Bacillus vallismortis BvABC5 with broad-spectrum antibacterial activity and application of bacillus vallismortis BvABC5

Abstract

The invention relates to the field of microorganisms, in particular to bacillus vallismortis BvABC5 with broad-spectrum antibacterial activity and application of the bacillus vallismortis BvABC5. According to the invention, a strain of bacillus vallismortis is separated and identified, and it is found that the strain has relatively broad-spectrum antibacterial activity; indoor bioassay and field tests are carried out by taking the prunus cerasifera pall pathogenic variety as a tested pathogenic bacterium, and results show that the strain can significantly inhibit the prunus cerasifera pall pathogenic variety from infecting peach leaves and has a good control effect on peach bacterial shot hole.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to Bacillus valerianus BvABC5 with broad-spectrum antibacterial activity and applications thereof. Background Art

[0002] Bacterial diseases are common diseases in agricultural production. Take peach bacterial puncture disease as an example. The disease is caused by the tree-borne xanthomonas arboricola pv. pruni. In the past 10 years, it has broken out many times in the main peach producing areas in the north and south of my country, causing serious economic losses and gradually becoming a major disease in peach production.

[0003] Bacillus spp. are an important resource microbial resource, encompassing a diverse range of biocontrol strains with diverse functions, including antagonism, inducing plant disease resistance, nutrient and spatial competition, and bacteriolysis. These strains demonstrate significant application value in crop disease control. For example, Bacillus subtilis, Bacillus pumilus, Bacillus megaterium, Bacillus licheniformis, and Bacillus amyloliquefaciens demonstrate excellent control against wheat sheath blight, tomato gray mold, tobacco black shank, cotton Verticillium wilt, and rice blast. However, the biocontrol capabilities of Bacillus spp. are primarily focused on controlling fungal diseases, and strains with strong efficacy against bacterial diseases are relatively scarce. Summary of the Invention

[0004] The purpose of the present invention is to provide a Bacillus thuringiensis strain.

[0005] Another object of the present invention is to provide applications of the above-mentioned Bacillus subtilis strain.

[0006] Another object of the present application is to provide a fungicide for preventing and controlling bacterial diseases.

[0007] According to the Bacillus vallismortis strain BvABC5 of the present application, its deposit number is CGMCC No. 32262. The present invention collected soil samples from the Gobi Desert in Baicheng County, Xinjiang Uygur Autonomous Region, isolated and purified to obtain a pure strain. After morphological and molecular marker identification, it was determined to be Bacillus vallismortis, and the strain was named BvABC5.

[0008] The present invention provides the application of the Bacillus valerianus strain BvABC5 for preventing and controlling bacterial diseases of crops.

[0009] The strain of the present application is used to develop a fungicide for preventing and treating bacterial diseases. As a single product or a combination product with chemical agents, it is suitable for preventing and treating bacterial diseases such as peach bacterial perforation disease, and has good application prospects.

[0010] The strain was used to conduct a plate confrontation experiment with plant pathogenic bacteria. The results showed that BvABC5 significantly inhibited the growth of Xanthomonas arboricola pv.pruni, Xanthomonas arboricola pv.juglandis, Ralstonia solanacearum, Pectobactrium carotovorum, Xanthomonas campestris pv.campestris, Pseudomonas syringae pv.maculicola, Pseudomonas syringae pv.tomato, and Acidovorax syringae. citrulli, showing a relatively broad-spectrum antibacterial activity; with the tree-borne xanthomonas plum pv. v. as the test pathogen, indoor bioassays were conducted using detached peach leaves, and the results showed that BvABC5 significantly inhibited the infection of the tree-borne xanthomonas plum pv. v.; spraying the mixture of BvABC5 strain and fermentation liquid in the field can effectively prevent and control peach bacterial shot hole disease, and its prevention effect is better than that of chemical fungicides such as zhongshengmycin and kasugamycin, and it has good compatibility with the insecticides spirotetramat, emamectin, lufenuron, chlorflucythrin, and the fungicidal pesticide difenoconazole, and their mixed use does not affect each other. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The colony morphology of the Bacillus thuringiensis strain BvABC5 of the present invention after culturing on LB medium for 7 days is shown;

[0012] Figure 2 The results of the plate confrontation experiment of the Bacillus subtilis strain BvABC5 of the present invention against pathogenic bacteria such as Ralstonia solanacearum and Acidophilus citrinum are shown;

[0013] Figure 3 The results of the plate confrontation experiment of the Bacillus dysvalleyi strain BvABC5 of the present invention against the xanthomonas aeruginosa pv.

[0014] Figure 4 It is shown that the Bacillus valerianus strain BvABC5 of the present invention significantly inhibits the infection of the tree-borne Xanthomonas plum pathovar in detached peach leaves.

[0015] The Bacillus vallismortis strain BvABC5 was deposited in the General Microbiology Center of China Culture Collection Administration, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. The classification name is Bacillus vallismortis, the deposit date is October 18, 2024, and the deposit number is CGMCC No. 32262. DETAILED DESCRIPTION

[0016] Example 1. Isolation, purification and identification of BvABC5 strain

[0017] The strain was isolated from a Gobi Desert soil sample collected in Baicheng County, Xinjiang Uygur Autonomous Region (2022). Take 10 g of soil sample, add 100 mL of sterile water, stir evenly, filter through three layers of gauze, collect the filtrate, and place it in an 80°C water bath for 20 minutes; transfer it to a clean bench, let it stand to room temperature, and draw the suspension. -1 , 10 -2 , 10 -3 , 10 -4 Dilute in series, take 100 μL and spread evenly on LB solid medium (10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, 15 g of agar, add distilled water to 1000 mL, pH 7.0), place in a 28 ° C incubator, and culture for 24-72 hours; according to the morphological classification of the obtained colonies, pick single colonies of different morphologies, streak on LB solid medium, and continue to culture; continue to pick single colonies, streak culture, repeat 3 times; select well-developed single colonies, inoculate into LB liquid medium (10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, add distilled water to 1000 mL, pH 7.0), culture at 28 ° C, 220 rpm for 12 hours, which is the purified strain, and store at -80 ° C. The preserved BvABC5 was inoculated on LB solid medium and cultured at 28 ° C for 7 days. It was observed that the colonies gradually grew and expanded, with a light brown feather-like surface, wavy expansion, and the formation of ring bands ( Figure 1Colonies were picked and inoculated into LB liquid medium and cultured at 28°C, 220 rpm for 16 h. The bacteria were collected by high-speed centrifugation, and genomic DNA was extracted and quantified using the CTAB method. 20 ng of genomic DNA was used as a template for PCR amplification using the universal bacterial 16S rDNA primers 27F (5'-GAGAGTTTGATCCTGGCTCAG-3') / 1492R (5'-TACGGTTACCTTGTTACGACTT-3'). The PCR product was sequenced to obtain a 1306-base 16S rDNA fragment (SEQ ID No: 1). Blast analysis in the NCBI database identified the strain as Bacillus vallismortis, which belongs to the phylum Firmicutes, class Bacilli, orders Bacillales, and family Bacillaceae.

[0018] SEQ ID No: 1

[0019] GAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATACAAGTCGA

[0020] GCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGG

[0021] GTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGCTTGT

[0022] TTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGC

[0023] GGCGCATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCAACGATGCGTAGCCGACCT

[0024] GAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAG

[0025] CAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATG

[0026] AAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGC

[0027] GGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGT

[0028] AATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTT

[0029] TCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGA

[0030] ACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATG

[0031] TGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGA

[0032] AAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTG

[0033] CTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCT

[0034] GGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGG

[0035] TGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCT

[0036] GACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTG

[0037] TCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATC

[0038] TTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAA

[0039] GGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAAT

[0040] GGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCA

[0041] GTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGAT

[0042] CAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAG

[0043] AGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGTAACAAGGTAACCGTA.

[0044] Example 2. Antibacterial analysis of BvABC5 strain against important plant pathogenic bacteria

[0045] The preserved BvABC5, as well as common important plant pathogens such as Ralstonia solanacearum and Acidophilus citrinum, were streaked onto LB solid medium and cultured at 28°C for 24 hours. Single colonies of different plant pathogens were picked and inoculated into LB liquid medium and cultured at 28°C and 220 rpm for 16 hours. The cells were collected by centrifugation (5000 rpm, 3 minutes) and resuspended in sterile water (OD 600 =1.0), draw 2 μL, and Figure 2 As shown in Figure A, the cells were inoculated into LB solid medium and cultured at 28°C for 24 h. Meanwhile, a single colony of BvABC5 was picked and inoculated into LB liquid medium and cultured at 28°C and 220 rpm for 16 h. The cells were collected by centrifugation (5000 rpm, 3 min) and resuspended in sterile water to an OD of 600=1.0; 2 μL of bacterial solution was aspirated and inoculated on both sides of the pathogenic bacteria cultured for 24 hours, with sterile water as the control. Each treatment was repeated 3 times. The plate was transferred to a 28°C incubator, inverted, and cultured in the dark. The growth of pathogens was observed and recorded after 7 days. The results showed that BvABC5 significantly inhibited the growth of important plant pathogenic bacteria such as Xanthomonas arborescens pv. walnut, Pseudomonas syringae pv. maculatus, Pseudomonas syringae pv. tomato, Ralstonia solanacearum, Pectinobacterium carotovora, Xanthomonas campestris pv. campestris, and Acidophilus citrinum. Figure 2 B), showing broad-spectrum antibacterial activity.

[0046] The BvABC5 and the pv. arborescens pv. were streaked onto LB solid medium and cultured at 28°C for 24 h. A single colony of the pv. arborescens pv. was picked and inoculated into LB liquid medium and cultured at 28°C and 220 rpm for 16 h. The cells were collected by centrifugation (5000 rpm, 3 min) and resuspended in sterile water (OD 600 =1.0); 2 μL of bacterial solution was drawn and streaked onto LB solid medium in a grid pattern, and cultured at 28°C for 24 h. At the same time, a single colony of BvABC5 was picked and inoculated into LB liquid medium, cultured at 28°C and 220 rpm for 16 h; the cells were collected by centrifugation (5000 rpm, 3 min) and resuspended in sterile water to an OD of 600 = 1.0; 2 μL of bacterial solution was inoculated into the center of the grid of a 24-hour-old tree-grown xanthomonas plum pathogen. Sterile water was used as a control. Each treatment was repeated three times. The plates were transferred to a 28°C incubator, inverted, and incubated in the dark. After 7 days, the growth of the pathogen was observed and recorded. The results showed ( Figure 3 ), BvABC5 showed obvious lytic activity against the tree-borne xanthomonas plum pathogenic variant and inhibited the normal growth of the pathogenic bacteria.

[0047] Example 3. Indoor bioassay results of BvABC5 strain against tree-growing xanthomonas plum pathovar

[0048] Take the preserved BvABC5 and the psoralen (Xap) of P. arborescens and streak them on LB solid medium respectively, and culture them at 28℃ for 16 hours. Pick the colonies and inoculate them into LB liquid medium, and culture them at 28℃ and 220rpm for 16 hours. Centrifuge the BvABC5 culture (5000rpm, 3min), collect the cells, and resuspend them in sterile water to an OD of 600 =0.2.

[0049] Two-month-old peach seedlings cultured in a greenhouse were selected. Mature leaves were selected and 10 μL of BvABC5 bacterial solution was injected into 3-4 locations on the underside of the leaf along the same side of the vein using a sterile syringe without the needle. Simultaneously, sterile water was injected into the corresponding location on the other side as a control. The treated peach seedlings were transferred to a greenhouse and cultured for another 12 hours. The bacterial solution of the affine Xanthomonas plum pv. spp. was centrifuged (5000 rpm, 3 minutes), the cells collected, and resuspended in sterile water to an OD of 0. 600 =1.0; Place three layers of wet filter paper in a sterile glass culture dish (200mm×50mm). Cut peach leaves that had been infiltrated with BvABC5 for 12 hours and place them on the filter paper. 3μL of Xap solution was aspirated and inoculated onto the surface of the leaf tissue infiltrated with BvABC5 or sterile water, and then slightly scratched. Transfer to a light incubator at 25℃ and incubate in the dark for 24 hours. Then, continue incubation and observation for 9 days under 16h light / 8h dark. The results showed that the water-soaked lesions caused by Xap on the leaf tissue infiltrated with BvABC5 were significantly smaller than those on the leaf tissue infiltrated with sterile water ( Figure 4 Figure A in the figure shows that the average area of ​​the lesions is 11.33 mm. 2 , the latter is 57.50mm 2 , the difference was extremely significant (p<0.001)( Figure 4 Figure B in the figure shows that BvABC5 effectively inhibits the infection of Xap on peach leaves.

[0050] Example 4. Field efficacy of BvABC5 against peach bacterial scald

[0051] Take the preserved BvABC5 strain, streak it on LB solid medium, and culture it at 28℃ for 24h. Pick the colony and inoculate it into 50mL LB liquid medium, and culture it at 28℃ and 220rpm for 16h. Take 5mL of seed culture liquid and transfer it to 1L LB liquid medium, and continue to culture it at 28℃ and 220rpm for 24h. Collect the culture liquid and dilute the BvABC5 culture liquid with water at a ratio of 1:5 to an OD of 600 =0.3-0.4. Subsequently, according to Table 1, a fungicide (40% difenoconazole) and an insecticide (10% chlorfenapyr, 2.5% lambda-cyhalothrin) were added to prepare Agent A. Simultaneously, Agent B (containing a bactericide, insecticide, and fungicide) and Agent C (containing an insecticide and fungicide) were prepared according to Table 1 as controls. Eighteen susceptible peach trees of the late 9th variety (8 years old) were selected and divided into three groups (6 trees / group) to receive Agent A, Agent B, and Agent C, respectively. Spraying began at the end of flowering using an electric backpack sprayer, with a dosage of approximately 4 L per tree. Four sprays were repeated every 10 days.

[0052] Table 1. Pharmaceutical Mixing

[0053]

[0054]

[0055] Ten days after discontinuing treatment, 25 leaves from each peach tree were randomly selected from the four directions (east, west, south, and north). The bacterial shoot disease index for each peach tree was calculated and analyzed according to Table 2. The results showed that the index for peach trees sprayed with agent A (containing the BvABC5 solution) was 0.56, while that for agents B (containing the combination of kasugamycin and zhongshengmycin) was 1.89, and that for agent C (without a bactericide) was as high as 5.20. Therefore, field spraying with BvABC5 is effective in controlling bacterial shoot disease in peaches, and is more effective than the bactericide combination of kasugamycin and zhongshengmycin. Furthermore, according to the tests, BvABC5 exhibits good compatibility with the insecticides chlorfenapyr and cyhalothrin, as well as the antifungal agent difenoconazole, allowing them to be used together without interfering with each other's effects.

[0056] Table 2. Grading standards for peach bacterial puncture

[0057]

[0058] Disease index = ∑(disease level × number of diseased leaves at each level) / (9 × total number of leaves) × 100

[0059] The above embodiments are only used to understand the technical solutions of the present application and do not limit the scope of protection of the present application.

Claims

1. Bacillus vallismortis strain BvABC5, characterized in that The deposit number of the Bacillus vallismortis strain BvABC5 is CGMCC No.32262.

2. Use of the Bacillus vallismortis strain BvABC5 according to claim 1.

3. The use according to claim 2, characterized in that The Bacillus vallismortis strain BvABC5 is used to inhibit pathogenic bacteria.

4. The use according to claim 3, characterized in that The pathogenic bacteria are Xanthomonas arboricola pv.pruni, Xanthomonas arboricola pv.juglandis, Ralstonia solanacearum, Pectobactrium carotovorum, Xanthomonas campestris pv.campestris, Pseudomonas syringae pv.maculicola, Pseudomonas syringaepv.Tomato or Acidovorax citrulli.

5. A fungicide for preventing and treating bacterial diseases, characterized in that: The active ingredient of the fungicide comprises the spores of the Bacillus vallismortis strain BvABC5 according to claim 1.

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