CN114350548A - Biocontrol microbial inoculum for inhibiting streptococcus agalactiae and preparation method and application thereof - Google Patents
Biocontrol microbial inoculum for inhibiting streptococcus agalactiae and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a biocontrol microbial inoculum for inhibiting streptococcus agalactiae, which comprises the following components in parts by weight: 20-40 parts of a boron-resistant lysine bacillus fermentation liquid and 8-12 parts of an auxiliary material, wherein the auxiliary material is at least one of water, a culture medium and glycerol, and the boron-resistant lysine bacillus fermentation liquid is obtained by performing secondary fermentation culture on the boron-resistant lysine bacillus. The biocontrol microbial inoculum for inhibiting the streptococcus agalactiae can effectively inhibit the growth of the streptococcus agalactiae, has an inhibition rate of over 49.5 percent in 36 hours of fermentation liquor, and can be used for preventing or treating infection diseases caused by the streptococcus agalactiae, particularly for effectively preventing or treating tilapia streptococcicosis infected by the streptococcus agalactiae. The boron-resistant lysine bacillus can reduce the infection of the streptococcus agalactiae to a great extent, has no influence on the ecological environment, is expected to be used as a high-quality chassis microorganism for synthetic biology research, and brings a new direction for the prevention and treatment of the streptococcus agalactiae.
Description
Technical Field
The invention relates to the technical field of biocontrol bacterium application, in particular to a biocontrol bacterium agent for inhibiting streptococcus agalactiae and a preparation method and application thereof.
Background
Streptococcus agalactiae (Streptococcus agalactiae), also known as Group B Streptococcus (GBS), is an opportunistic pathogen and has a very strong pathogenic capacity in humans. The main infected people are the elderly and people with low immunity, especially the newborn. Streptococcus agalactiae is easy to colonize in the vagina of a pregnant woman, thereby causing intrauterine infection, is a main cause of diseases such as neonatal septicemia, pneumonia, meningitis and endocarditis, and has high lethality rate. Meanwhile, streptococcus agalactiae is also a main pathogenic bacterium causing streptococcicosis of tilapia. The current treatment methods are as follows:
antibiotic therapy is currently the predominant treatment. Strategies to prevent neonatal and maternal GBS disease include prenatal antibiotic prophylaxis and vaccination of the mother with GBS vaccine during the late stages of pregnancy. The antibiotic prevention and treatment is preferably penicillin G, and secondly, antibiotics such as erythromycin, chloramphenicol, clindamycin, ofloxacin, vancomycin, fluoroquinolones, etc. may be selected, but these antibiotics are only useful for treatment in cases where they are allergic to penicillin or cephalosporin and a predisposition for the organism has been established. However, current studies indicate that GBS is increasing in resistance to clindamycin, erythromycin and fluoroquinolone drugs, and vancomycin resistance has also been reported, which presents a significant problem in the treatment of patients allergic to penicillin or cephalosporin.
Thus, in recent years scientists have been working to find and develop new alternatives to traditional antibiotic therapies. Certain of these microorganisms produce secondary metabolites with broad spectrum activity, rapid killing kinetics, low levels of induced resistance and low toxicity to the host are believed to be useful in the treatment of Streptococcus agalactiae infections.
Disclosure of Invention
In view of the above, the present invention aims to provide a biocontrol microbial inoculum for inhibiting streptococcus agalactiae, a preparation method and applications thereof, and to solve the above problems.
The technical scheme of the invention is realized as follows:
a biocontrol microbial inoculum for inhibiting streptococcus agalactiae comprises the following components in parts by weight: 20-40 parts of boron-resistant lysine bacillus fermentation liquor and 8-12 parts of auxiliary materials; wherein the B-resistant lysine bacillus fermentation liquor is obtained by secondary fermentation culture of B-resistant lysine bacillus (Lysinibacillus boronolans).
Further, the boron-resistant lysine bacillus fermentation liquid is obtained by adding polyglutamic acid into boron-resistant lysine bacillus and performing secondary fermentation culture.
Further, the preparation method of the boron-resistant lysine bacillus fermentation liquor comprises the following steps:
(1) b-lysine-resistant bacillus 1-2 ring is taken and inoculated to a seed culture solution, activated and cultured for 12-16h at the temperature of 36-38 ℃ at 180r/min for 120-;
(2) according to the volume ratio of 2: 5, inoculating the activated bacteria liquid to the enrichment culture liquid, and carrying out enrichment culture at 120-600The value is 4.8-2.5, and a proliferation bacterial liquid is obtained;
(3) according to the volume ratio of 2: 7, inoculating the enrichment bacterium liquid to a fermentation culture liquid, performing fermentation culture at the temperature of 35-36 ℃ for 4-6h, adding 0.02-0.04 wt% of polyglutamic acid based on the fermentation culture liquid, and performing fermentation culture at the temperature of 35-36 ℃ for 6-8h to obtain a fermentation bacterium liquid;
(4) centrifuging the zymophyte liquid at 9000r/min of 7000-.
Further, the seed culture solution comprises the following components: 4.8-5.2g/L yeast extract, 9.5-10.5g/L tryptone and 9.5-10.5g/L sodium chloride, and the pH value is 7.0-7.2;
further, the propagation medium comprises the following components: 4.8-5.2g/L yeast extract, 9.5-10.5g/L tryptone, 9.5-10.5g/L sodium chloride and 2.2-3.2g/L polyglutamic acid, and the pH value is 7.0-7.2;
further, the fermentation broth comprises the following components: 4.8-5.2g/L yeast extract, 9.5-10.5g/L tryptone, 9.5-10.5g/L sodium chloride, 0.8-1.8g/L polyglutamic acid and 0.2-0.5g/L bergenin, and has a pH value of 6.7-7.0.
Further, the auxiliary material is at least one of water, culture medium and glycerol.
Further, the medium is an LB medium.
Further explaining, the biological control agent also comprises 12 to 16 parts of carrier; wherein the carrier is one or more of solvent, filler and binder.
Further, the preparation method of the biocontrol microbial inoculum for inhibiting streptococcus agalactiae comprises the following steps: mixing the boron-resistant lysine bacillus fermentation liquor and auxiliary materials, adding a carrier, stirring, spray-drying and crushing to obtain the biocontrol microbial inoculum.
Further, the biocontrol microbial inoculum is used for preventing or treating diseases caused by the streptococcus agalactiae.
Further indicates that the biocontrol microbial inoculum is applied to preventing or treating streptococcicosis of tilapia.
Further, the biocontrol microbial inoculum can be mixed with a medium, wherein the medium can be a carrier carrying Streptococcus agalactiae (Streptococcus agalactiae), such as any one or more of seeds, plants, soil for plant growth and culture medium for plant cultivation.
Compared with the prior art, the invention has the beneficial effects that:
the biocontrol microbial inoculum for inhibiting the streptococcus agalactiae is prepared from the boron-resistant lysine bacillus fermentation liquor, can effectively inhibit the growth of the streptococcus agalactiae, has an inhibition rate of over 49.5 percent, and has the effects of preventing or treating diseases caused by the streptococcus agalactiae, particularly tilapia streptococcosis caused by the streptococcus agalactiae.
In addition, the invention adopts a stepwise mode of activation, multiplication and secondary fermentation culture to carry out a culture mode of the boron-resistant lysine bacillus, combines a specific multiplication culture solution and a specific fermentation culture solution containing polyglutamic acid with certain concentration, is favorable for regulating and accumulating various secondary metabolites generated by the boron-resistant lysine bacillus in the culture process, and adds the polyglutamic acid to be cooperated with a culture medium in the secondary fermentation process, thereby further improving the activity of the boron-resistant lysine bacillus, leading the various secondary metabolites to jointly play a better sterilization effect and obviously improving the inhibition effect of the boron-resistant lysine bacillus fermentation liquor on the growth of streptococcus agalactiae.
The boron-resistant lysine bacillus has broad-spectrum antibacterial property, is harmless to the environment and human health, can grow on a common LB culture medium, is easy to culture, has short growth period, can be used for preparing a large amount of bactericides for inhibiting streptococcus agalactiae in a short time, and has the characteristics of low cost and high benefit; the fermentation liquor of the B-lysine-resistant bacillus contains various antibacterial secondary metabolites, and compared with a single bactericidal substance, the bactericidal effect of the combined exertion of the various secondary metabolites is better; according to an embodiment of the invention, the boron-resistant lysine bacillus can be mixed with other bacteria and fungi with biological control functions to prepare a preparation with good inhibition effect on various pathogenic bacteria.
The biocontrol microbial inoculum for inhibiting the streptococcus agalactiae prepared by the invention has the advantages of simple preparation method, no toxicity, no pollution, no residue and obvious effect, can be used as a high-quality chassis microorganism for synthetic biology research, and brings a new direction for the prevention and treatment of the streptococcus agalactiae.
Drawings
FIG. 1 is a graph showing the effect of a plate inhibition experiment of a B-resistant lysine bacillus fermentation broth on growth of Streptococcus agalactiae; wherein, panel A is experimental group 1-1, and panel B is control group.
Detailed Description
In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.
The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.
The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.
The LB culture medium (Luria-Bertani culture medium, abbreviated as LB culture medium) is a commonly used bacterial culture medium and is a semi-synthetic culture medium.
The OD (optical density, abbreviated as OD) referred to herein means the optical density absorbed by the test object, and the unit of detection is represented by the OD value. By measuring the OD (using OD) at 600nm of B-lysine-resistant Bacillus culture fluid600Expressed), the concentration of the B.borotolerant Bacillus culture broth can be measured to estimate the growth of B.borotolerant Bacillus.
Example 1 bacteriostatic Effect of B-resistant lysine-Bacillus fermentation broth
The inhibition effect of the B-resistant lysine bacillus fermentation liquor on the growth of the streptococcus agalactiae is measured by a medium-containing method.
Experimental group 1:
experimental groups 1-1:
the seed culture solution comprises the following components: 5g/L yeast extract, 10g/L tryptone and 10g/L sodium chloride, pH 7.0;
the proliferation culture solution comprises the following components: 5.2g/L yeast extract, 10.5g/L tryptone, 10.5g/L sodium chloride and 2.8g/L polyglutamic acid, pH 7.0;
the fermentation culture solution comprises the following components: 5.2g/L yeast extract, 10.5g/L tryptone, 10.5g/L sodium chloride, 1.2g/L polyglutamic acid and 0.3g/L bergenin, pH 6.8; preparing seed culture solution, proliferation culture solution and fermentation culture solution according to the above components, sterilizing at high temperature, and cooling to 55 deg.C;
experimental groups 1-2:
the seed culture solution comprises the following components: 5g/L yeast extract, 10g/L tryptone and 10g/L sodium chloride, pH 7.2;
the proliferation culture solution comprises the following components: 4.8g/L yeast extract, 9.5g/L tryptone, 9.5g/L sodium chloride and 3.2g/L polyglutamic acid, pH 7.2;
the fermentation culture solution comprises the following components: 4.8g/L yeast extract, 9.5g/L tryptone, 9.5g/L sodium chloride, 1.8g/L polyglutamic acid and 0.5g/L bergenin, pH 7.0; preparing seed culture solution, proliferation culture solution and fermentation culture solution according to the above components, sterilizing at high temperature, and cooling to 55 deg.C;
the preparation steps of the boron-resistant lysine bacillus fermentation liquid of the experimental group 1-1 and the experimental group 1-2 are as follows:
(1) inoculating loop 1 of the single colony of the B-lysine resistant bacillus to a seed culture solution, and performing activation culture at the temperature of 37 ℃ at 160r/min for 14h to obtain an activated bacterial solution;
(2) according to the volume ratio of 2: 5, inoculating the activated bacterial liquid to a proliferation culture solution, and performing proliferation culture at 160r/min and 37 ℃ until the bacterial liquid OD600The value is 2.5, and a proliferation bacterial liquid is obtained;
(3) according to the volume ratio of 2: 7, inoculating the enrichment bacterium liquid to a fermentation culture liquid, performing fermentation culture at 35 ℃ for 5 hours, adding 0.03 wt% of polyglutamic acid based on the fermentation culture liquid, and performing fermentation culture at 35 ℃ for 7 hours to obtain a fermentation bacterium liquid;
(4) centrifuging the zymocyte liquid at 8000r/min for 10min, collecting supernatant, and filtering with 0.22 μm filter membrane to obtain the B-resistant lysine bacillus zymocyte liquid.
Experimental group 2:
(1) seed culture solution and proliferation culture solution prepared by the experimental group 1-1 are adopted;
(2) inoculating loop 1 of the single colony of the B-lysine resistant bacillus to a seed culture solution, and performing activation culture at the temperature of 36 ℃ at 160r/min for 14h to obtain an activated bacterial solution;
(3) according to the volume ratio of 2: 5, inoculating the activated bacterial liquid to a proliferation culture solution, and performing proliferation culture at 160r/min and 36 ℃ until the bacterial liquid OD600The value is 4.8, and a proliferation bacterial liquid is obtained;
(4) centrifuging the proliferated bacteria liquid at 8000r/min for 10min, collecting supernatant, and filtering with 0.22 μm filter membrane to obtain the boron-resistant lysine bacillus fermentation liquid.
Experimental group 3:
(1) seed culture solution and fermentation culture solution prepared by the experimental group 1-1 are adopted;
(2) inoculating loop 1 of the single colony of the B-lysine resistant bacillus to a seed culture solution, and performing activation culture at the temperature of 38 ℃ at 160r/min for 14h to obtain an activated bacterial solution;
(3) according to the volume ratio of 2: 7, inoculating the activated bacterium liquid to a fermentation culture solution, performing fermentation culture at 35 ℃ for 5 hours, adding 0.03 wt% of polyglutamic acid based on the fermentation culture solution, and performing fermentation culture at 35 ℃ for 7 hours to obtain a fermentation bacterium liquid;
(4) centrifuging the zymocyte liquid at 8000r/min for 10min, collecting supernatant, and filtering with 0.22 μm filter membrane to obtain the B-resistant lysine bacillus zymocyte liquid.
Experimental group 4:
the difference from the experimental group 1-1 is that the seed culture solution prepared by the experimental group 1-1 is used for replacing the propagation culture solution and the fermentation culture solution, and the specific steps are as follows:
(1) inoculating loop 1 of the single colony of the B-lysine resistant bacillus to a seed culture solution, and performing activation culture at the temperature of 37 ℃ at 160r/min for 14h to obtain an activated bacterial solution;
(2) according to the volume ratio of 2: 5, inoculating the activated bacterial liquid to a new seed culture liquid, and culturing at the temperature of 37 ℃ at 160r/min until the bacterial liquid OD600The value is 2.5, and a culture bacterium solution 1 is obtained;
(3) according to the volume ratio of 2: 7, inoculating the culture bacterial liquid 1 to a new seed culture liquid, culturing for 5 hours at 35 ℃, adding 0.03 wt% of polyglutamic acid based on the new seed culture liquid, and culturing for 7 hours at 35 ℃ to obtain a culture bacterial liquid 2;
(4) centrifuging the culture solution 2 at 8000r/min for 10min, collecting supernatant, and filtering with 0.22 μm filter membrane to obtain B-resistant lysine bacillus fermentation solution.
Experimental group 5:
the difference from the experimental group 1-1 is that the step (4) does not add polyglutamic acid, and the specific steps are as follows: according to the volume ratio of 2: and 7, inoculating the enrichment bacterial liquid to the fermentation culture liquid, and performing fermentation culture at 35 ℃ for 12 hours to obtain the fermentation bacterial liquid.
The experimental method comprises the following steps: selecting single colony of Streptococcus agalactiae, inoculating to LB liquid culture medium, culturing at 37 deg.C overnight at 150r/min, and diluting the bacteria solution to OD600The value was 0.1OD600Sucking 1mL, centrifuging at 6000rpm for 10min, discarding supernatant, washing precipitate with 1mL PBS buffer solution for 3 times, centrifuging at 6000rpm for 3min, discarding supernatant, and collecting precipitateAdding 500 mu L of the B-resistant lysine bacillus of the experimental group 1-5 for fermentation, placing the mixture in a constant-temperature incubator at 37 ℃ for incubation for 10-12h, and replacing the B-resistant lysine bacillus fermentation liquid with PBS buffer solution in the control group. Respectively sucking 5 mu L of bacterial suspension of an experimental group and a control group, vertically dropping the bacterial suspension into the central point of an LB flat plate, placing the LB flat plate in a constant-temperature incubator at 37 ℃ for static culture for 18-36h, observing the colony diameter of the streptococcus agalactiae, measuring the colony diameter by adopting a cross method, and calculating the bacteriostasis rate, wherein the bacteriostasis rate (%) is (the diameter of the control group colony-the diameter of the experimental group)/the diameter of the control group colony multiplied by 100 percent, and the results are shown in the following table 1 and figure 1:
as can be seen from the above table, the test groups 1-1 and 1-2 showed an inhibition rate of 45.5% or more for the growth of Streptococcus agalactiae, and as can be seen from FIG. 1, the growth of Streptococcus agalactiae on the culture dish was significantly inhibited by B.borotolerant lysine.
The experimental groups 2 and 3 do not undergo the processes of activation, proliferation and fermentation culture, and the growth inhibition effect of the boron-resistant lysine bacillus fermentation liquor on streptococcus agalactiae is weakened, so that the boron-resistant lysine bacillus is gradually subjected to a stepped culture mode of activation, proliferation and fermentation culture, and secondary metabolites of the boron-resistant lysine bacillus are favorably regulated and accumulated; the experiment group 4 adopts the single seed culture solution to obtain the boron-resistant lysine bacillus fermentation liquid, the inhibition rate on the growth of streptococcus agalactiae is lower, and the stepped culture mode is combined with the specific proliferation culture solution and the specific fermentation culture solution, so that the boron-resistant lysine bacillus fermentation liquid is more favorable for generating various antibacterial secondary metabolites, and the various secondary metabolites jointly play a better bactericidal effect; the experimental group 5 shows that the addition of polyglutamic acid in the fermentation culture process can play a role in regulating and controlling the product of the fermentation of the boron-resistant lysine bacillus together with the culture medium, is beneficial to regulating the stable period of the growth of the boron-resistant lysine bacillus, can enhance the activity of the fermentation liquid, enables the boron-resistant lysine bacillus fermentation liquid to reach the bacteriostasis rate of 49.5% after 36 hours, and remarkably promotes the bactericidal performance of the boron-resistant lysine bacillus fermentation liquid.
Example 2 prevention and treatment experiment of Tilapia mossambica streptococcicosis
A biocontrol microbial inoculum for inhibiting streptococcus agalactiae consists of 30 parts of boron-resistant lysine bacillus fermentation liquor prepared by an experimental group 1-1, 10 parts of water and 14 parts of corn protein powder;
the preparation method of the biocontrol microbial inoculum comprises the following steps: mixing the boron-resistant lysine bacillus fermentation liquor with water, adding kaolin, stirring, spray drying and crushing to obtain a biocontrol microbial inoculum;
selecting 40 healthy tilapia mossambica tails with the weight of 80-90g, dividing the tilapia mossambica tails into 2 groups, feeding tilapia mossambica feed for 2 times every day, changing water every 5d, adjusting water quality, mixing and feeding a biocontrol agent and tilapia mossambica feed in a ratio of 1:200 to form a test group, and mixing and feeding florfenicol and tilapia mossambica feed in a ratio of 1:2000 to form a medicine group; after feeding for 10 days, injecting 0.2mL dose of Streptococcus agalactiae suspension into abdominal cavity of tilapia, continuously feeding for 7 days, and observing tilapia condition, wherein the Streptococcus agalactiae suspension is obtained by selecting Streptococcus agalactiae single colony, inoculating into LB liquid culture medium, culturing at 37 deg.C overnight at 150r/min, and diluting with normal saline until total bacteria number is 1 × 108Collecting accumulated liquid in the inner membrane of the gill cap of the dead tilapia mossambica in a CFU/mL mode, inoculating the collected liquid to a culture dish for culture at 37 ℃, observing the infection condition of streptococcus agalactiae by gram staining, and displaying that the dead tilapia mossambica are infected with the streptococcus agalactiae by microscopic examination results. The results of the control experiments are given in table 2 below:
| item | Number of deaths | Mortality (%) |
| Test group | 2 | 10 |
| Drug group | 5 | 25 |
As can be seen from the table above, the biocontrol bactericide prepared by the invention greatly reduces the mortality rate of tilapia infected with streptococcus agalactiae, can effectively inhibit the growth of streptococcus agalactiae and enhance the immunocompetence of tilapia, has good treatment effect and is superior to drug treatment.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. The biocontrol microbial inoculum for inhibiting streptococcus agalactiae is characterized by comprising the following components in parts by weight: 20-40 parts of boron-resistant lysine bacillus fermentation liquor and 8-12 parts of auxiliary materials; the boron-resistant lysine bacillus fermentation liquid is obtained by performing secondary fermentation culture on boron-resistant lysine bacillus.
2. The biocontrol microbial inoculum for inhibiting streptococcus agalactiae according to claim 1, wherein the boron-resistant lysine bacillus fermentation broth is obtained by adding polyglutamic acid to boron-resistant lysine bacillus and performing secondary fermentation culture.
3. The biocontrol microbial inoculum for inhibiting streptococcus agalactiae according to claim 1 or 2, wherein the preparation method of the boron-resistant lysine bacillus fermentation broth comprises the following steps:
(1) b-lysine-resistant bacillus 1-2 ring is taken and inoculated to a seed culture solution, activated and cultured for 12-16h at the temperature of 36-38 ℃ at 180r/min for 120-;
(2) push buttonThe volume ratio is 2: 5, inoculating the activated bacteria liquid to the enrichment culture liquid, and carrying out enrichment culture at 120-600The value is 4.8-2.5, and a proliferation bacterial liquid is obtained;
(3) according to the volume ratio of 2: 7, inoculating the enrichment bacterium liquid to a fermentation culture liquid, performing fermentation culture at the temperature of 35-36 ℃ for 4-6h, adding 0.02-0.04 wt% of polyglutamic acid based on the fermentation culture liquid, and performing fermentation culture at the temperature of 35-36 ℃ for 6-8h to obtain a fermentation bacterium liquid;
(4) centrifuging the zymophyte liquid at 9000r/min of 7000-.
4. The biocontrol bacterial agent for inhibiting streptococcus agalactiae according to claim 3, wherein said seed culture solution comprises the following components: 4.8-5.2g/L yeast extract, 9.5-10.5g/L tryptone and 9.5-10.5g/L sodium chloride, and the pH value is 7.0-7.2;
the proliferation culture solution comprises the following components: 4.8-5.2g/L yeast extract, 9.5-10.5g/L tryptone, 9.5-10.5g/L sodium chloride and 2.2-3.2g/L polyglutamic acid, and the pH value is 7.0-7.2;
the fermentation culture solution comprises the following components: 4.8-5.2g/L yeast extract, 9.5-10.5g/L tryptone, 9.5-10.5g/L sodium chloride, 0.8-1.8g/L polyglutamic acid and 0.2-0.5g/L bergenin, and has a pH value of 6.7-7.0.
5. The biocontrol microbial inoculum for inhibiting streptococcus agalactiae according to claim 1, wherein the adjuvant is at least one of water, culture medium and glycerol.
6. The biocontrol microbial inoculum for inhibiting Streptococcus agalactiae according to claim 5, wherein said medium is LB medium.
7. The biocontrol microbial inoculum for inhibiting streptococcus agalactiae according to claim 1 or 2, further comprising 12-16 parts of a carrier; the carrier is one or more of a solvent, a filler and a binder.
8. The method for preparing the biocontrol microbial inoculum for inhibiting streptococcus agalactiae according to any one of claims 1 to 7, characterized by comprising the following steps: mixing the boron-resistant lysine bacillus fermentation liquor and auxiliary materials, adding a carrier, stirring, spray-drying and crushing to obtain the biocontrol microbial inoculum.
9. The use of a biocontrol bacterial agent for inhibiting streptococcus agalactiae according to claim 8, wherein said biocontrol bacterial agent is in the prevention or treatment of a disease in which streptococcus agalactiae causes an infection.
10. The use of a biocontrol bacterial agent for inhibiting streptococcus agalactiae according to claim 8, wherein said biocontrol bacterial agent is used in the prevention or treatment of streptococcicosis in tilapia.
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| CN111100806A (en) * | 2019-10-11 | 2020-05-05 | 海南大学 | Areca-nut root rot bactericide prepared by taking boron-resistant lysine bacillus as underpan cells |
| CN111100805A (en) * | 2019-10-11 | 2020-05-05 | 海南大学 | Rice sheath blight disease bactericide prepared by using boron-resistant lysine bacillus as chassis cells |
| CN111187726A (en) * | 2019-10-11 | 2020-05-22 | 海南大学 | Rice blast bactericide prepared by using lysine-resistant bacillus borreliensis as chassis cells |
| CN113416670A (en) * | 2021-06-10 | 2021-09-21 | 江苏省农业科学院 | Boron-resistant lysine bacillus with bacteriostatic ability and application thereof |
| CN113652367A (en) * | 2021-07-29 | 2021-11-16 | 海南大学 | Dragon fruit canker bactericide and application thereof |
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| CN111100806A (en) * | 2019-10-11 | 2020-05-05 | 海南大学 | Areca-nut root rot bactericide prepared by taking boron-resistant lysine bacillus as underpan cells |
| CN111100805A (en) * | 2019-10-11 | 2020-05-05 | 海南大学 | Rice sheath blight disease bactericide prepared by using boron-resistant lysine bacillus as chassis cells |
| CN111187726A (en) * | 2019-10-11 | 2020-05-22 | 海南大学 | Rice blast bactericide prepared by using lysine-resistant bacillus borreliensis as chassis cells |
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