CN114657097B - Bacillus belgii LGT-1 capable of efficiently antagonizing ralstonia solanacearum and application thereof - Google Patents
Bacillus belgii LGT-1 capable of efficiently antagonizing ralstonia solanacearum and application thereof Download PDFInfo
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Abstract
The invention discloses Bacillus beiLeisi LGT-1 for efficiently antagonizing ralstonia solanacearum and application thereof. The Bacillus beilis LGT-1 is classified and named as Bacillus beilaisiBacillus velezensisThe preservation number is CGMCC No.24342, and the nucleotide sequence of 16S rDNA is shown in SEQ ID No. 1. The Bacillus belgii LGT-1 has strong bacterial wilt antagonistic capability, the diameter of a bacteriostatic zone for pathogenic bacteria of tobacco bacterial wilt reaches 47.9mm, and the permanent planting of ralstonia solanacearum can be directly prevented; meanwhile, the strain has high activity and simple culture method, can be directly used for preventing and treating tobacco bacterial wilt, can also be prepared into a plurality of preparations of microbial inoculum for preparing soil pathogenic bacteria preventing and treating agent, and can reduce the drug resistance of tobacco to pathogenic bacteria. Therefore, the Bacillus belgii LGT-1 has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a Bacillus belgii LGT-1 capable of efficiently antagonizing Ralstonia solanacearum and application thereof.
Background
The tobacco bacterial wilt is bacterial soil-borne plant disease caused by ralstonia solanacearum, can parasitize more than 50 families and hundreds of plants, and causes serious loss to the production of many crops and cash crops. Since the tobacco bacterial wilt is reported in China for the first time, the occurrence range and the harm degree of the tobacco bacterial wilt are continuously spread and aggravated, huge economic loss is brought to the tobacco industry every year, and once field tobacco plants are infected with the bacterial wilt, the diseased plants can be rapidly attacked and wilted to die along with favorable attack conditions of high temperature and high humidity. At present, the prevention and treatment of tobacco bacterial wilt is mainly chemical prevention and treatment, and other prevention and treatment methods (including disease-resistant variety screening, agricultural measure management, chemical prevention and treatment and the like) are used for assistance. Although chemical control has better control effect on tobacco bacterial wilt, the drug resistance of pathogenic bacteria can be improved, the diversity of biological communities in tobacco field soil can be reduced, and the quality of the tobacco field soil environment can be influenced.
In recent years, the quality of roasted grass and the quality of tobacco field soil are more and more emphasized, and the reduction of food for chemical prevention and control and the use of the chemical prevention and control are inevitable trends, so that the development and the popularization and the application of the tobacco bacterial wilt prevention and control agent have very important significance for soil improvement and quality improvement of flue-cured tobacco.
Disclosure of Invention
The invention aims to provide Bacillus beijerinckii LGT-1 capable of efficiently antagonizing ralstonia solanacearum and application thereof. The Bacillus belgii LGT-1 has strong effect of inhibiting tobacco bacterial wilt, can be directly used or fermented into a microbial inoculum, and is used for preparing the microbial inoculum for preventing and treating tobacco bacterial wilt.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention provides a Bacillus belgii LGT-1 for efficiently antagonizing ralstonia solanacearum, which is classified and named as Bacillus belgii velezensis, and the preservation number is CGMCC No.24342.
Furthermore, the nucleotide sequence of 16S rDNA of the Bacillus beleisi LGT-1 is shown in SEQ ID No. 1.
Furthermore, the gyrA nucleotide sequence of the Bacillus belgii LGT-1 is shown in SEQ ID No.2, and the gyrB nucleotide sequence thereof is shown in SEQ ID No. 3.
The invention also provides application of the Bacillus beiLeisi LGT-1 in preparing a control microbial inoculum for inhibiting soil pathogenic bacteria.
Furthermore, the content of viable bacteria contained in the control microbial inoculum is not less than 10 9 CFU/mL of Bacillus belgii LGT-1 or its inoculum.
Further, the preparation method of the Bacillus belgii LGT-1 microbial inoculum comprises the following steps:
(1) Inoculating activated Bacillus belgii LGT-1 into a seed culture medium, and culturing at 25-35 ℃ for 20-24 h to prepare LGT-1 seed liquid;
(2) Transferring the LGT-1 seed liquid into a fermentation tank, and carrying out fermentation culture for 40-50 h at 25-35 ℃ to prepare LGT-1 fermentation liquid;
(3) And (3) transferring the LGT-1 fermentation liquor into the fermentation tank again, fermenting and culturing for 40-50 h at the temperature of 25-35 ℃, directly packaging or centrifuging the prepared secondary fermentation liquor, taking the precipitate for drying, and packaging to obtain the control microbial inoculum.
Furthermore, the inoculation amount of the LGT-1 seed liquid or the LGT-1 fermentation liquid transferred into the fermentation tank is 1% -5%.
Preferably, the inoculation amount of the LGT-1 seed liquid or the LGT-1 fermentation liquid transferred into the fermentation tank is 2 percent.
Further, the formula of the seed culture medium is as follows: peptone 10 g.L -1 5 g.L yeast powder -1 Sodium chloride 10 g.L -1 And the pH value is 7.2-7.4.
Further, the formula of the fermentation medium in the fermentation tank is as follows: soluble starch 6 g.L -1 ～12g·L -1 Soy peptone 10 g.L -1 ～15g·L -1 ，CaCl 2 5g·L -1 ～10g·L -1 The pH value is 7-7.5.
Preferably, the formula of the fermentation medium in the fermentation tank is as follows: soluble starch 10 g.L -1 Soy peptone 14 g.L -1 ，CaCl 2 6g·L -1 The pH was 7.5.
Preferably, the preparation method of the Bacillus belgii LGT-1 microbial inoculum comprises the following steps:
(1) Inoculating activated Bacillus belgii LGT-1 into a seed culture medium, and culturing at 30 ℃ at 200r/min for 24h to prepare LGT-1 seed liquid;
(2) Transferring the LGT-1 seed liquid into a fermentation tank with the inoculation amount of 2%, and carrying out fermentation culture at 30 ℃ for 48h at 180r/min to prepare LGT-1 fermentation liquid;
(3) And (3) transferring the LGT-1 fermentation liquor into the fermentation tank again with the inoculation amount of 2%, fermenting and culturing at 30 ℃ for 48h at 180r/min to obtain LGT-1 secondary fermentation liquor, directly packaging or centrifuging, taking the precipitate, drying, and packaging to obtain the Bacillus beilaisi LGT-1 microbial inoculum.
Further, the soil pathogenic bacteria are tobacco bacterial wilt bacteria.
Furthermore, the Bacillus belgii LGT-1 obviously inhibits the activity of tobacco bacterial wilt germs.
The invention also provides application of the Bacillus beiLeisi LGT-1 in preparing a preparation for reducing or inhibiting the drug resistance of tobacco pathogenic bacteria.
Compared with the prior art, the invention has the following beneficial effects and advantages:
the invention separates and screens a bacillus bacterial wilt bacterium (Ralstonia solanacearum) highly effective antagonistic bacillus Bellisae LGT-1 from rhizosphere soil of healthy plants in soil infected by bacterial wilt in the tobacco-applying area in Hubei province. The bacterial strain has the characteristic of efficiently inhibiting the activity of tobacco bacterial wilt, the diameter of an inhibition zone for pathogenic bacteria of the tobacco bacterial wilt reaches 47.9mm, and the bacterial strain can directly prevent the permanent planting of the bacterial wilt; meanwhile, the strain has high activity and simple culture method, can be directly used for preventing and treating tobacco bacterial wilt, can also be prepared into a plurality of preparations of microbial inoculum for preparing soil pathogenic bacteria preventing and treating agent, and can reduce the drug resistance of tobacco to pathogenic bacteria. Therefore, the application prospect is wide.
Drawings
FIG. 1 is a characteristic diagram of a colony of Bacillus beleisi LGT-1;
FIG. 2 is a 16S rRNA phylogenetic tree of Bacillus beleisi LGT-1;
FIG. 3 is a gyrA phylogenetic tree of Bacillus beleisi LGT-1;
FIG. 4 is a gyrB phylogenetic tree of Bacillus beilesiensis LGT-1;
FIG. 5 shows the effect of different media on the viable count of the LGT-1 fermentation broth of Bacillus beiLeisi;
FIG. 6 shows the effect of different C-source media on the viable count of Bacillus beiLensis LGT-1;
FIG. 7 is a graph showing the effect of different N-source media on the viable count of Bacillus beiensis LGT-1;
FIG. 8 is a graph showing the effect of inorganic salt species on the viable count of Bacillus belief LGT-1;
FIG. 9 shows the effect of different fermentation temperatures on the number of viable bacteria of Bacillus beijerinckii LGT-1;
FIG. 10 is a graph showing the effect of different initial fermentation pH on the viable count of Bacillus beilis LGT-1;
FIG. 11 shows the effect of different inoculum sizes on the number of viable bacteria of Bacillus beiLensis LGT-1;
FIG. 12 is a graph showing the effect of different liquid contents on the number of viable bacteria of Bacillus beijerinckii LGT-1;
FIG. 13 shows the antagonistic effect of Bacillus beiLensis LGT-1 on Ralstonia solanacearum.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the contents in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless defined otherwise, 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 invention belongs.
Example 1: isolation, screening and identification of Strain LGT-1
1. Isolation and screening of Strain LGT-1
In the planting base of tobacco planted in Enshi of Hubei province, healthy plants are selected from the land infected by bacterial wilt, 100g of rhizosphere soil attached to roots is taken, placed in a sterilized self-sealing bag and taken back to a laboratory. A conventional dilution plate method is adopted for strain separation, a tobacco bacterial wilt pathogenic bacterium (Ralstonia solanacearum) is taken as an indicator bacterium, antagonistic strains are screened, and a strain with strong antagonism is obtained and named as LGT-1.
2. Identification of Strain LGT-1
1. Morphological identification
The strain was inoculated on an LB plate (tryptone 1%, yeast extract 0.5%, naCl 1%, agar powder 18%, pH = 7), incubated at 30 ℃ for 12-48h, and the colony characteristics were observed. The strains cultured for 12h were picked and gram-stained, and the strains cultured for 48h were picked and spore-stained.
The results are shown in figure 1, the colony is colorless and transparent in the initial stage, has smooth, moist, sticky and irregular edge on the surface, and is milky white in the later stage, and the surface has obvious bulges and folds. The thalli are rod-shaped, produce spores and are gram-positive.
2. Physiological and biochemical identification
The screened strains are subjected to physiological and biochemical measurement by referring to Bergey's Manual of bacteria identification and general Manual of bacteria systematic identification. The results are shown in Table 1, and combined with morphological observations, LGT-1 was initially identified as a Bacillus strain.
Table 1: characterization of physiological and biochemical reaction characteristics of LGT-1
Note: "+" is positive and "-" is negative.
3. Molecular biological identification
The genomic DNA of the strain LGT-1 was extracted using a bacterial genome extraction kit purchased from Ai Kerui (Shanghai) Ltd, and 16S rRNA gene sequences and gyrA and gyrB gene sequences were amplified using universal primers. The reaction conditions are shown in Table 2.
Table 2: gene sequence amplification reaction conditions
The PCR amplification product is sent to a limited engineering company of bioengineering (Shanghai) to detect, and the 16S rRNA fragment, the gyrA fragment, the 986bp and the 1028bp gyrB gene fragment of the strain LGT-1 are obtained.
The 16SrDNA gene sequence of the LGT-1 obtained by sequencing is shown as SEQ ID No. 1; the gyrA gene sequence obtained by sequencing is shown as SEQ ID No. 2; the sequence of the gyrB gene obtained by sequencing is shown as SEQ ID No. 3.
Submitting the 16SrDNA, gyrA and gyrB gene sequences to NCBI for online comparison, wherein the similarity of LGT-1 and Bacillus beiLeisi is 96.89%, 99.17% and 98.73%, respectively, and a phylogenetic tree is constructed by utilizing MEGA X software. As shown in fig. 2-4, LGT-1 was clustered with bacillus belgii. And determining the strain LGT-1 as Bacillus velezensis by combining the morphological characteristics and physiological and biochemical identification of the strain.
Strain preservation is carried out on a strain LGT-1, and the preservation unit of the Bacillus beiLeisi LGT-1: china general microbiological culture Collection center (CGMCC); address: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation date is as follows: 2022, month 01, 19; the preservation number of the Bacillus velezensis is as follows: CGMCC No.24342.
Example 2: culture of Bacillus beilesiensis LGT-1
1. Preparation of seed liquid
Test strains: the Bacillus belgii LGT-1 strain is provided by the laboratory of the research center for photosynthetic carbon sequestration productivity of Qingdao agricultural university.
Test medium: LGT-1 strain seed medium: peptone 10 g.L -1 Yeast powder 5 g.L -1 Sodium chloride 10 g.L -1 And the pH value is 7.2-7.4.
Activation of LGT-1 Strain: the LGT-1 strain preserved in a laboratory is transferred into a flat solid culture medium and cultured for 24h at the constant temperature of 30 ℃ in a constant temperature incubator for standby.
Preparing a seed solution: inoculating a ring of activated LGT-1 strain into a 100mL triangular flask containing 40mL seed culture medium, and culturing at 30 deg.C for 200r min -1 Shaking and culturing for 24h under the condition.
Initial conditions of shake flask fermentation: inoculating seed solution of LGT-1 strain into 100mL triangular flask containing 40mL fermentation medium at 30 deg.C and 200 r.min -1 Shaking and culturing for 24h under the condition of (1). And carrying out a single-factor test, a culture medium orthogonal optimization test and a fermentation condition optimization test.
LGT-1 fermentation liquor viable count: the viable count was determined by dilution plate counting.
2. Screening of basal Medium
4 different culture media (see table 3) are selected, the LGT-1 strain is subjected to shake flask fermentation in different basic culture media, and the viable count of the LGT-1 strain fermentation liquor is respectively determined.
Table 3: media composition
The experimental results are shown in FIG. 5, the viable bacteria number per unit volume is the highest, 10.20X 10 under the shake flask fermentation culture of the strain LGT-1 in the No.2 culture medium 8 cfu·mL -1 The number of viable bacteria in the fermentation liquid per unit volume of the No.1 culture medium is 8.62 multiplied by 10 8 cfu·mL -1 And the number of viable bacteria in the fermentation liquid of No.3 culture medium per unit volume is 7.83 multiplied by 10 8 cfu·mL -1 And the number of viable bacteria in unit volume corresponding to the No. 4 culture medium fermentation liquid is 8.96 multiplied by 10 8 cfu·mL -1 . Therefore, medium No.2 was selected as the basal medium for shake flask fermentation of Bacillus beiLensis strain LGT-1.
3. Single factor test
(1) Carbon source species and concentration screening
Respectively using equal amounts of glucose, corn flour, maltose, soluble starch, sucrose and mannitol to substitute carbon source (C source) in basic culture medium, and culturing with a base without C sourceThe medium was Control (CK), other fermentation conditions were unchanged, shake flask fermentations were performed with 3 replicates per treatment. And (4) determining the viable count of different C-source fermentation liquors by adopting a viable count method, and screening the optimal C source. On the basis of screening out the optimal C source species, 0.4%,0.6%,0.8%,1.0%,1.2%,1.4% namely 4,6,8, 10, 12, 14 g.L are set under the same conditions -1 Shake flask fermentation was performed on 6 media with different concentrations of C source to screen out the optimal C source concentration.
As a result, as shown in FIG. 6, the number of viable bacteria of Bacillus beijerinckii LGT-1 was greatly affected by different C sources, and the maximum number of viable bacteria per unit volume was 17.53X 10 when soluble starch was used as the C source of the medium 8 cfu·m L -1 Therefore, soluble starch was selected as the C source for the B.beiensis LGT-1 shake flask fermentation medium.
(2) Screening of Nitrogen Source species and concentrations thereof
On the basis of C source screening, replacing a nitrogen source (N source) in a fermentation medium by equivalent peptone, beef extract, urea, ammonium sulfate, soybean peptone, corn steep liquor dry powder and yeast powder, and performing shake flask fermentation for 3 times with the medium without the N source as a Control (CK) and other fermentation conditions unchanged. And calculating the viable count of different N-source fermentation liquors by a viable count method, and screening the optimal N source. On the basis of screening out the optimal N source, 0.4%,0.6%,0.8%,1.0%,1.2%,1.4% namely 4,6,8, 10, 12, 14 g.L is set under the same conditions -1 Shake flask fermentation is carried out on 6 culture media with different concentrations of N sources to screen out the optimal concentration of the N source.
As shown in FIG. 7, the number of viable bacteria per unit volume was significantly affected when Bacillus belgii LGT-1 was used with different N sources as the medium components. Wherein the maximum viable bacteria number is 17.5 × 10 when soybean peptone is used as single N source 8 cfu·m L -1 Thus, soy peptone was chosen as the N source for the B.beiensis LGT-1 shake flask fermentation medium.
(3) Screening of inorganic salt species and concentrations thereof
Adding 0.6 percent of ZnSO on the basis of optimizing a C source and an N source respectively 4 ，NaCl，CaCl 2 ，MnSO 4 ，MgSO 4 KCl was used instead of inorganic salts in the fermentation medium, and the medium without inorganic salts was used as a Control (CK), and other fermentation conditions were not changed, and each treatment was repeated 3 times. And calculating the viable count of different inorganic salt fermentation liquids by adopting a viable count method, and screening the optimal inorganic salt. On the basis of the selection of the best inorganic salt, different concentrations of macroelement inorganic salt (0.2%, 0.3%,0.4%,0.5%,0.6%,0.7%, i.e. 2,3,4,5,6,7 g.L) were examined under the same conditions -1 ) The number of viable bacteria in the fermentation liquor, and the concentration of the optimal inorganic salt is screened out.
As shown in FIG. 8, the number of viable bacteria in the fermentation broth per unit volume of Bacillus beiLeisi LGT-1 was significantly affected by the inorganic salt species, and CaCl was used 2 When the fermentation broth is inorganic salt, LGT-1 can utilize the inorganic salt best, and the number of viable bacteria in the fermentation broth per unit volume reaches the maximum value, namely 20.06 multiplied by 10 8 cfu·mL -1 By ZnSO 4 ，MnSO 4 ，MgSO 4 When the fermentation liquor is inorganic salt, the number of viable bacteria in unit volume of the fermentation liquor is less than that of a control group without the inorganic salt. Thus, caCl was selected 2 Inorganic salt used as a shake flask fermentation medium of the Bacillus belgii LGT-1.
4. Fermentation medium orthogonal test optimization
The optimal C source of the culture medium obtained by screening is soluble starch, the optimal N source is soybean peptone, and the optimal inorganic salt is CaCl 2 . Mixing soluble starch (A), soybean peptone (B) and CaCl 2 (C) Three factors the factors and levels for the orthogonal test were designed according to L9 (33) and are shown in Table 4, see Table 4. Designed according to an orthogonal experiment at 30 ℃ and 200 r.min -1 And carrying out shake culture for 24h under the condition of pH 7.2, comparing the viable count of different combinations of fermentation liquor by taking the viable count of the fermentation liquor as an optimization index, and determining the optimal culture medium formula.
Table 4: orthogonality factor and level
The results are shown in Table 5, orthogonal testAs a result, the N-source soybean peptone has the greatest influence on the number of viable bacteria in the fermentation broth of Bacillus beiLeisi LGT-1 per unit volume, and the inorganic salt CaCl 2 The corresponding combination of the C source soluble starch is a No. 4 culture medium A2B1C2, and the number of viable bacteria in unit volume of fermentation liquid is 32.23 multiplied by 10 8 cfu·mL -1 . Therefore, the fermentation medium formula of the Bacillus belgii LGT-1 is determined to be 10 g.L of soluble starch -1 Soy peptone 14 g.L -1 ，CaCl 2 6g·L -1 。
Table 5: results of fermentation Medium Quadrature experiments
5. Shake flask fermentation condition optimization
On the basis of optimization of a culture medium, the fermentation temperature, the initial pH, the inoculation amount and the liquid loading amount of shake flask fermentation are optimized. Each test group is provided with 3 times of repetition, other conditions are ensured to be unchanged during one factor optimization period, and the viable count of shake flask fermentation liquor under different conditions is counted. Fermentation temperature: inoculating LGT-1 strain, and performing shake fermentation at 25, 28, 31, 34 and 37 deg.C; initial pH: adjusting the initial pH value of the culture medium to 6.5,7.0,7.5,8.0,8.5 by using HCl and NaOH respectively; inoculation amount: inoculating the LGT-1 strain according to the inoculation amounts of 1%,2%,3%,4% and 5% in volume fraction; liquid loading amount: the LGT-1 strain is inoculated into culture media with liquid loading amounts of 10%,20%,30%,40% and 50% respectively according to volume fraction (namely 100mL of triangular flasks are respectively filled with the culture media with 10, 20, 30, 40 and 50 mL) for shake flask fermentation.
The fermentation temperature is shown in FIG. 9, the maximum viable bacteria amount per unit volume in the fermentation broth obtained by shaking the flask at 28 deg.C is 37.4 × 10 8 cfu·m L -1 (ii) a Initial pH of fermentation in fermentation as shown in FIG. 10The initial pH of 7.5 shows that the fermentation liquid has the highest bacteria content and the viable bacteria number is 37.03X 10 8 cfu·m L -1 (ii) a As shown in FIG. 11, the number of viable bacteria in the fermentation broth per unit volume reached a maximum value of 34.17X 10 at 2% inoculation amount 8 cfu·m L -1 (ii) a As shown in FIG. 12, when the liquid loading amount of the culture medium is 20%, the viable bacteria count per unit volume of the fermentation liquid is significantly higher than that of other liquid loading amounts, and the maximum effective bacteria content is 37.4 × 10 8 cfu·m L -1 。
Example 3: bacteriostatic activity of Bacillus beilis LGT-1
1. Pathogenic bacteria activation
Inoculating tobacco bacterial wilt pathogenic bacteria to an NA plate for activation, and fermenting and culturing for 48h by NB for later use.
2. Antagonism experiment
Diluting the cultured tobacco bacterial wilt pathogenic bacteria into bacterial suspension according to the amount of 1 percent. 50 μ L of LGT-1 seed solution prepared in example 2 was centrifuged at 12000rpm for 10min, the supernatant was added to a perforated LB plate of 5mm, and a dilution of the pathogen was uniformly sprayed on the plate for culture and observation.
As shown in FIG. 13, the diameter of the zone of inhibition of Bacillus beleisi LGT-1 against pathogenic bacteria of tobacco bacterial wilt reached 47.9mm.
Example 4: preparation of Bacillus beilesiensis LGT-1 microbial inoculum
(1) Selecting a single colony of Bacillus belgii LGT-1, inoculating the single colony into a triangular flask filled with 10-30 mL of LB liquid culture medium (tryptone 1%, yeast extract 0.5%, naCl 1%, pH = 7.2-7.4), carrying out shaking culture at the temperature of 30 ℃ and 200r/min for about 24 hours until the OD600 value is more than 0.6, and preparing a seed solution;
(2) Transferring the seed liquid into a fermentation tank for fermentation at an inoculation amount of 2% (fermentation medium formula: 10 g.L soluble starch) -1 Soy peptone 14 g.L -1 ，CaCl 2 6g·L -1 1000mL of distilled water, adjusting the pH value to 7.5), culturing at 30 ℃ for about 48 hours at 180r/min to prepare fermentation liquor;
(3) Different microbial inoculum formulations are prepared according to different application aspects:
a) Liquid preparation: fermenting the fermentation liquid in 2% inoculum size in fermentation tank at 30 deg.C for 180r/min for 48 hr, and stopping fermentation until the fermented bacterial liquid contains 10 bacterial liquid per ml 9 ～10 10 And (4) counting the number of the live bacteria, and packaging the fermentation liquor by using a plastic bottle or a plastic bag to obtain the Bacillus beilesensis liquid microbial inoculum.
b) Solid powder preparation: fermenting the fermentation broth in a fermentation tank at 30 deg.C for 180r/min for 48 hr, stopping fermentation, centrifuging, adding charcoal powder to the obtained thallus to adsorb thallus, wherein the thallus number per gram of microbial inoculum is 10 12 ～10 14 And (4) counting the number of the live bacteria, and hermetically packaging the live bacteria by using a plastic bag to obtain the bacillus belief solid microbial agent.
The prepared microbial inoculum can be used as a biological control preparation for controlling phytopathogen, the bacterial strain in the microbial inoculum has high activity, and the culture method is simple.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described in the foregoing embodiments, or equivalents may be substituted for some of the features thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Sequence listing
<110> Qingdao university of agriculture
<120> Bacillus belgii LGT-1 with high-efficiency ralstonia solanacearum antagonism and application thereof
<141> 2022-03-25
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1041
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
aggggggtgc tataatgcaa gtcgagcgga cagatgggag cttgctccct gatgttagcg 60
gcggacgggt gagtaacacg tgggtaacct gcctgtaaga ctgggataac tccgggaaac 120
cggggctaat accggatggt tgtctgaacc gcatggttca gacataaaag gtggcttcgg 180
ctaccactta cagatggacc cgcggcgcat tagctagttg gtgaggtaac ggctcaccaa 240
ggcgacgatg cgtagccgac ctgagagggt gatcggccac actgggactg agacacggcc 300
cagactccta cgggaggcag cagtagggaa tcttccgcaa tggacgaaag tctgacggag 360
caacgccgcg tgagtgatga aggttttcgg atcgtaaagc tctgttgtta gggaagaaca 420
agtgccgttc aaatagggcg gcaccttgac ggtacctaac cagaaagcca cggctaacta 480
cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg tccggaatta ttgggcgtaa 540
agggctcgca ggcggtttct taagtctgat gtgaaagccc ccggctcaac cggggagggt 600
cattggaaac tggggaactt gagtgcagaa gaggagagtg gaattccacg tgtagcggtg 660
aaatgcgtag agatgtggag gaacaccagt ggcgaaggcg actctctggt ctgtaactga 720
cgctgaggag cgaaagcgtg gggagcgaac aggattagat accctggtag tccacgccgt 780
aaacgatgag tgctaagtgg ttagggggtt tccgcccctt agtgctgcag ctaacgcatt 840
aagcactccg cctgggggag tacggtcgca agactgaact caagggaatt gacggggccg 900
cacagcgggg ggagcatgtg gtttattcga agcacgcaag aaccttacca ggcttggatc 960
ttgcacccct aaaaaaagac gcccttcggg gaaaggaaag gggggggggt ggtggccccc 1020
ctctcccggg agagtgatgt g 1041
<210> 2
<211> 986
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tagtcatgca tgagcgttat cgtatcccgg gcgcttccgg atgtgcgtga cggtctgaag 60
ccggttcaca gacggatttt gtacgcgatg aatgatttag gcatgaccag tgacaaacca 120
tataaaaaat ctgcccgtat tgtcggtgaa gttatcggta agtaccaccc gcacggtgac 180
tcagcggttt acgaatcaat ggtcagaatg gcgcaggatt ttaactaccg ctacatgctt 240
gttgacggac acggcaactt cggttcggtt gacggcgact cagcggccgc gatgcgttac 300
acagaagcga gaatgtcaaa aatcgcaatg gaaatcctac gggacattac gaaagatacg 360
attgattatc aagataacta tgacggcgca gaaagagaac ctgtcgtcat gccttcgaga 420
tttccgaatc tgctcgtcaa cggagctgcc ggtattgcgg tcggaatggc gacaaatatt 480
cctccgcatc agcttgggga agtcattgaa ggcgtgcttg ccgtaagtga gaatcctgag 540
attacaaacc aggagctgat ggaatacatc ccgggcccgg attttccgac tgcaggtcag 600
attttaggcc ggagcggcat ccgcaaggca tatgaatccg gacggggatc cattacgatc 660
cgggctaagg ctgaaatcga agagacatca tcgggaaaag aaagaattat tgtcacagaa 720
cttccttatc aggtgaacaa agcgagatta attgaaaaaa tcgcagatct tgtccgggac 780
aaaaaaatcg aaggaattac ggatctgcgt gacgaatccg accgtaacgg aatgagaatc 840
gtcattgaga ttcgccgtga cgctaatgct cacgtcattt tgaataacct gtacaaacaa 900
acggccctgc agacgtcttt cggaatcaac ctgctggcgc tcgttgacgg acagccgaag 960
gtctaagctg acacgcgggg gccgaa 986
<210> 3
<211> 1028
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgcagtcga ggagcggata taaagtatcc ggcggtcttc acggtgtagg ggcgtctgtc 60
gtaaacgcct tgtcgaccac tcttgacgtt acggttcatc gtgacggaaa aatccactat 120
caggcgtacg agcgcggtgt acctgtggcc gatcttgaag tgatcggtga tactgataag 180
accggaacga ttacgcactt cgttccggat ccggaaattt tcaaagaaac aaccgtatac 240
gactatgacc tgctttcaaa ccgtgtccgg gaattggcct tcctgacaaa aggcgtaaac 300
atcacgattg aagacaaacg tgaaggacaa gaacggaaaa acgagtacca ctacgaaggc 360
ggaatcaaaa gctatgttga gtacttaaac cgttccaaag aagtcgttca tgaagagccg 420
atttatatcg aaggcgagaa agacggcata acggttgaag ttgcgttgca atacaacgac 480
agctatacaa gcaacattta ttctttcaca aataacatca acacatacga aggcgggacg 540
cacgaagccg gatttaaaac cggtctgacc cgtgtcataa acgactatgc aagaagaaaa 600
gggattttca aagaaaatga tccgaattta agcggggatg atgtgagaga agggctgact 660
gccattattt caattaagca ccctgatccg caattcgaag ggcagacgaa aacgaagctc 720
ggcaactccg aagcgagaac gatcactgat acgctgtttt cttctgcgct ggaaacattc 780
cttcttgaaa atccggactc agcccgcaaa atcgttgaaa aaggtttaat ggccgcaaga 840
gcgcggatgg cagcgaaaaa agcacgggaa ttgacccggc gcaaaagtgc gcttgagatt 900
tcaatctgcc gggcaactgg cggactgttc ttctaaaatc cgagcattcc gagctgttat 960
cgtaaagggg gctctgcggg cggatcagcg aacaggggcg ggatcgcctt tccaagcctt 1020
ctggccgt 1028
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
<210> 6
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
cagtcaggaa atgcgtacgt cctt 24
<210> 7
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
caaggtaatg ctccaggcat tgct 24
<210> 8
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
gaagtcatca tgaccgttct gcaygcnggn ggnaarttyg a 41
<210> 9
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
agcagggtac gcatgtgcga gccrtcnacr tcngcrtcng tcat 44
Claims (7)
1. The Bacillus subtilis LGT-1 capable of efficiently antagonizing ralstonia solanacearum is characterized by being named as Bacillus subtilis velezensis in a classification mode, and the preservation number is CGMCC No.24342.
2. The use of Bacillus belgii LGT-1 according to claim 1 for the preparation of a control fungicide for the inhibition of soil pathogenic bacteria, wherein said soil pathogenic bacteria is tobacco bacterial wilt.
3. The application of claim 2, wherein the control microbial inoculum contains viable bacteria with the content of not less than 10 9 CFU/mL of Bacillus belgii LGT-1 or its inoculum.
4. The use according to claim 2, characterized in that the Bacillus belgii LGT-1 preparation is prepared by:
(1) Inoculating activated Bacillus belgii LGT-1 into a seed culture medium, and culturing at 25-35 ℃ for 20-24 h to prepare LGT-1 seed liquid;
(2) Transferring the LGT-1 seed liquid into a fermentation tank, and carrying out fermentation culture for 40-50 h at 25-35 ℃ to prepare LGT-1 fermentation liquid;
(3) And (3) transferring the LGT-1 fermentation liquor into the fermentation tank again, carrying out fermentation culture for 40-50 h at the temperature of 25-35 ℃, directly packaging or centrifuging the prepared LGT-1 secondary fermentation liquor, taking the precipitate, drying, and packaging to obtain the Bacillus beilaisi LGT-1 microbial inoculum.
5. The use according to claim 4, wherein the formulation of the fermentation medium in the fermenter is: soluble starch 6 g.L -1 ～12g·L -1 Soy peptone 10 g.L -1 ～15g·L -1 ，CaCl 2 5g·L -1 ～10g·L -1 The pH value is 7-7.5, and the rest is water.
6. The use according to claim 4, wherein the LGT-1 seed solution or LGT-1 fermentation broth is transferred to the fermentor in an amount of 1% to 5%.
7. Use of Bacillus belgii LGT-1 in the preparation of a formulation for reducing or inhibiting the resistance of a tobacco-pathogenic bacterium, according to claim 1, wherein the tobacco-pathogenic bacterium is the bacterium tobacco wilt.
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