF BH07 of claim 1 in a microbial culture medium, collecting the culture, and treating a plant with the culture; the plant being a grape. The pathogen of the plant disease is at least one of Coniella vitis, Colletotrichum aenigma, Fusarium oxysporum, Pestalotiopsis clavispora, Diaporthe eres, Alternaria viticola, Botryosphaeria dothidea, and Botrytis cinerea.

Images