CN111117936B - Bacillus amyloliquefaciens TBA03 and application thereof - Google Patents
Bacillus amyloliquefaciens TBA03 and application thereof Download PDFInfo
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- CN111117936B CN111117936B CN202010093203.5A CN202010093203A CN111117936B CN 111117936 B CN111117936 B CN 111117936B CN 202010093203 A CN202010093203 A CN 202010093203A CN 111117936 B CN111117936 B CN 111117936B
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Abstract
The invention provides a bacillus amyloliquefaciens TBA03 and application thereof, belonging to the technical field of microorganism and biological control. The classification of the bacillus amyloliquefaciens TBA03 is named as bacillus amyloliquefaciens (A) (Bacillus amyloliquefaciens) The strain is preserved in China general microbiological culture Collection center on 12 and 9 months in 2019, and the preservation number is CGMCC NO. 19108. The Bacillus amyloliquefaciens TBA03 is Ralstonia solanacearum (B.solani:)Ralstonia solanacearum) The antagonistic bacteria can effectively prevent and treat the diseases of the ralstonia solanacearum, and provides an environment-friendly, ecological and safe biological prevention and treatment method for relieving the diseases of the ralstonia solanacearum.
Description
Technical Field
The invention belongs to the technical field of microorganism and biological control, and particularly relates to a bacillus amyloliquefaciens TBA03 and application thereof.
Background
Tobacco is a leaf crop. As a big tobacco producing country, China has the tobacco planting area, the tobacco yield and the tobacco sales volume all living in the first place in the world. Tobacco is a crop which is forbidden to be continuously planted, if the tobacco is continuously planted on the same plowing land, the phenomena of yield reduction, tobacco quality deterioration and growth condition deterioration are caused, and the yield and the quality of the tobacco are seriously influenced. According to statistics, the direct and indirect economic loss caused by continuous tobacco cropping reaches 40 billion yuan RMB every year, and the sustainable development of the tobacco industry in China is seriously threatened. Therefore, how to relieve the continuous cropping obstacle of tobacco is an important problem to be solved urgently at present, and becomes a hot spot of research of the same lines at home and abroad.
At present, chemical reagent method, rotation and intercropping method and microbial subtraction method are mainly adopted for treating the continuous cropping obstacle of tobacco at home and abroad. Chemical reagents such as fertilizer and the like added into the continuous cropping tobacco soil have the advantages of quick effect, convenience, high economic benefit and the like, but the effective period is short, and the environment is polluted by excessive or long-term use, so that the plant quality is influenced; the rotation and intercropping method has a considerable effect on eliminating continuous cropping obstacles, but the method is rarely applied at present due to the shortage of soil resources; the microorganism elimination method has the advantages of inhibiting the growth of harmful microorganisms, improving the growth characteristics of tobacco, lightening tobacco diseases, along with environmental protection, ecology, safety and the like by screening and adding beneficial microorganisms into soil.
According to reports, the soil microbial structure is one of important factors influencing tobacco diseases and causing continuous cropping obstacles of tobaccos. The utilization of antagonistic bacteria for plant disease control is considered to be a scientific, reasonable, efficient and environment-friendly measure. Bacillus amyloliquefaciens of the inventionBacillus amyloliquefaciens) TBA03 can obviously inhibit the growth of ralstonia solanacearum in tobacco soil, and can achieve the purpose of relieving the tobacco bacterial wilt disease.
Disclosure of Invention
The invention aims to provide a bacillus amyloliquefaciens strain TBA03 and application thereof in relieving tobacco bacterial wilt diseases. Studies have shown that the Bacillus amyloliquefaciens strain TBA03 of the present invention has a inoculation concentration of 4.0X 108When the soil is air-dried by CFU/kg, the purpose of remarkably relieving the tobacco bacterial wilt disease can be achieved.
In order to achieve the purpose, the invention adopts the following technical scheme:
a bacillus amyloliquefaciens TBA03 is classified and named as: bacillus amyloliquefaciens (A)Bacillus amyloliquefaciens) The strain is preserved in China general microbiological culture Collection center (CGMCC) in 2019, 12 and 9 days, the preservation number is CGMCC NO.19108, the address is the institute of microbiology of China academy of sciences No. 3 of Navy, No.1 institute of Western No.1, North Chen of the Chaoyang district, Beijing city, and the strain is antagonistic bacterium of the pathogenic bacterium Ralstonia solanacearum.
Bacillus amyloliquefaciens TBA03 for preventing and treating ralstonia solanacearum (L.) (Ralstonia solanacearum) The use of (1).
A microbial preparation containing the bacillus amyloliquefaciens TBA 03.
A microbial preparation containing the bacillus amyloliquefaciens TBA03 is used for preventing and treating Ralstonia solanacearum (L.) KuntzeRalstonia solanacearum) The use of (1).
Bacillus amyloliquefaciens (A)Bacillus amyloliquefaciens) The screening protocol for TBA03 is shown in FIG. 1. Firstly, collecting the root soil of the continuous cropping flue-cured tobacco K326 in 30 years in Fuliang shed village of Yuxi city, Yunnan province, obtaining the pathogenic bacteria of the continuous cropping tobacco soil as ralstonia solanacearum (Ralstonia solanacearum) through high-throughput sequencing and analysisRalstonia). Subsequently, bacterial wilt pathogens were enriched and cultured using a selective medium for bacterial wilt pathogens (SMSA medium). Designing a ralstonia solanacearum specific primer, carrying out PCR amplification to obtain pure ralstonia solanacearum, adding the pure ralstonia solanacearum into conventional rice soil, verifying the pathogenicity of the ralstonia solanacearum to flue-cured tobacco K326 seedlings, obtaining ralstonia solanacearum causing tobacco ralston diseases, screening antagonistic bacteria having the optimal antagonistic action on the ralstonia solanacearum by adopting a plate antagonistic method, carrying out morphological and 16S nuclear RNA gene (16S rDNA) related identification work on the screened antagonistic bacteria, and finally obtaining the bacillus amyloliquefaciens TBA 03.
The method comprises the following specific steps:
(1) soil collection: at the end of 08 months in 2018, collecting the K326 rhizosphere soil of continuous cropping flue-cured tobacco in Fuliang shed countryside of Yuxi city in Yunnan province and the control soil without planting tobacco in the vicinity of a tobacco area in 30 years respectively, dividing the soil sample by a quartering method, storing the collected soil sample in a refrigerator at the temperature of-80 ℃, and preparing the collected soil for analyzing the structural diversity of the microbial community of the next soil and screening pathogenic bacteria.
(2) And (3) analyzing structural diversity of the soil microbial community: genomic DNAs of non-tobacco-planted Control Soil (CS) and continuous cropping flue-cured tobacco rhizosphere soil (CCS) were extracted, respectively, the purity and concentration of the extracted DNAs were checked by agarose gel electrophoresis, and PCR amplification of CS and CCS genomic DNAs was performed using specific primers (341F: CCTAYGGGRBGCASCAG and 543R: ATTACCGCGGCTGCTGG) with Barcode. The DNA sample is sent to Beijing Ovwison Gene science and technology limited, the Illumina Miseq PE300 high-throughput sequencing platform is utilized for sequencing, and the bacterial 16S rDNA V3-V4 region is selected for microbial diversity detection. Sequencing results show that the ralstonia solanacearum is an important pathogenic bacterium which has higher flora quantity and causes continuous cropping obstacle of tobaccos in the area.
(3) Enrichment, separation and purification of pathogenic bacteria of Ralstonia solanacearum: weighing 10 g of K326 rhizosphere soil of continuous cropping flue-cured tobacco in Fuliang shed village of Yuxi city, Yunnan province in 30 years, dissolving in a triangular flask filled with 90 mL of sterile water, and fully oscillating and shaking uniformly to obtain 10 g of K326 rhizosphere soil-1And (4) diluting the solution. Take 10 mL of 10-1Adding the diluted soil suspension into another 90 mL sterile water triangular flask, and shaking thoroughly to obtain 10-2Diluting the solution to 10-3And (4) diluting the solution. 50 mu L10 is sucked-3The diluted soil dilution is coated on an SMSA selective culture medium and is placed in a constant temperature incubator at 30 ℃ for 72 hours. When clear and selectable bacterial colonies grow on the culture medium, the clear bacterial colonies on the SMSA solid culture medium are picked into a centrifuge tube filled with 1 mL of SMSA liquid culture medium, cultured overnight by a shaking table (200 rpm) at 30 ℃, placed in a refrigerator at 4 ℃ and stored for later use. The SMSA medium is prepared according to the formulation of Elphinstone J G et al (Elphinstone J G., Hennessy J., Wilson J K., Stead D E. Sensitivity of differential methods for the detection ofRalstonia solanacearumin potato tuber extracts. Eppo Bulletin, 1996, 26, 663-678)。
Preparing an SMSA culture medium: 10 g of peptone and L g casamino acids were weighed, and 5 mL of glycerol and 995 mL of 18.2 M.OMEGA.ultrapure water were added to prepare 1L of a liquid medium. If a solid medium is prepared, 15 g of agar is added to 1L of liquid medium. Autoclaving at 121 deg.C for 15 min, cooling to 50 deg.C, and adding bacitracin 25 mg (1250U), polymyxin B sulfate 100 mg (600000U), penicillin 0.5 mg (825U), chloramphenicol 5 mg, crystal violet 5 mg, and triphenyltetrazolium chloride 50 mg (TTC) per liter of culture medium.
(4) Identification of pathogenic bacteria of Ralstonia solanacearum:
reference is made to the description of Kubota R et al (Kubota R., Vine B g., Alvarez a m.,JenkinsDM. Detection of Ralstonia solanacearum by Loop-Mediated Isothermal Amplification [J]phytopathology, 2008, 98, 1045), design of Flic virulence gene of Ralstonia solanacearumSpecific primers (F: 5'-TGGCGGTTGCGGTCTA-3' and R: 5'-GATCAGGTCTTCCGGCTCTA-3') were used to PCR amplify the flic gene. The PCR amplification system (20. mu.L) was: taq PCR Master Mix 10. mu.L, upstream and downstream primers 1. mu.L each, sterile water 5. mu.L, DNA template 3. mu.L. The amplification procedure was: pre-denaturation at 95 ℃ for 5 min, denaturation at 95 ℃ for 30 sec, annealing at 55 ℃ for 30 sec, extension at 72 ℃ for 1 min, 30 cycles, and extension at 72 ℃ for 10 min. The PCR product was subjected to purity detection by 1% (m/v) agarose gel electrophoresis, and then sequenced by Biotech, Inc. of Burkh, Fuzhou, and subjected to BLAST comparison analysis on NCBI (http:// www.ncbi.nlm.nih.gov /) database to identify the pathogen as Ralstonia solanacearumRalstonia solanacearum)。
(5) Antagonistic bacterium Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Enrichment, separation and purification of TBA 03:
s1: collecting the K326 rhizosphere soil of the continuous cropping flue-cured tobacco in 30 years in Fuliang shed countryside of Yuxi city, Yunnan province by a five-point sampling method, and storing the collected soil in a refrigerator at the temperature of-80 ℃ for later use;
s2: grinding and sieving the collected soil, weighing 10 g of soil, dissolving in 90 mL of sterile water, fully shaking, placing in 80 ℃ water bath, and treating for 10 min (to kill thallus which cannot form spores) to obtain a soil suspension;
s3: diluting the soil suspension in a gradient manner to obtain soil suspensions with different dilutions;
s4: draw dilution 10-1、10-2And 10-3Spreading the suspension onto broth (LB) solid medium, placing in an incubator at 30 deg.C, and culturing for 24 hr;
s5: selecting clear and distinguishable single colonies with different forms on a culture medium, carrying out enrichment culture for 12 h, placing in a refrigerator at 4 ℃, and storing for later use;
s6: the ralstonia solanacearum enriched, separated and purified and stored in a refrigerator at 4 ℃ is diluted by 10 times with sterile water to prepare a ralstonia solanacearum suspension. Sucking 50 mu L of strain suspension, coating the strain suspension on an LB plate, uniformly placing 2 pieces of filter paper sheets with the diameter of 7 mm on the LB plate, wherein 1 piece of filter paper is point-connected with bacterial liquid which is enriched and cultured in 20 mu L S5, the other piece of filter paper is point-connected with 20 mu L of sterilized water as a contrast, placing the filter paper sheets in an incubator at 30 ℃, culturing for 3 days, and selecting a strain of bacteria with strong antagonistic effect on the Ralstonia solanacearum, and naming the strain as TBA 03. Transferring the screened strong antagonistic strain into 5 mL LB liquid medium for amplification culture, performing shaking table (200 rpm) overnight culture at 30 ℃, placing in a refrigerator at 4 ℃ and storing for later use.
LB was purchased from Qingdao Gaokoubo Biotech GmbH, Okinawa, and comprises the following components: 10.0 g/L of tryptone, 5.0 g/L of yeast extract powder and 10.0 g/L of sodium chloride. Adding 25 g LB into each liter of ultrapure water, and autoclaving at 121 ℃ for 15 min for later use. If a solid medium is prepared, 15 g of agar is added per liter of LB liquid medium.
(6) Antagonistic bacterium Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Identification of TBA 03:
PCR amplification of the 16S nuclear somatic RNA gene (16S rDNA) of TBA03 was performed using bacterial universal primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3'). The PCR amplification system (20. mu.L) was: taq PCR Master Mix 10. mu.L, upstream and downstream primers 1. mu.L each, sterile water 5. mu.L, DNA template 3. mu.L. The amplification procedure was: pre-denaturation at 95 ℃ for 5 min, denaturation at 95 ℃ for 30 sec, annealing at 55 ℃ for 30 sec, extension at 72 ℃ for 1 min, 30 cycles, and extension at 72 ℃ for 10 min. The PCR product is subjected to purity detection by 1% (m/v) agarose gel electrophoresis, sent to Fuzhou Bersank Biotechnology Ltd for sequencing, subjected to BLAST comparison analysis on NCBI (http:// www.ncbi.nlm.nih.gov /) database to construct a phylogenetic tree, and identified as the antagonistic bacterium which is the bacillus amyloliquefaciens (Bacillus amyloliquefaciens) ((Bacillus amyloliquefaciens))Bacillus amyloliquefaciensTBA03) with accession number: CGMCC number 19108.
The bacteriological characteristics are as follows: the thallus is rod-shaped, has a long straight shape, two blunt ends, a length of 2-3 mu m and a width of 0.6-0.7 mu m, is a facultative aerobic bacterium which is a milky, round and protruding colony on an LB culture medium and is suitable for a growth temperature of 20-50 ℃ and a pH of 3.0-9.0 for gram-positive bacteria.
Deposit of bacillus amyloliquefaciens TBA 03: bacillus amyloliquefaciens TBA03 was stored at-80 ℃ using a glycerol cryovial.
The invention has the advantages that:
the utilization of antagonistic bacteria for preventing and treating plant diseases is a scientific, reasonable, efficient and environment-friendly measure. The bacillus amyloliquefaciens TBA03 separated and screened by the method can obviously inhibit the growth of pathogenic bacteria of Ralstonia solanacearum and reduce the harm of tobacco bacterial wilt. The bacterial strain is obtained by separating and screening continuous cropping tobacco soil, has stronger resistance and adaptability to the tobacco soil environment, and plays an important role in relieving the tobacco bacterial wilt.
Drawings
FIG. 1: technical route diagrams of the present invention.
FIG. 2: the relative abundance of pathogenic bacteria in the 30-year continuous cropping flue-cured tobacco K326 rhizosphere soil is on the genus level, CCS represents the 30-year continuous cropping flue-cured tobacco K326 rhizosphere soil in Fuliang village in Yuxi City of Yunnan province, and CS represents the control soil without planting tobacco near a tobacco area; the abscissa represents the soil and the ordinate represents the relative abundance of the microorganisms.
FIG. 3: the plate of the bacillus amyloliquefaciens TBA03 and the Ralstonia solanacearum is opposite. A: inoculation of bacillus amyloliquefaciens TBA03, B: a sterile water control group was inoculated.
FIG. 4: the invention relates to an evolutionary tree of bacillus amyloliquefaciens TBA 03.
FIG. 5: scanning electron micrographs of the bacillus amyloliquefaciens TBA03 of the present invention.
FIG. 6: the bacillus amyloliquefaciens TBA03 disclosed by the invention is used for preventing and controlling the bacterial wilt from infecting tobacco seedlings. A: control soil without added pathogenic bacteria of ralstonia solanacearum and bacillus amyloliquefaciens TBA03, B: simultaneously inoculating ralstonia solanacearum (4.0X 10)8CFU/kg soil) and Bacillus amyloliquefaciens TBA03 (4.0X 10)8CFU/kg soil), C: inoculation with pathogenic bacterial of Ralstonia solanacearum only (4.0X 10)8CFU/kg soil).
Detailed Description
In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the following examples are only examples of the present invention and do not represent the scope of the present invention defined by the claims.
Example 1 Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Screening and identification of TBA03
1. Pathogenic bacterial of Ralstonia solanacearum (A)Ralstonia solanacearum) Screening of (2)
(1) Soil collection: at the end of 08 months in 2018, collecting the K326 rhizosphere soil of continuous cropping flue-cured tobacco in Fuliang shed countryside of Yuxi city in Yunnan province and the control soil without planting tobacco in the vicinity of a tobacco area in 30 years respectively, dividing the soil sample by a quartering method, storing the collected soil sample in a refrigerator at the temperature of-80 ℃, and preparing the collected soil for analyzing the structural diversity of the microbial community of the next soil and screening pathogenic bacteria.
(2) And (3) analyzing structural diversity of the soil microbial community: genomic DNAs of non-tobacco-planted Control Soil (CS) and continuous cropping flue-cured tobacco rhizosphere soil (CCS) were extracted, respectively, the purity and concentration of the extracted DNAs were checked by agarose gel electrophoresis, and PCR amplification of CS and CCS genomic DNAs was performed using specific primers (341F: CCTAYGGGRBGCASCAG and 543R: ATTACCGCGGCTGCTGG) with Barcode. The DNA sample is sent to Beijing Ovwison Gene science and technology limited, the Illumina Miseq PE300 high-throughput sequencing platform is utilized for sequencing, and the bacterial 16S rDNA V3-V4 region is selected for microbial diversity detection. Sequencing results show that the ralstonia solanacearum is an important pathogenic bacterium with a high flora number and causing continuous cropping obstacle of tobaccos in the area (figure 2).
(3) Enrichment, separation and purification of pathogenic bacteria of Ralstonia solanacearum:
weighing 10 g of K326 rhizosphere soil of continuous cropping flue-cured tobacco in Fuliang shed village of Yuxi city, Yunnan province in 30 years, dissolving in a triangular flask filled with 90 mL of sterile water, and fully oscillating and shaking uniformly to obtain 10 g of K326 rhizosphere soil-1And (4) diluting the solution. Take 10 mL of 10-1Adding the diluted soil suspension into another 90 mL sterile water triangular flask, and shaking thoroughly to obtain 10-2Diluting the solution to 10-3And (4) diluting the solution. 50 mu L10 is sucked-3The diluted soil dilution is coated on an SMSA selective culture medium and is placed in a constant temperature incubator at 30 ℃ for 72 hours. When clear and selectable bacterial single colonies grow on the culture medium, SM is addedA single bacterial colony clearly visible on the SA solid culture medium is picked into a centrifuge tube filled with 1 mL of SMSA liquid culture medium, cultured overnight by a shaker (200 rpm) at 30 ℃, placed in a refrigerator at 4 ℃ and stored for later use. The SMSA medium is prepared according to the formulation of Elphinstone J G et al (Elphinstone J G., Hennessy J., Wilson J K., Stead D E. Sensitivity of differential methods for the detection ofRalstonia solanacearum in potato tuber extracts. Eppo Bulletin, 1996, 26, 663-678.)。
Preparing an SMSA culture medium: 10 g of peptone and L g casamino acids were weighed, and 5 mL of glycerol and 995 mL of 18.2 M.OMEGA.ultrapure water were added to prepare 1L of a liquid medium. If a solid medium is prepared, 15 g of agar is added to 1L of liquid medium. Autoclaving at 121 deg.C for 15 min, cooling to 50 deg.C, and adding bacitracin 25 mg (1250U), polymyxin B sulfate 100 mg (600000U), penicillin 0.5 mg (825U), chloramphenicol 5 mg, crystal violet 5 mg, and triphenyltetrazolium chloride 50 mg (TTC) per liter of culture medium.
(4) Identification of pathogenic bacteria of Ralstonia solanacearum:
reference is made to the description of Kubota R et al (Kubota R., Vine B g., Alvarez a m.,JenkinsDM. Detection of Ralstonia solanacearum by Loop-Mediated Isothermal Amplification [J]phytopathology, 2008, 98, 1045), designing primers specific to the flic pathogenic gene of Ralstonia solanacearum (F: 5'-TGGCGGTTGCGGTCTA-3' and R: 5'-GATCAGGTCTTCCGGCTCTA-3'), PCR-amplifying the flic gene. The PCR amplification system (20. mu.L) was: taq PCR Master Mix 10. mu.L, upstream and downstream primers 1. mu.L each, sterile water 5. mu.L, DNA template 3. mu.L. The amplification procedure was: pre-denaturation at 95 ℃ for 5 min, denaturation at 95 ℃ for 30 sec, annealing at 55 ℃ for 30 sec, extension at 72 ℃ for 1 min, 30 cycles, and extension at 72 ℃ for 10 min. The PCR product was subjected to purity detection by 1% (m/v) agarose gel electrophoresis, and then sequenced by Biotech, Inc. of Burkh, Fuzhou, and subjected to BLAST comparison analysis on NCBI (http:// www.ncbi.nlm.nih.gov /) database to identify the pathogen as Ralstonia solanacearumRalstonia solanacearum)。
2. Bacillus amyloliquefaciens (A)Bacillus amyloliquefaciens) Screening for TBA03
The Bacillus amyloliquefaciens (A) of the inventionBacillus amyloliquefaciens) TBA03 is obtained by separating from 30 years continuous cropping flue-cured tobacco K326 rhizosphere soil in Fuliang shed village in Yuxi city, Yunnan province.
(1) Selection of TBA03 Medium
LB broth (purchased from Qingdao Gaokoubo Biotech, Inc.) was used, which had the following composition: tryptone 10.0 g/L, yeast extract 5.0 g/L, sodium chloride 10.0 g/L, each 1L ultrapure water with 25 g LB broth powder (if preparing solid medium, each 1L liquid medium need to add 15 g agar). Sterilizing at 121 deg.C under high pressure for 15 min.
(2) TBA03 enrichment, isolation and purification
S1: collecting the K326 rhizosphere soil of the continuous cropping flue-cured tobacco in 30 years in Fuliang shed countryside of Yuxi city, Yunnan province by a five-point sampling method, and storing the collected soil in a refrigerator at the temperature of-80 ℃ for later use;
s2: grinding and sieving the collected soil, weighing 10 g of soil, dissolving in 90 mL of sterile water, fully shaking, placing in 80 ℃ water bath, and treating for 10 min (to kill thallus which cannot form spores) to obtain a soil suspension;
s3: diluting the soil suspension by 10 times in a gradient manner to obtain soil suspensions with different dilutions;
s4: draw dilution 10-1、10-2And 10-3Spreading the soil suspension liquid on LB broth solid culture medium in 50 μ L, placing in 30 deg.C incubator, and culturing for 24 hr;
s5: selecting clear and distinguishable single colonies with different forms on a culture medium, carrying out enrichment culture for 12 h, and placing in a refrigerator at 4 ℃ for later use;
s6: the screened Ralstonia solanacearum (L.), (Ralstonia solanacearum) Transferring into SMSA liquid culture medium, diluting the suspension after overnight culture (30 deg.C, 200 rpm) by 10 times with sterile water, sucking 50 μ L of strain suspension, spreading onto LB plate, and uniformly placing 2 pieces of filter paper with diameter of 7 mm on LB plate, wherein 1 piece of filter paper is spotted with the enriched culture solution in 20 μ L S5, and another piece of filter paper is spotted with 20 μ L of sterilized water as controlAnd placing the strain in an incubator at 30 ℃ for 3 d, and selecting a bacterium with strong antagonistic effect on the Ralstonia solanacearum, wherein the bacterium is named as TBA 03. Transferring the screened strong antagonistic strain into 5 mL LB liquid medium for amplification culture, performing shaking table (200 rpm) overnight culture at 30 ℃, placing in a refrigerator at 4 ℃ and storing for later use.
LB was purchased from Qingdao Gaokoubo Biotech GmbH, Okinawa, and comprises the following components: 10.0 g/L of tryptone, 5.0 g/L of yeast extract powder and 10.0 g/L of sodium chloride. Adding 25 g LB into each liter of ultrapure water, and autoclaving at 121 ℃ for 15 min for later use. If a solid medium is prepared, 15 g of agar is added per liter of LB liquid medium.
(3) Detection of bacteriostatic effect of TBA03
The ralstonia solanacearum enriched, separated and purified in the above steps and stored in a refrigerator at 4 ℃ is diluted 10 times with sterile water to prepare a suspension. Sucking 50 mu L of Ralstonia solanacearum suspension, coating the Ralstonia solanacearum suspension on an LB flat plate, and screening antagonistic bacteria of the Ralstonia solanacearum by adopting a flat plate opposite method. First, 2 pieces of filter paper with a diameter of 7 mm were placed evenly on a plate, wherein 20. mu.L of the enriched culture TBA03 suspension was spotted on 1 piece of filter paper, and 20. mu.L of sterilized water was spotted on the other piece of filter paper as a control. Parallel 3 sets. The plates were placed in an incubator at 30 ℃ and cultured for 3 days, and it was found that Bacillus amyloliquefaciens TBA03 was able to inhibit the growth of Ralstonia solanacearum efficiently (FIG. 3).
(4) Identification of TBA03
PCR amplification of the 16S nuclear somatic RNA gene (16S rDNA) of TBA03 was performed using bacterial universal primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3'). The PCR amplification system (20. mu.L) was: taq PCR Master Mix 10. mu.L, upstream and downstream primers 1. mu.L each, sterile water 5. mu.L, DNA template 3. mu.L. The amplification procedure was: pre-denaturation at 95 ℃ for 5 min, denaturation at 95 ℃ for 30 sec, annealing at 55 ℃ for 30 sec, extension at 72 ℃ for 1 min, 30 cycles, and extension at 72 ℃ for 10 min. The PCR product was subjected to purity detection by 1% (m/v) agarose gel electrophoresis, sequenced by Fuzhou Bersank Biotech Ltd, subjected to BLAST comparison analysis in NCBI (http:// www.ncbi.nlm.nih.gov /) database to construct a phylogenetic tree (FIG. 4), and identified as Bacillus amyloliquefaciensBacteria (A), (B)Bacillus amyloliquefaciens TBA03)。
The bacteriological characteristics of the strain of bacillus amyloliquefaciens TBA03 are as follows: the thallus is rod-shaped, has a long straight shape, two blunt ends, a length of 2-3 mu m and a width of 0.6-0.7 mu m, is a facultative aerobic bacterium which is a milky, round and protruding colony on an LB culture medium and is suitable for a growth temperature of 20-50 ℃ and a pH of 3.0-9.0 of gram-positive bacteria (figure 5).
Deposit of bacillus amyloliquefaciens TBA 03: bacillus amyloliquefaciens TBA03 was stored at-80 ℃ using a glycerol cryovial.
Bacillus amyloliquefaciens TBA03, designated by the classification: bacillus amyloliquefaciens (A)Bacillus amyloliquefaciens) TBA03, deposited in China general microbiological culture Collection center on 12.9.2019, with the deposit number: CGMCC number 19108.
Example 2 evaluation of the effectiveness of Bacillus amyloliquefaciens TBA03 in controlling invasion of pathogenic bacteria Rayleigh solanacearum on cured tobacco K326 seedlings
Transplanting K326 tobacco seedlings in 4-5 leaf stage into small square pots (1 plant/pot) with the length multiplied by 7 cm multiplied by 8 cm, adding 250 g of conventional rice field soil into each pot, and performing the following three treatments on the 5 th day after transplanting. Treatment 1: no bacteria is added; and (3) treatment 2: inoculating 1 mL of the solution with the concentration of 1 × 10 to the root of the tobacco by a root irrigation method8CFU/mL of Ralstonia solanacearum suspension. After 1 d of inoculation, the tobacco roots were further inoculated with 1 mL of 1X 108CFU/mL of a Bacillus amyloliquefaciens TBA03 bacterial suspension; and (3) treatment: inoculating 1 mL of the solution at a concentration of 1X 10 to the root of tobacco only8CFU/mL Ralstonia solanacearum suspension. The above were 3 treatments, each of which was repeated 3 times (table 1). And (4) placing the culture medium in an illumination incubator at 24-25 ℃ for culture, and continuously observing the infection condition of pathogenic bacteria. After culturing for 30 days, the K326 tobacco seedlings are found to grow well in the experiment treatment group 1 (figure 6A) without adding pathogenic bacteria and antagonistic bacteria, while in the experiment treatment group 3 (figure 6C) with only adding pathogenic bacteria, the K326 tobacco seedlings are short and small in plants and yellow and withered in leaves, which indicates that the Ralstonia solanacearum obviously inhibits the growth of the K326 tobacco seedlings; however, in the experimental treatment group 2 (FIG. 6B) with the simultaneous addition of pathogenic bacteria and antagonistic bacteria, K326 tobacco plants grew without excessThe test treatment group 1 is good, but is obviously superior to the pathogenic bacterium poisoning treatment group 3, and shows that the bacillus amyloliquefaciens TBA03 obviously relieves the poisoning effect of the ralstonia solanacearum on K326 tobacco plants. In conclusion, the screened bacillus amyloliquefaciens TBA03 (with the preservation number of CGMCC NO.19108) can effectively prevent and control the toxicity of the pathogenic bacteria of Ralstonia solanacearum to the tobacco, and has wide application prospect in the field.
Table 1 test design table
The above description is only a preferred embodiment of the present invention, and any modification and modification made within the scope of the claims of the present invention are covered by the present invention.
SEQUENCE LISTING
<110> Fujian agriculture and forestry university
<120> bacillus amyloliquefaciens TBA03 and application thereof
<130> 7
<160> 7
<170> PatentIn version 3.3
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<212> DNA
<213> Artificial sequence 341F
<400> 1
cctaygggrb gcascag 17
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<212> DNA
<213> Artificial sequence 543R
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attaccgcgg ctgctgg 17
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<213> Artificial sequence F
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tggcggttgc ggtcta 16
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gatcaggtct tccggctcta 20
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ggttaccttg ttacgactt 19
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atacatgcaa gtcgagcgga cagatgggag cttgctccct gatgttagcg gcggacgggt 60
gagtaacacg tgggtaacct gcctgtaaga ctgggataac tccgggaaac cggggctaat 120
accggatggt tgtttgaacc gcatggttca gacataaaag gtggcttcgg ctaccactta 180
cagatggacc cgcggcgcat tagctagttg gtgaggtaac ggctcaccaa ggcgacgatg 240
cgtagccgac ctgagagggt gatcggccac actgggactg agacacggcc cagactccta 300
cgggaggcag cagtagggaa tcttccgcaa tggacgaaag tctgacggag caacgccgcg 360
tgagtgatga aggttttcgg atcgtaaagc tctgttgtta gggaagaaca agtgccgttc 420
aaatagggcg gcaccttgac ggtacctaac cagaaagcca cggctaacta cgtgccagca 480
gccgcggtaa tacgtaggtg gcaagcgttg tccggaatta ttgggcgtaa agggctcgca 540
ggcggtttct taagtctgat gtgaaagccc ccggctcaac cggggagggt cattggaaac 600
tggggaactt gagtgcagaa gaggagagtg gaattccacg tgtagcggtg aaatgcgtag 660
agatgtggag gaacaccagt ggcgaaggcg actctctggt ctgtaactga cgctgaggag 720
cgaaagcgtg gggagcgaac aggattagat accctggtag tccacgccgt aaacgatgag 780
tgctaagtgt tagggggttt ccgcccctta gtgctgcagc taacgcatta agcactccgc 840
ctggggagta cggtcgcaag actgaaactc aaaggaattg acgggggccc gcacaagcgg 900
tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accaggtctt gacatcctct 960
gacaatccta gagataggac gtccccttcg ggggcagagt gacaggtggt gcatggttgt 1020
cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgatctt 1080
agttgccagc attcagttgg gcactctaag gtgactgccg gtgacaaacc ggaggaaggt 1140
ggggatgacg tcaaatcatc atgcccctta tgacctgggc tacacacgtg ctacaatgga 1200
cagaacaaag ggcagcgaaa ccgcgaggtt aagccaatcc cacaaatctg ttctcagttc 1260
ggatcgcagt ctgcaactcg actgcgtgaa gctggaatcg ctagtaatcg cggatcagca 1320
tgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca cgagagtttg 1380
taacacccga agtcggtgag gtaacctttt aggagccagc cgc 1423
Claims (4)
1. A strain of Bacillus amyloliquefaciens TBA03 is characterized in that: the classification of the bacillus amyloliquefaciens TBA03 is named as bacillus amyloliquefaciens (A) (Bacillus amyloliquefaciens) The strain is preserved in China general microbiological culture Collection center on 12 and 9 months in 2019, and the preservation number is CGMCC NO. 19108.
2. The use of the bacillus amyloliquefaciens TBA03 as a pesticide in the control ofRalstonia solanacearum (L.), (L.nicotianae)Ralstonia solanacearum) The use of (1).
3. A microbial preparation comprising the strain of Bacillus amyloliquefaciens TBA03 of claim 1.
4. A method for controlling Ralstonia solanacearum with a microbial preparation according to claim 3Ralstonia solanacearum) The use of (1).
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| CN105296386A (en) * | 2015-10-22 | 2016-02-03 | 武汉骏安生物科技有限公司 | Endophytic bacillus amyloliquefaciens capable of generating a large number of antagonistic tobacco bacterial wilt active materials |
| WO2016156164A1 (en) * | 2015-03-27 | 2016-10-06 | Industrias Químicas Del Vallés, S.A. | A strain of bacillus amyloliquefaciens and its use in the control of diseases caused by bacteria and fungi in plants |
| CN108690821A (en) * | 2017-12-07 | 2018-10-23 | 中国热带农业科学院环境与植物保护研究所 | One plant height imitates bacillus amyloliquefaciens and its microbial inoculum and application |
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| CN104711209A (en) * | 2015-02-09 | 2015-06-17 | 湖南省烟草公司永州市公司 | Bacillus amyloliquefaciens B011 for preventing and treating tobacco bacterial wilt and application thereof |
| WO2016156164A1 (en) * | 2015-03-27 | 2016-10-06 | Industrias Químicas Del Vallés, S.A. | A strain of bacillus amyloliquefaciens and its use in the control of diseases caused by bacteria and fungi in plants |
| CN105296386A (en) * | 2015-10-22 | 2016-02-03 | 武汉骏安生物科技有限公司 | Endophytic bacillus amyloliquefaciens capable of generating a large number of antagonistic tobacco bacterial wilt active materials |
| CN108690821A (en) * | 2017-12-07 | 2018-10-23 | 中国热带农业科学院环境与植物保护研究所 | One plant height imitates bacillus amyloliquefaciens and its microbial inoculum and application |
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| Title |
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| Antibacterial activity against Ralstonia solanacearum of the lipopeptides secreted from the Bacillus amyloliquefaciens strain FJAT-2349;M C Chen等;《J Appl Microbiol》;20190227;第126卷(第5期);1519-1529，参见摘要 * |
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