Brucella with antibacterial activity and application thereof

Abstract

The invention discloses a Brucella with antibacterial activity and application of the Brucella. The invention discloses Burkholderia sp.HNIV0701, and the preservation number of the Burkholderia sp.HNIV0701 is GDMCC No: 66007. The Burkholderia sp.HNIV0701 disclosed by the invention has a stable, efficient and broad-spectrum antibacterial property, has an inhibition effect on a plurality of plant pathogenic bacteria such as Botryosphaeria dothidea, Phytophthora nicotianae, Fusarium solani, Alternaria alternata, Thanatepora cucurbitae, Ralstonia solanacearum and the like, and also can be used for inhibiting Staphylococcus aureus and / or Listeria monocytogenes, so that the Burkholderia sp.HNIV0701 can be used for inhibiting a plurality of plant pathogenic bacteria such as the Botryosphaeria dothidea, the Phytophthora nicotianae, the Fusarium solanum, the Alternaria solanacearum and the like. The Brucella HNIV0701 can hydrolyze organic phosphorus and can be used for soil improvement. The Brucella HNIV0701 has nitrogen fixation capability and has the potential of promoting plant growth.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and in particular relates to a Buckminster bacterium with antibacterial activity and application thereof. Background Art

[0002] Agricultural production is a major issue affecting the national economy and people's livelihoods, and the occurrence of plant diseases is one of the key factors restricting agricultural production. Chemical pest control, as the primary means of controlling plant diseases, has led to a series of environmental and social issues, including pesticide residues and non-target damage. With the improvement of agricultural productivity in my country, green pest control is being vigorously promoted in agriculture. Biological control, as an environmentally friendly pest control method, is gaining increasing attention.

[0003] Using antagonistic bacteria attached to plants to control plant diseases and insect pests is an effective way, and screening antagonistic strains with good effects and stable activity is a prerequisite for ensuring the success of biological control. The main biocontrol bacteria currently studied are Burkholderia spp., Pseudomonas spp., Bacillus spp., etc. Plant rhizosphere bacteria exist in large numbers around the root system of plants. These bacteria can colonize on the surface of plants and have a stable living environment. Some can antagonize pathogens, and some can induce plant disease resistance. This type of microorganism is non-toxic to humans and animals, is environmentally friendly, and can be developed as a resource for biocontrol bacteria for plant diseases. The microbial germplasm resources in the environment are very rich, and less than 1% of the microorganisms can be cultured in the laboratory. There are approximately 7.3×10 29 There are a large number of "difficult to culture / uncultured" microbial germplasm resources in the environment that need to be developed. New microbial germplasm resources have the potential for biosynthesis of a variety of secondary metabolites. The development and utilization of "difficult to culture / uncultured" microbial germplasm resources in the environment are currently hot topics and difficulties in research at home and abroad, and are also important resources for the development of disease control strains. Summary of the Invention

[0004] The present invention provides a new Burkholderia sp. HNIV0701 species with antibacterial activity. Comparison analysis with the EzBioCloud database showed that the 16S rDNA of the active strain had a maximum nucleotide identity of greater than 98.7% with the 16S rDNA of multiple Burkholderia spp. type strains in the database, indicating that the strain belongs to the genus Burkholderia. Whole-genome average nucleotide identity analysis showed that the ANI of strain HNIV0701 with strains with high 16S rDNA similarity was less than 95%, indicating that strain HNIV0701 is a new species of Burkholderia.

[0005] The Burkholderia sp. HNIV0701 of the present invention was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on March 13, 2025, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Postal Code: 510070, Deposit Number: GDMCC No: 66007.

[0006] The second object of the present invention is to provide a bacterial agent containing the above-mentioned Bucknerella HNIV0701.

[0007] The third object of the present invention is to provide the use of the above-mentioned Buckminster bacteria HNIV0701 or bacterial agent in the preparation of antibacterial drugs.

[0008] Preferably, the antibacterial agent is an antibacterial or antifungal agent, and the bacteria may be Fusarium solani, Alternaria alternate, Thanatephorus cucumeris, Ralstonia solanacearum, Staphylococcus aureus and / or Listeria monocytogenes; the fungus may be Botryosphaeria dothidea or Phytophthora parasitica.

[0009] The fourth object of the present invention is to provide the use of Buckeye bacteria HNIV0701, its culture, its fermentation liquid or its isolated active ingredients in the prevention and / or treatment of grape canker, tobacco black shank, tobacco Fusarium root rot, tobacco brown spot, apple leaf spot, wheat sheath blight, cotton damping-off, tobacco target spot, tobacco bacterial wilt and / or tomato bacterial wilt.

[0010] Buckeye bacteria HNIV0701 has the ability to dissolve phosphorus and fix nitrogen, can enhance the absorption of elements such as phosphorus, nitrogen, and iron by plants, and has the effect of promoting plant growth. Therefore, the fifth object of the present invention is to provide an application in the preparation of a preparation for promoting plant growth.

[0011] The Burkholderia sp. HNIV0701 of the present invention exhibits stable, highly effective, and broad-spectrum antimicrobial properties, inhibiting a variety of plant pathogens, including Botryosphaeria dothidea, Phytophthora parasitica, Fusarium solani, Alternaria alternate, Thanatephorus cucumeris, and Ralstonia solanacearum. It also inhibits the growth of foodborne pathogens, such as Staphylococcus aureus and Listeria monocytogenes. Burkholderia sp. HNIV0701 can hydrolyze organic phosphorus and can be used for soil improvement. Burkholderia sp. HNIV0701 also has nitrogen-fixing properties and has the potential to promote plant growth.

[0012] Burkholderia sp.HNIV0701 was deposited in Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on March 13, 2025, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Postal Code: 510070, Deposit Number: GDMCC No: 66007. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 A "trap" device for uncultured / difficult-to-culture bacteria;

[0014] Figure 2 This is the phylogenetic tree of Buckeye bacteria HNIV0701 based on 16S rDNA sequence analysis;

[0015] Figure 3 This is the inhibitory spectrum of B. buxiformis HNIV0701;

[0016] Figure 4 To test the phosphate solubilization ability of B.

[0017] Figure 5 It is a test of the nitrogen fixation ability of Buckeye bacteria HNIV0701. DETAILED DESCRIPTION

[0018] In order to deepen the understanding of the present invention, the present invention will be further described in detail below with reference to the embodiments.

[0019] Example 1: Isolation of active strain Burkholderia sp. HNIV0701

[0020] To isolate uncultured / difficult-to-cultivate bacteria from soil, we fabricated a "trap" device based on previous literature. A 0.45 μm pore size cellulose membrane was attached to the bottom of a steel ring (outer diameter 4.0 cm, inner diameter 2.5 cm, thickness 0.5 cm). Then, 2.5 ml of 0.5% soft agar containing vitamins (folic acid, 2.0 mg / L; pyridoxine hydrochloride, 10.0 mg / L; riboflavin, 5.0 mg / L; biotin, 2.0 mg / L; thiamine, 5.0 mg / L; niacin, 5.0 mg / L; calcium pantothenate, 5.0 mg / L; vitamin B12, 0.1 mg / L; and para-aminobenzoic acid, 5.0 mg / L) was added. After the soft agar solidified, another 0.22 μm pore size cellulose membrane was attached to the top of the steel ring. The trap was placed in a Petri dish (15 cm in diameter) containing moist soil (200-300 g) ( Figure 1 After 21 days at room temperature in the dark, the soft agar was removed and chopped, then ground with a sterile pestle and suspended in sterile water. The suspension was serially diluted, and 100 μL was spread onto an R2A plate and incubated at 28°C for 2 days. Colonies were picked based on their morphology and streaked for purification. The resulting strain was stored in a -80°C freezer. The strain numbered HNIV0701 is the Burkholderia sp. HNIV0701 of the present application.

[0021] Example 2: Identification of Burkholderia sp. HNIV0701 strain

[0022] 16S rDNA sequence analysis of active strains: The 16S rDNA of the target strain was amplified using the extracted total DNA as a template and universal 16S rDNA primers (1492R: GGTTACCTTGTTACGACTT, 27F: AGAGTTTGATCCTGG CTCA). PCR amplification conditions included 30 cycles of pre-denaturation at 95°C for 5 minutes, denaturation at 94°C for 1 minute, annealing at 58°C for 30 seconds, extension at 72°C for 1.5 minutes, and constant temperature at 72°C for 10 minutes, followed by storage at 4°C. The PCR amplification product was sent to a sequencing company (BGI) for sequencing, resulting in the 16S rDNA sequence of the strain (the nucleotide sequence is shown in SEQ ID NO. 1). Whole-genome sequencing of the strain was commissioned by Beijing ThermoLily Biotechnology Co., Ltd.

[0023] Comparison of 16S rDNA sequences of active strains and construction of phylogenetic tree: The 16S rDNA sequences of the strains obtained by sequencing were submitted to the EzBioCloud database for analysis. The results showed that the nucleotide identity of the 16S rDNA sequence of strain HNIV0701 was higher than 98.7% with the 16S rDNA nucleotides of 26 type strains of Burkholderia spp. (Table 1). Based on the 16S rDNA sequence information, the neighbor-joining phylogenetic tree was constructed using Mega5 software. The results showed that strain HNIV0701 was closely related to Burkholderia pyrrocinia DSM10685. T , Burkholderia stabilis ATCC BAA-67 T , Burkholderia stagnalis LMG28156 T Located on the same small branch ( Figure 2 ), indicating that strain HNIV0701 belongs to the genus Burkholderia. To further confirm its taxonomic status, we selected the genome of a model strain with a 16S rDNA identity greater than 98.7% with that of strain HNIV0701 and performed an average nucleotide identity analysis with the genome of strain HNIV0701. The results showed that strain HNIV0701 and Burkholderia pyrrocinia DSM10685 T The highest ANIb value was 93.35% (Table 2), which was lower than the threshold of 95% for identification as the same species, indicating that strain HNIV0701 was a new species of the genus Bucknerella.

[0024] Table 1. 16S rDNA nucleotide sequence identity between Burkholderia sp. HNIV0701 and the model strain

[0025]

[0026]

[0027] Table 2. ANIb analysis of Burkholderia sp. HNIV0701 and closely related strains

[0028]

[0029]

[0030] The strain HNIV0701 was named Burkholderia sp. HNIV0701 was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on March 13, 2025, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Postal Code: 510070, Deposit Number: GDMCC No: 66007.

[0031] Example 3, Determination of the Inhibitory Spectrum of Buckeye Bacteria HNIV0701

[0032] The antifungal ability test of Buckeye's fungus HNIV0701 is as follows: a 0.5 cm-sized pathogenic fungus cake is inoculated into the center of a PDA plate, 5 μL of Buckeye's fungus HNIV0701 bacterial solution is spotted 3 cm around it, and cultured at 28°C. After 3-5 days, the inhibitory ability of Buckeye's fungus HNIV0701 against plant pathogenic fungi is observed and recorded.

[0033] The antibacterial activity of Buckeye bacteria HNIV0701 was tested as follows: Buckeye bacteria HNIV0701 was cultured in LB at 28°C for 24 hours. 5 μL of the culture was spotted onto a pre-incubated PDA plate and incubated at 28°C for 48 hours. Pathogenic bacteria were cultured in LB for 24 hours, then added to a 1% agar-containing LB solid medium melted at approximately 50°C. The agar was then poured onto a plate pre-inoculated with Buckeye bacteria HNIV0701 to create a double-layer plate. After 24 hours, the antibacterial activity of Buckeye bacteria HNIV0701 was observed and recorded.

[0034] The results showed that HNIV0701 not only had an inhibitory effect on a variety of plant pathogenic fungi such as Botryosphaeria dothidea, Phytophthora parasitica, Fusarium solani, Alternaria alternate, Thanatephorus cucumeris and Ralstonia solanacearum, but also inhibited the growth of foodborne pathogens such as Staphylococcus aureus and Listeria monocytogenes. Figure 3 ).

[0035] Example 4: Phosphate-solubilizing ability test of Buckeye bacteria HNIV0701

[0036] The formula of the phosphate solubilization ability test medium National Botanical Research Institute's phosphate growth medium (NBRIP) (1L) is as follows: glucose, 10g; Ca3(PO4)2 5g; MgCl2·6H2O 5g; MgSO4·7H2O 0.25g, KCl 0.2g, (NH4)2SO4 0.1g, and the pH was adjusted to 7.0 before sterilization.

[0037] Prepare phosphate solubilization medium and pour it onto plates. 5 μL of cultured B. edulis HNIV0701 was inoculated onto NBRIP phosphate solubilization test medium and cultured at 28°C for about a week. The larger the diameter of the phosphate solubilization ring, the stronger the phosphate solubilization ability. The results showed that B. edulis HNIV0701 could produce a phosphate solubilization ring on the NBRIP phosphate solubilization test medium ( Figure 4 ), indicating that Bucknerella HNIV0701 can hydrolyze organic phosphorus and can be used for soil improvement.

[0038] Example 5: Detection of Nitrogen Fixation Ability of Buckeye Bacteria HNIV0701

[0039] The formula of nitrogen fixation ability test medium (1 L) is as follows: Glucose 20 g, K2HPO4 0.25 g, MgSO4 0.122 g, NaCl 0.005 g, FeSO4·7H2O 0.0005 g, and the pH is adjusted to 7.0 before sterilization.

[0040] The overnight culture of Buckeye bacteria HNIV0701 was washed twice with sterile water and inoculated into nitrogen fixation test medium at a volume ratio of 1:100 and cultured at 28°C for about a week. Strains with nitrogen fixation ability will grow in the nitrogen fixation test medium. The results showed that Buckeye bacteria HNIV0701 grew in the nitrogen fixation test medium ( Figure 5 ), indicating that Bucknerella HNIV0701 has nitrogen fixation ability and has the potential to promote plant growth.

[0041] SEQ ID NO.1

[0042]

Claims

1. Burkholderia sp. HNIV0701, deposit number: GDMCC No: 66007.

2. A bacterial agent, characterized in that The invention contains the Buckeye bacteria HNIV0701 as claimed in claim 1 as an active ingredient.

3. Use of the Buckeye bacteria HNIV0701 described in claim 1 or the bacterial agent described in claim 2 in the preparation of antibacterial drugs.

4. The use according to claim 3, characterized in that: The antibacterial property is anti-bacteria or anti-fungus.

5. The use according to claim 4, characterized in that: The bacteria are Ralstonia solanacearum, Staphylococcus aureus and / or Listeria monocytogenes.

6. The use according to claim 4, characterized in that: The fungi are Fusarium solani, Alternaria alternate, Thanatephorus cucumeris, Botryosphaeria dothidea and / or Phytophthora parasitica.

7. Use of Buckeye bacteria HNIV0701, its culture, its fermentation liquid or its separated active ingredients according to claim 1 in drugs for preventing and / or treating grape canker, tobacco black shank, tobacco Fusarium root rot, tobacco brown spot, apple leaf spot, wheat sheath blight, cotton damping-off, tobacco target spot, tobacco bacterial wilt and / or tomato bacterial wilt.

8. Use of the Buckeye bacteria HNIV0701 according to claim 1 in the preparation of a plant growth promoting preparation.

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