CN114657097A - Bacillus beleisi LGT-1 capable of efficiently antagonizing ralstonia solanacearum and application thereof - Google Patents
Bacillus beleisi 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 hasThe bacterial wilt antagonistic capacity is strong, the diameter of a inhibition 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 disease 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 assisted. 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 soil environment quality of the tobacco field can be influenced.
In recent years, the quality of the roasted grass and the quality of the tobacco field soil are more and more emphasized, and the reduction of food and use for chemical prevention and control is a necessary trend, so that the development and popularization and 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 a bacterial wilt prevention and control microbial inoculum for tobacco.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention provides a Bacillus subtilis LGT-1 for efficiently antagonizing ralstonia solanacearum, which is classified and named as Bacillus subtilis velezensis, and the preservation number is CGMCC No. 24342.
Further, the 16S rDNA nucleotide sequence of the Bacillus belgii LGT-1 is shown in SEQ ID No. 1.
Further, 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 belgii LGT-1 in preparation of 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 109CFU/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-1Yeast powder 5 g.L-1Sodium chloride 10 g.L-1And 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-1Soy peptone 10 g.L-1～15g·L-1，CaCl2 5g·L-1～10g·L-1The pH value is 7 to 7.5.
Preferably, the formula of the fermentation medium in the fermentation tank is as follows: soluble starch 10 g.L-1Soy peptone 14 g.L-1，CaCl2 6g·L-1The 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 ℃ and 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 by using the inoculation amount of 2%, fermenting and culturing for 48h at 30 ℃ 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.
Further, the Bacillus belgii LGT-1 obviously inhibits the activity of tobacco bacterial wilt pathogen.
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 ralstonia solanacearum; 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 colony of Bacillus belgii LGT-1;
FIG. 2 is a 16S rRNA phylogenetic tree of Bacillus beleisi LGT-1;
FIG. 3 is a gyrA phylogenetic tree of Bacillus belgii 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 beijerinckii 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
First, separating 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.
II, 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 bacteria 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 reactions of LGT-1
Note: "+" is positive and "-" is negative.
3. Molecular biological identification
Genomic DNA of strain LGT-1 was extracted using a bacterial genome extraction kit purchased from Aikori (Shanghai) GmbH, 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 and the gyrB gene fragment of the obtained strain LGT-1 are 1041bp, 986bp and 1028bp respectively.
The 16SrDNA gene sequence of the LGT-1 obtained by sequencing is shown as SEQ ID No. 1; the sequence of the gyrA gene obtained by sequencing is shown as SEQ ID No. 2; the sequence of the gyrB gene obtained by sequencing is shown in 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 (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
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-1Yeast powder 5 g.L-1Sodium chloride 10 g.L-1And 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-1Shaking and culturing for 24h under the condition.
Initial conditions of shake flask fermentation: inoculating seed solution of LGT-1 strain into a 100mL triangular flask containing 40mL fermentation medium at 30 deg.C and 200r min-1Shaking 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.
Second, screening of basic culture 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 medium8cfu·mL-1The number of viable bacteria in the fermentation liquid per unit volume of the No.1 culture medium is 8.62 multiplied by 108cfu·mL-1And the number of viable bacteria in the fermentation liquid of No.3 culture medium per unit volume is 7.83 multiplied by 108cfu·mL-1And the number of viable bacteria in unit volume corresponding to the No. 4 culture medium fermentation liquid is 8.96 multiplied by 108cfu·mL-1. Therefore, medium No.2 was selected as the basal medium for shake flask fermentation of Bacillus belgii strain LGT-1.
Three, single factor test
(1) Selection of carbon Source types and concentrations thereof
Respectively replacing a carbon source (C source) in a basic culture medium by equal amounts of glucose, corn flour, maltose, soluble starch, sucrose and mannitol, taking the basic culture medium without the C source as a Control (CK), and performing shake flask fermentation under the same other fermentation conditions, wherein each treatment is repeated for 3 times. 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%, i.e., 4, 6, 8, 10, 12, 14 g.L, were set under the same conditions-1Shake flask fermentation is carried out on 6 culture media with different concentrations of C source so as to screen out the optimal concentration of the C source.
As shown in FIG. 6, the number of viable bacteria of Bacillus beilaiensis LGT-1 was greatly influenced by different C sources, and the maximum value of the number of viable bacteria per unit volume was 17.53X 10 when soluble starch was used as the C source of the medium8cfu·m L-1Therefore, soluble starch was selected as the C source for the B.beiensis LGT-1 shake flask fermentation medium.
(2) Selection of nitrogen source species and concentration thereof
On the basis of C source screening, respectively using peptone, beef extract and water extract with equal quantity,Urea, ammonium sulfate, soybean peptone, corn steep liquor dry powder and yeast powder replace nitrogen sources (N sources) in a fermentation medium, the medium without the N source is used as a Control (CK), other fermentation conditions are unchanged, and shake flask fermentation is carried out, wherein each treatment is repeated for 3 times. 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% of the optimal N source, namely 4, 6, 8, 10, 12, 14 g.L, are set under the same conditions-1Shake 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, when different N sources were used as the medium components, Bacillus belgii LGT-1 significantly affected the number of viable bacteria per unit volume. Wherein the maximum viable bacteria number is 17.5 × 10 when soybean peptone is used as single N source8cfu·m L-1Thus, 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 respectively4，NaCl，CaCl2，MnSO4，MgSO4KCl was used as a Control (CK) instead of the inorganic salt in the fermentation medium, and the other fermentation conditions were not changed and repeated 3 times per treatment. 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 screening out the optimal inorganic salt, the different concentrations (0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%) of the macroelement inorganic salt, i.e. 2, 3, 4, 5, 6, 7 g.L, were examined under the same conditions-1) And (4) screening out the optimal concentration of the inorganic salt according to the viable count in the fermentation liquor.
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 used2When 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 108cfu·mL-1By ZnSO4，MnSO4，MgSO4In the case of an inorganic salt, the salt,the number of viable bacteria in unit volume of fermentation liquid is less than that in the control group without inorganic salt. Thus, CaCl was selected2Inorganic salt used as a shake flask fermentation medium of the Bacillus belgii LGT-1.
Fourthly, 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 CaCl2. Mixing soluble starch (A), soybean peptone (B) and CaCl2(C) Three factors the factors and levels for the orthogonal test were designed according to L9(33), as shown in Table 4, see Table 4. Designed according to an orthogonal experiment at 30 ℃ and 200 r.min-1And 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
As shown in Table 5, the results of the orthogonal test revealed that N-source soybean peptone had the greatest influence on the number of viable bacteria in a unit volume of fermentation broth of Bacillus beilaisi LGT-1, and further, inorganic salt CaCl2The corresponding combination of C source soluble starch is No. 4 culture medium A2B1C2, and the number of viable bacteria in unit volume of fermentation liquid is 32.23 × 108cfu·mL-1. Therefore, the fermentation medium formula of the Bacillus belgii LGT-1 is determined to be 10 g.L of soluble starch-1Soy peptone 14 g.L-1，CaCl2 6g·L-1。
Table 5: results of fermentation Medium Quadrature experiments
Optimization of shake flask fermentation conditions
On the basis of optimizing the culture medium, the fermentation temperature, the initial pH, the inoculation amount and the liquid loading amount of the 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 respectively; initial pH: adjusting the initial pH values of the culture medium to 6.5, 7.0, 7.5, 8.0 and 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 × 108cfu·m L-1(ii) a The initial pH of the fermentation is shown in FIG. 10, and the fermentation broth has the highest bacteria content per unit volume at the initial pH of 7.5, and the viable bacteria count is 37.03X 108cfu·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% inoculation8cfu·m L-1(ii) a As shown in FIG. 12, when the medium was filled at 20%, the number of viable bacteria per unit volume of the fermentation liquid was significantly higher than that of the other liquid contents, and the maximum effective bacteria content was 37.4X 108cfu·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 5mm perforated LB plate, and a dilution of the pathogenic bacteria 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 47.9 mm.
Example 4: preparation of Bacillus beilesiensis LGT-1 microbial inoculum
(1) Selecting a single colony of Bacillus beleisi 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%, and pH 7.2-7.4), performing shaking culture at the temperature of 30 ℃ at 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，CaCl2 6g·L-11000mL 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 ml9～1010And (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 1012～1014And (4) counting the number of the live bacteria, and hermetically packaging the live bacteria by using a plastic bag to obtain the bacillus beilesensis solid microbial inoculum.
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 various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Sequence listing
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Claims (10)
1. The Bacillus belgii LGT-1 for efficiently antagonizing ralstonia solanacearum is characterized by being named as Bacillus belgii in classificationBacillus velezensisThe preservation number is CGMCC No. 24342.
2. The Bacillus belgii LGT-1 according to claim 1, wherein the 16S rDNA nucleotide sequence of the Bacillus belgii LGT-1 is as shown in SEQ ID No. 1.
3. The Bacillus belgii LGT-1 of claim 1, wherein the Bacillus belgii LGT-1 isgyrAThe nucleotide sequence is shown as SEQ ID No.2, whichgyrBThe nucleotide sequence is shown as SEQ ID No. 3.
4. Use of Bacillus belgii LGT-1 according to any one of claims 1 to 3 for the preparation of a control fungicide for the inhibition of soil pathogenic bacteria.
5. The application of claim 4, wherein the control microbial inoculum contains viable bacteria with the content of not less than 109CFU/mL of Bacillus belgii LGT-1 or its inoculum.
6. The use according to claim 4, 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, fermenting and culturing 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.
7. The use according to claim 6, wherein the formulation of the fermentation medium in the fermenter is: soluble starch 6 g/L-1~12 g/L-1Soy peptone 10 g/L-1~15 g/L-1，CaCl2 5 g/L-1~10 g/L-1The pH value is 7 to 7.5.
8. The use according to claim 6, wherein the LGT-1 seed solution or LGT-1 fermentation broth is transferred to the fermentor at an inoculum size of 1% to 5% each.
9. The use according to claim 4, wherein the soil pathogenic bacteria is tobacco bacterial wilt.
10. Use of Bacillus belgii LGT-1 according to any one of claims 1 to 3 for the preparation of a formulation for reducing or suppressing the resistance of pathogenic bacteria in tobacco.
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| CN118222458A (en) * | 2024-05-06 | 2024-06-21 | 青岛农业大学 | Bacillus bailii F046, microbial agent and application thereof |
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