CN113373092A - Compound microbial preparation and preparation method thereof - Google Patents
Compound microbial preparation and preparation method thereof Download PDFInfo
- Publication number
- CN113373092A CN113373092A CN202110743497.6A CN202110743497A CN113373092A CN 113373092 A CN113373092 A CN 113373092A CN 202110743497 A CN202110743497 A CN 202110743497A CN 113373092 A CN113373092 A CN 113373092A
- Authority
- CN
- China
- Prior art keywords
- microbial preparation
- bacillus subtilis
- preparation
- bacterial
- powder
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/28—Streptomyces
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K17/00—Soil-conditioning materials or soil-stabilising materials
- C09K17/14—Soil-conditioning materials or soil-stabilising materials containing organic compounds only
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2101/00—Agricultural use
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
Abstract
The invention provides a compound microbial preparation and a preparation method thereof, wherein bacillus subtilis and streptomyces roseoflavus are compounded into the compound microbial preparation, the prevention and treatment effect is realized by secreting resistant substances, heavy parasitizing action, inducing host cells to generate a defense structure, competition action and other disease prevention mechanisms, the two strains also have synergistic interaction, can effectively and stably prevent and treat soil-borne disease pathogenic bacteria such as gray mold, powdery mildew, banana wilt, soybean root rot and the like, and can also be used for soil improvement of crop fields such as vegetables, flowers, melons, fruits, forest trees, Chinese herbal medicines and the like after continuous cropping.
Description
Technical Field
The invention relates to the technical field of biological preparations, in particular to a compound microbial preparation and a preparation method thereof.
Background
At present, the prevention and control of plant diseases are mainly based on chemical prevention and control, however, long-term use of chemical prevention and control agents not only causes pollution, but also causes various diseases to have higher and higher resistance, and the chemical prevention and control agents kill harmful bacteria, kill probiotics in plants and in soil by mistake, destroy immune systems of the plants and cause the plant diseases to be more difficult to control.
In recent years, as the pollution problem of chemical pesticides has become more serious, biological control has been increasingly emphasized because of its advantages of no pollution, no residue, difficulty in generating drug resistance, safety to ecological environment and human health, and the like, and the application of biological control technology in plant disease control is receiving much attention. The biological control technology is to utilize the biological control microorganisms existing in the nature, and utilize the living bodies or metabolites thereof to inhibit the quantity and activity of pathogenic microorganisms, thereby reducing the occurrence of diseases or slowing the occurrence rate of the diseases and reducing the severity of the diseases.
At present, most of researches on biological control agents are single bacterial strains, and CN103131657A, namely, a Bacillus subtilis strain and a biological control preparation and application thereof, discloses application of the Bacillus subtilis strain in the biological control preparation, but the single bacterial strain has a single disease prevention mechanism and low and unstable control effect. The composite bacteria preparation has various bacterial strains, so that the preservation and the application of the composite bacteria preparation are limited by various conditions.
Disclosure of Invention
Aiming at the problems, the invention provides a compound microbial preparation and a preparation method thereof.
The invention relates to a compound microbial preparation, which comprises the following components: bacillus subtilis and Streptomyces roseoflavus.
Further, the amount of live bacteria contained in the complex microbial preparation is 106-109cfu/g。
Further, the CFU ratio of the bacillus subtilis to the streptomyces roseoflavus in the compound microbial preparation is (4-7): (1-2).
Further, the complex microbial preparation further comprises: a solidifying agent.
Further, the solidifying agent is at least one of diatomite, kaolin, bentonite and mica powder.
The invention also provides a preparation method of the compound microbial preparation, which comprises the following steps:
(1) respectively inoculating bacillus subtilis and streptomyces roseoflavus to respective activation culture mediums for activation culture, and respectively inoculating the bacillus subtilis and the streptomyces roseoflavus to respective fermentation culture mediums for fermentation culture to obtain respective zymophyte liquid;
(2) respectively filtering zymocyte liquid, collecting mycoplasm, drying, crushing to obtain bacterial powder, and mixing the bacterial powder of each strain to obtain composite bacterial powder;
(3) mixing the bacteria liquid filtered in the step (2), adding 75-95% v/v ethanol solution with the volume 4-7 times that of the bacteria liquid, and performing ultrasonic extraction for 5-10min at the ultrasonic power of 200-230W to obtain composite bacteria liquid;
(4) and mixing the compound bacterial liquid with a solidifying agent, drying, and adding compound bacterial powder to obtain the compound microbial preparation.
Further, the activation medium may be any of various conventional activation media capable of culturing Bacillus subtilis and Streptomyces roseoflavus.
Further, each liter of the bacillus subtilis fermentation medium comprises the following components: 2-5g/L ammonium sulfate, 1-4g/L calcium carbonate, 0.2-0.5g/L magnesium sulfate, 0.1-0.5/L sodium chloride, 3-6g/L peptone, 10-15g/L corn starch, 10-15g/L malt flour and the balance of water.
Further, each liter of the streptomyces roseoflavus fermentation medium comprises the following components: 0.2-0.5g/L dipotassium hydrogen phosphate, 0.3-0.8g/L ammonium nitrate, 2-4g/L calcium carbonate, 5-10g/L potassium sodium tartrate, 0.2-0.8g/L sodium citrate, 10-20g/L starch, 8-12g/L peanut cake powder, 14-22g/L corn steep liquor and the balance of water.
Furthermore, the invention also provides application of the compound microbial preparation in preventing and treating soil-borne disease pathogenic bacteria and/or preparing soil conditioners.
Further, the soil-borne disease pathogenic bacteria include, but are not limited to, Botrytis cinerea (Botrytis cinerea), Sphaerotheca fuliginea (Sphaerotheca fuliginea), Fusarium oxysporum (Fusarium oxysporum), Verticillium dahliae Kleb, and Rhizoctonia solani (Rhizoctonia solani).
Compared with the prior art, the invention has the beneficial effects that:
the invention compounds bacillus subtilis and streptomyces roseoflavus into a compound microbial preparation, realizes the prevention and control effect by secreting resistant substances, heavy parasitizing action, inducing host cells to generate a plurality of disease prevention mechanisms such as a defense structure, a competitive action and the like, has a synergistic interaction effect between the two strains, can effectively and stably prevent and control soil-borne disease pathogenic bacteria such as gray mold, powdery mildew, banana vascular wilt, soybean root rot and the like, and can also be used for soil improvement of crop fields such as vegetables, flowers, melons, fruits, forest trees, Chinese herbal medicines and the like after continuous cropping.
The invention carries out fermentation culture after activating strains, after the fermentation is finished, the mycoplasm and the zymocyte liquid are separated, the mycoplasm is prepared into dry powder, active substances in the zymocyte liquid are mixed and extracted, the extracted compound bacteria liquid is matched with a solid forming agent to prepare a powdery preparation, and finally the powdery preparation is mixed with the bacteria powder, so that the bacteria powder can be effectively preserved.
Detailed Description
In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.
The commercial product number of the bacillus subtilis used in the invention is ACCC 03221, and the commercial product number of the streptomyces roseoflavus is ACCC 40415.
The product number of the streptomyces lavendulae used in the invention is ACCC 40024.
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.
Example 1
A complex microbial preparation comprising: the bacillus subtilis, the streptomyces roseoflavus and the diatomite have a CFU ratio of 5: 2.
the amount of viable bacteria contained in the composite microbial preparation is 108cfu/g。
A preparation method of a compound microbial preparation comprises the following steps:
(1) respectively inoculating bacillus subtilis and streptomyces roseoflavus into a PDA culture medium, performing activation culture for 6h at 28 ℃, performing activation culture, then inoculating the bacillus subtilis into a bacillus subtilis fermentation culture medium, and performing culture for 72h under the conditions of 32-35 ℃ and pH7 to obtain a bacillus subtilis zymocyte solution; inoculating streptomyces roseoflavus into a streptomyces roseoflavus fermentation culture medium, and culturing for 144h at 30-32 ℃ to obtain streptomyces roseoflavus fermentation broth;
the bacillus subtilis fermentation medium comprises the following components in each liter: 3g/L ammonium sulfate, 2g/L calcium carbonate, 0.3g/L magnesium sulfate, 0.5/L sodium chloride, 35g/L peptone, 12g/L corn starch, 12g/L malt flour and the balance of water;
the Streptomyces roseoflavus fermentation medium comprises the following components in each liter: 0.3g/L dipotassium hydrogen phosphate, 0.5g/L ammonium nitrate, 3g/L calcium carbonate, 6g/L potassium sodium tartrate, 0.5g/L sodium citrate, 15g/L starch, 10g/L peanut cake powder, 18g/L corn steep liquor and the balance of water;
(2) respectively filtering zymocyte liquid, collecting mycoplasm, drying, crushing to obtain bacterial powder, and mixing the bacterial powder of each strain to obtain composite bacterial powder;
(3) mixing the bacteria liquid filtered in the step (2), adding 80% v/v ethanol solution 6 times the volume of the bacteria liquid, and performing ultrasonic extraction for 6min at the ultrasonic power of 230W to obtain composite bacteria liquid;
(4) and mixing the compound bacterial liquid with diatomite, drying, and adding compound bacterial powder to obtain the compound microbial preparation.
Example 2
A complex microbial preparation comprising: the bacillus subtilis, the streptomyces roseoflavus and the mica powder have a CFU ratio of 4: 1.
the amount of viable bacteria contained in the composite microbial preparation is 106cfu/g。
A preparation method of a compound microbial preparation comprises the following steps:
(1) respectively inoculating bacillus subtilis and streptomyces roseoflavus into a PDA culture medium, performing activation culture for 6h at 28 ℃, performing activation culture, then inoculating the bacillus subtilis into a bacillus subtilis fermentation culture medium, and performing culture for 72h under the conditions of 32-35 ℃ and pH7 to obtain a bacillus subtilis zymocyte solution; inoculating streptomyces roseoflavus into a streptomyces roseoflavus fermentation culture medium, and culturing for 144h at 30-32 ℃ to obtain streptomyces roseoflavus fermentation broth;
the bacillus subtilis fermentation medium comprises the following components in each liter: 2g/L ammonium sulfate, 1g/L calcium carbonate, 0.2g/L magnesium sulfate, 0.4/L sodium chloride, 6g/L peptone, 15g/L corn starch, 10g/L malt flour and the balance of water;
the Streptomyces roseoflavus fermentation medium comprises the following components in each liter: 0.5g/L dipotassium hydrogen phosphate, 0.3-0.8g/L ammonium nitrate, 2g/L calcium carbonate, 5g/L potassium sodium tartrate, 0.2g/L sodium citrate, 10g/L starch, 12g/L peanut cake powder, 22g/L corn steep liquor and the balance of water;
(2) respectively filtering zymocyte liquid, collecting mycoplasm, drying, crushing to obtain bacterial powder, and mixing the bacterial powder of each strain to obtain composite bacterial powder;
(3) mixing the bacteria liquid filtered in the step (2), adding 75% v/v ethanol solution which is 7 times of the volume of the bacteria liquid, and performing ultrasonic extraction for 5min at the ultrasonic power of 210W to obtain composite bacteria liquid;
(4) and mixing the compound bacterial liquid with mica powder, drying, and adding the compound bacterial powder to obtain the compound microbial preparation.
Example 3
A complex microbial preparation comprising: the bacillus subtilis, the streptomyces roseoflavus and the diatomite have a CFU ratio of 5: 2.
the amount of viable bacteria contained in the composite microbial preparation is 108cfu/g。
A preparation method of a compound microbial preparation comprises the following steps:
(1) respectively inoculating bacillus subtilis and streptomyces roseoflavus into a PDA culture medium, performing activation culture for 6h at 28 ℃, performing activation culture, then inoculating the bacillus subtilis into a bacillus subtilis fermentation culture medium, and performing culture for 72h under the conditions of 32-35 ℃ and pH7 to obtain a bacillus subtilis zymocyte solution; inoculating streptomyces roseoflavus into a streptomyces roseoflavus fermentation culture medium, and culturing for 144h at 30-32 ℃ to obtain streptomyces roseoflavus fermentation broth;
the bacillus subtilis fermentation medium comprises the following components in each liter: 3g/L ammonium sulfate, 2g/L calcium carbonate, 0.3g/L magnesium sulfate, 0.5/L sodium chloride, 35g/L peptone, 12g/L corn starch, 12g/L malt flour and the balance of water;
the Streptomyces roseoflavus fermentation medium comprises the following components in each liter: 0.3g/L dipotassium hydrogen phosphate, 0.5g/L ammonium nitrate, 3g/L calcium carbonate, 6g/L potassium sodium tartrate, 0.5g/L sodium citrate, 15g/L starch, 10g/L peanut cake powder, 18g/L corn steep liquor and the balance of water;
(2) respectively filtering zymocyte liquid, collecting mycoplasm, drying, crushing to obtain bacterial powder, and mixing the bacterial powder of each strain to obtain composite bacterial powder;
(3) mixing the bacteria liquid filtered in the step (2) to obtain a composite bacteria liquid;
(4) and mixing the compound bacterial liquid with diatomite, drying, and adding compound bacterial powder to obtain the compound microbial preparation.
Example 4
A complex microbial preparation comprising: the bacillus subtilis, the streptomyces roseoflavus and the diatomite have a CFU ratio of 5: 2.
the amount of viable bacteria contained in the composite microbial preparation is 108cfu/g。
A preparation method of a compound microbial preparation comprises the following steps:
(1) respectively inoculating bacillus subtilis and streptomyces roseoflavus into a PDA culture medium, performing activation culture for 6h at 28 ℃, performing activation culture, then inoculating the bacillus subtilis into a bacillus subtilis fermentation culture medium, and performing culture for 72h under the conditions of 32-35 ℃ and pH7 to obtain a bacillus subtilis zymocyte solution; inoculating streptomyces roseoflavus into a streptomyces roseoflavus fermentation culture medium, and culturing for 144h at 30-32 ℃ to obtain streptomyces roseoflavus fermentation broth;
the bacillus subtilis fermentation medium comprises the following components in each liter: 3g/L ammonium sulfate, 2g/L calcium carbonate, 0.3g/L magnesium sulfate, 0.5/L sodium chloride, 35g/L peptone, 12g/L corn starch, 12g/L fish meal and the balance of water;
the Streptomyces roseoflavus fermentation medium comprises the following components in each liter: 0.3g/L dipotassium hydrogen phosphate, 0.5g/L ammonium nitrate, 3g/L calcium carbonate, 6g/L potassium sodium tartrate, 0.5g/L sodium citrate, 15g/L starch, 10g/L peanut cake powder, 18g/L corn steep liquor and the balance of water;
(2) respectively filtering zymocyte liquid, collecting mycoplasm, drying, crushing to obtain bacterial powder, and mixing the bacterial powder of each strain to obtain composite bacterial powder;
(3) mixing the bacteria liquid filtered in the step (2), adding 80% v/v ethanol solution 6 times the volume of the bacteria liquid, and performing ultrasonic extraction for 6min at the ultrasonic power of 230W to obtain composite bacteria liquid;
(4) and mixing the compound bacterial liquid with diatomite, drying, and adding compound bacterial powder to obtain the compound microbial preparation.
Example 5
A complex microbial preparation comprising: the bacillus subtilis, the streptomyces roseoflavus and the diatomite have a CFU ratio of 5: 2.
the amount of viable bacteria contained in the composite microbial preparation is 108cfu/g。
A preparation method of a compound microbial preparation comprises the following steps:
(1) respectively inoculating bacillus subtilis and streptomyces roseoflavus into a PDA culture medium, performing activation culture for 6h at 28 ℃, performing activation culture, then inoculating the bacillus subtilis into a bacillus subtilis fermentation culture medium, and performing culture for 72h under the conditions of 32-35 ℃ and pH7 to obtain a bacillus subtilis zymocyte solution; inoculating streptomyces roseoflavus into a streptomyces roseoflavus fermentation culture medium, and culturing for 144h at 30-32 ℃ to obtain streptomyces roseoflavus fermentation broth;
the bacillus subtilis fermentation medium comprises the following components in each liter: 3g/L ammonium sulfate, 2g/L calcium carbonate, 0.3g/L magnesium sulfate, 0.5/L sodium chloride, 35g/L peptone, 12g/L corn starch, 12g/L malt flour and the balance of water;
the Streptomyces roseoflavus fermentation medium comprises the following components in each liter: 0.3g/L dipotassium hydrogen phosphate, 0.5g/L ammonium nitrate, 3g/L calcium carbonate, 6g/L potassium sodium tartrate, 15g/L starch, 10g/L peanut cake powder, 18g/L corn steep liquor and the balance of water;
(2) respectively filtering zymocyte liquid, collecting mycoplasm, drying, crushing to obtain bacterial powder, and mixing the bacterial powder of each strain to obtain composite bacterial powder;
(3) mixing the bacteria liquid filtered in the step (2), adding 80% v/v ethanol solution 6 times the volume of the bacteria liquid, and performing ultrasonic extraction for 6min at the ultrasonic power of 230W to obtain composite bacteria liquid;
(4) and mixing the compound bacterial liquid with diatomite, drying, and adding compound bacterial powder to obtain the compound microbial preparation.
Comparative example 1
A microbial preparation comprising: bacillus subtilis and diatomite, wherein the microbial preparation contains 10 viable bacteria8cfu/g。
A method for preparing a microbial preparation, comprising the steps of:
(1) inoculating bacillus subtilis to a PDA culture medium, performing activation culture at 28 ℃ for 6h, performing activation culture, inoculating to a bacillus subtilis fermentation culture medium, and culturing at 32-35 ℃ and pH7 for 72h to obtain a bacillus subtilis fermentation broth;
the bacillus subtilis fermentation medium comprises the following components in each liter: 3g/L ammonium sulfate, 2g/L calcium carbonate, 0.3g/L magnesium sulfate, 0.5/L sodium chloride, 35g/L peptone, 12g/L corn starch, 12g/L malt flour and the balance of water;
(2) filtering the zymocyte liquid, collecting the mycoplasm, drying and crushing to obtain bacterial powder;
(3) adding the residual bacterial liquid filtered in the step (2) into 80% v/v ethanol solution 6 times the volume of the bacterial liquid, and performing ultrasonic extraction for 6min at the ultrasonic power of 230W to obtain the bacterial liquid;
(4) and (4) mixing the bacterial liquid extracted in the step (3) with diatomite, drying, and adding bacterial powder to obtain the compound microbial preparation.
Comparative example 2
A microbial preparation comprising: the microbial preparation contains 10 live bacteria in the amount of 108cfu/g。
A method for preparing a microbial preparation, comprising the steps of:
(1) respectively inoculating streptomyces roseoflavus into a PDA culture medium, performing activation culture for 6h at 28 ℃, performing activation culture, then inoculating into a streptomyces roseoflavus fermentation culture medium, and performing culture for 144h at 30-32 ℃ to obtain streptomyces roseoflavus zymogen liquid;
the Streptomyces roseoflavus fermentation medium comprises the following components in each liter: 0.3g/L dipotassium hydrogen phosphate, 0.5g/L ammonium nitrate, 3g/L calcium carbonate, 6g/L potassium sodium tartrate, 0.5g/L sodium citrate, 15g/L starch, 10g/L peanut cake powder, 18g/L corn steep liquor and the balance of water;
(2) filtering the zymocyte liquid, collecting the mycoplasm, drying and crushing to obtain bacterial powder;
(3) adding the residual bacterial liquid filtered in the step (2) into 80% v/v ethanol solution 6 times the volume of the bacterial liquid, and performing ultrasonic extraction for 6min at the ultrasonic power of 230W to obtain the bacterial liquid;
(4) and (4) mixing the bacterial liquid extracted in the step (3) with diatomite, drying, and adding bacterial powder to obtain the compound microbial preparation.
Comparative example 3
A complex microbial preparation comprising: bacillus subtilis and Streptomyces lavendulae (Streptomyces lavendulae) and diatomite, wherein the CFU ratio of the bacillus subtilis to the Streptomyces lavendulae is 5: 2.
the amount of viable bacteria contained in the composite microbial preparation is 108cfu/g。
A preparation method of a compound microbial preparation comprises the following steps:
(1) respectively inoculating bacillus subtilis and streptomyces lavendulae into a PDA culture medium, performing activation culture for 6h at 28 ℃, performing activation culture, then inoculating the bacillus subtilis into a bacillus subtilis fermentation culture medium, and performing culture for 72h under the conditions of 32-35 ℃ and pH7 to obtain a bacillus subtilis zymocyte solution; inoculating streptomyces lavendulae into a streptomyces lavendulae fermentation culture medium, and culturing at 30-32 ℃ for 144h to obtain a streptomyces lavendulae zymocyte liquid;
the bacillus subtilis fermentation medium comprises the following components in each liter: 3g/L ammonium sulfate, 2g/L calcium carbonate, 0.3g/L magnesium sulfate, 0.5/L sodium chloride, 35g/L peptone, 12g/L corn starch, 12g/L malt flour and the balance of water;
the Streptomyces lavendulae fermentation medium comprises the following components in percentage by liter: 0.3g/L dipotassium hydrogen phosphate, 0.5g/L ammonium carbonate, 3g/L calcium carbonate, 0.5g/L magnesium sulfate, 15g/L starch, 10g/L soybean meal, 18g/L corn steep liquor and the balance of water;
(2) respectively filtering zymocyte liquid, collecting mycoplasm, drying, crushing to obtain bacterial powder, and mixing the bacterial powder of each strain to obtain composite bacterial powder;
(3) mixing the bacteria liquid filtered in the step (2), adding 80% v/v ethanol solution 6 times the volume of the bacteria liquid, and performing ultrasonic extraction for 6min at the ultrasonic power of 230W to obtain composite bacteria liquid;
(4) and mixing the compound bacterial liquid with diatomite, drying, and adding compound bacterial powder to obtain the compound microbial preparation.
Test example 1 effects of use
Test strains: botrytis cinerea (Botrytis cinerea), Podospora cucumerina (Sphaerotheca fuliginea (Schlect. Ex Fr. poll.), Fusarium oxysporum (Fusarium oxysporum), Verticillium dahliae Kleb, Rhizoctonia solani (Rhizoctonia solani);
the determination method comprises the following steps: diluting and dissolving a bacterial preparation by using a proper amount of double distilled water to prepare a bacterial preparation solution, wherein the dilution multiple is 100 times, uniformly mixing the bacterial preparation solution with a PDA culture medium according to a volume ratio of 1:3 to prepare a PDA drug-containing culture medium, a blank control is to add the same amount of double distilled water into the culture medium, three times are arranged for each group, then a puncher with the diameter of 5mm is used for cutting bacterial cakes along the outer edge of hypha, the bacterial cakes are inoculated into the PDA drug-containing culture medium, the PDA drug-containing culture medium is placed in a constant-temperature incubator at the temperature of 27 +/-1 ℃ for culture for 5 days, the diameter of the bacterial colonies is measured, the average value is obtained, and the bacteriostasis rate is calculated by comparing the blank control, wherein the calculation formula is as follows:
the bacteriostatic rate (%) was [ (diameter of colonies in control blank group-diameter of colonies in treatment group)/(diameter of colonies in control blank group-diameter of cake) ] × 100%
TABLE 1
According to experimental data, the composite microbial preparation prepared by the invention can inhibit botrytis cinerea, hyphomycete, fusarium oxysporum, verticillium dahliae and rhizoctonia solani, and the antibacterial effect is better than that of a single microbial preparation.
Test example 2 controlling effect on cucumber Gray mold
Selecting a 'Jinyou No. 1' seedling cucumber seedling growing to the three-leaf center, and evaluating the control effect of the microbial preparation on cucumber gray mold by adopting a pot experiment method;
the test method comprises the following steps: after inoculating the cucumber seedlings with the botrytis cinerea for 24 hours, uniformly spraying a fungus preparation solution (same as the test example 1) on the front and back surfaces of the leaves, wherein the spraying amount is 10 mL/pot, spraying once every 7 days, culturing in an environment with the temperature of 27 +/-1 ℃ and the relative humidity of 90%, spraying clean water with the same amount as the fungus preparation solution on a blank group, repeating 5 pots for each treatment, and observing and grading the disease 7 days and 14 days after the fungus preparation is applied. The classification criteria are as follows:
level 0: no disease spots;
level 1:3 scabs exist on a single leaf;
and 3, level: 4-6 scabs exist on a single leaf;
and 5, stage: 7-10 scabs exist on a single leaf;
and 7, stage: 11-20 scabs exist in a single leaf, and the parts are densely divided into pieces;
and 9, stage: the single leaf patch has dense scab, and occupies more than 1/4 of leaf area;
disease index [ Σ (number of diseased leaves at each stage × representative value at that stage) ]/(total number of investigated leaves × 9) × 100;
the prevention and treatment effect is (blank disease index-treatment group disease index)/blank disease index multiplied by 100%;
TABLE 2
As can be seen from the above table, the composite microbial preparation of the present invention has a superior control effect on cucumber gray mold, wherein the control effect of the composite microbial preparation prepared in example 2 is the best.
Test example 3 controlling Effect on Banana wilt
Selecting a banana seedling with 5-7 leaves, and evaluating the control effect of the microbial preparation on banana wilt by adopting a pot experiment method;
the test method comprises the following steps: after 24 hours of inoculating fusarium oxysporum to banana seedlings, uniformly pouring a bacterial preparation solution (same as the experimental example 1) into soil of the banana seedlings, wherein the pouring amount is 30 mL/pot, the solution is sprayed once every 7 days and is placed in an environment with the temperature of 25 +/-2 ℃ and the relative humidity of 60% for culture, a blank group is poured with clean water with the same amount as the bacterial preparation solution, each treatment is repeated for 5 pots, and the disease is observed and classified 7 days and 14 days after the bacterial preparation is applied, and the classification standard is as follows:
level 0: the plant has no withered and yellow symptom;
level 1: the leaves of the plants have slight withered and yellow symptoms, tender leaves are intact, a small part of roots are slightly browned, and the stems are in water stain-like brown stain;
and 2, stage: the plant leaves have obvious withered and yellow symptoms, tender leaves are intact, the roots are browned, and the stems and the pseudostems are browned in a water stain shape;
and 3, level: the whole plant has withered and yellow symptoms, the stem and the pseudostem are browned and decayed and are connected, and a few petioles have red brown stains;
4, level: the plant withers and dies, and the root system is seriously browned and rotten;
disease index [ Σ (number of diseased leaves at each stage × representative value at that stage) ]/(total number of investigated leaves × 4) × 100;
the prevention and treatment effect is (blank disease index-treatment group disease index)/blank disease index multiplied by 100%;
TABLE 3
As can be seen from the above table, the composite microbial preparation of the present invention has superior control effect on rubber blight, wherein the control effect of the composite microbial preparation prepared in example 2 is the best.
Test example 4 field test
The test is carried out in Xiongying area of Haikou city in 3 months in 2020, a test field after 1 year of continuous cropping is selected, the test is carried out by adopting cucumber seedlings of 'Jinyou No. 1' growing to three leaves and one heart, the planting density is 1000 strains/mu, each group of tests is 0.5 mu, a bacterial preparation is applied before the cucumber seedlings are planted, the application amount is 1 kg/mu, the bacterial preparation is applied for 1 time every 10 days, the bacterial preparation is diluted 1000 times and then used, meanwhile, a blank control group without the bacterial preparation is set, the morbidity of cucumbers is observed and recorded in the planting process, the organic matter content of soil is measured after the cucumber seedlings are completely harvested in 5 months in the same year, the cucumber yield is recorded, and the test results are shown in Table 4;
TABLE 4
As can be seen from field test results, the yield of the cucumber is increased by 14.7-16.9% after the compound microbial preparation is applied, the morbidity is low, the organic matter content of the soil after continuous cropping is obviously improved compared with a blank control group, and the action effect of the compound microbial preparation is obviously better than that of a single microbial preparation and other compound microbial preparations.
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. A complex microbial preparation, comprising: bacillus subtilis and Streptomyces roseoflavus.
2. The complex microbial preparation according to claim 1, wherein the amount of viable bacteria contained in the complex microbial preparation is 106-109cfu/g。
3. The complex microbial preparation of claim 2, wherein the CFU ratio of bacillus subtilis to streptomyces roseoflavus in the complex microbial preparation is (4-7): (1-2).
4. The complex microbial preparation of claim 2, further comprising a solidifying agent.
5. The composite microbial preparation according to claim 4, wherein the solidifying agent is at least one of diatomite, kaolin, bentonite and mica powder.
6. The method for preparing a complex microbial preparation according to claim 4, comprising the steps of:
(1) respectively inoculating bacillus subtilis and streptomyces roseoflavus to respective activation culture mediums for activation culture, and respectively inoculating the bacillus subtilis and the streptomyces roseoflavus to respective fermentation culture mediums for fermentation culture to obtain respective zymophyte liquid;
(2) respectively filtering zymocyte liquid, collecting mycoplasm, drying, crushing to obtain bacterial powder, and mixing the bacterial powder of each strain to obtain composite bacterial powder;
(3) mixing the bacteria liquid filtered in the step (2), adding an ethanol solution with the volume 4-7 times that of the bacteria liquid, and performing ultrasonic extraction for 5-10min at the ultrasonic power of 200-230W to obtain a composite bacteria liquid;
(4) and mixing the compound bacterial liquid with a solidifying agent, drying, and adding compound bacterial powder to obtain the compound microbial preparation.
7. The method of preparing a complex microbial preparation according to claim 6, wherein said bacillus subtilis fermentation medium comprises, per liter: 2-5g/L ammonium sulfate, 1-4g/L calcium carbonate, 0.2-0.5g/L magnesium sulfate, 0.1-0.5/L sodium chloride, 3-6g/L peptone, 10-15g/L corn starch and 10-15g/L malt powder.
8. The method for preparing a complex microbial preparation according to claim 6, wherein each liter of the fermentation medium of Streptomyces roseoflavus comprises the following components: 0.2-0.5g/L dipotassium hydrogen phosphate, 0.3-0.8g/L ammonium nitrate, 2-4g/L calcium carbonate, 5-10g/L potassium sodium tartrate, 0.2-0.8g/L sodium citrate, 10-20g/L starch, 8-12g/L peanut cake powder and 14-22g/L corn steep liquor.
9. Use of the complex microbial preparation according to any one of claims 1 to 8 for controlling soil-borne disease pathogens and/or for preparing soil conditioners.
10. Use of a composite microbial preparation according to claim 9, wherein said soil-borne pathogenic bacteria include, but are not limited to, Botrytis cinerea (Botrytis cinerea), Sphaerotheca fuliginea (Sphaerotheca fuliginea), Fusarium oxysporum (Fusarium oxysporum), Verticillium dahliae Kleb, and Rhizoctonia solani (Rhizoctonia solani).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110743497.6A CN113373092B (en) | 2021-06-30 | 2021-06-30 | Composite microbial preparation and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110743497.6A CN113373092B (en) | 2021-06-30 | 2021-06-30 | Composite microbial preparation and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113373092A true CN113373092A (en) | 2021-09-10 |
| CN113373092B CN113373092B (en) | 2023-09-15 |
Family
ID=77580448
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110743497.6A Active CN113373092B (en) | 2021-06-30 | 2021-06-30 | Composite microbial preparation and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113373092B (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1565197A (en) * | 2003-07-09 | 2005-01-19 | 中国科学院沈阳应用生态研究所 | Plant rhizosphere growth promoting bacteria agent production method using streptomycete |
| CN103725630A (en) * | 2013-12-06 | 2014-04-16 | 大连三科生物工程有限公司 | Microbial agent with antagonism to potato common scab |
| CN103773711A (en) * | 2013-11-28 | 2014-05-07 | 中国农业科学院农业资源与农业区划研究所 | Bacillus amyloliquefaciens and microbial agent and uses thereof |
| CN104498369A (en) * | 2014-12-05 | 2015-04-08 | 中国农业科学院植物保护研究所 | Trichoderma koningii and bacterial agent containing same and application thereof in prevention of cylindrocarpon destructans |
| US20150135371A1 (en) * | 2013-11-08 | 2015-05-14 | The Regents Of The University Of California | Synergy-based biocontrol of plant pathogens |
| CN104686582A (en) * | 2013-12-06 | 2015-06-10 | 大连三科生物工程有限公司 | Preparation method of microorganism bacterium agent for resisting potato scab |
| CN107382553A (en) * | 2017-09-14 | 2017-11-24 | 海南金雨丰生物工程有限公司 | A kind of marine alga, Trichoderma harzianum and bacillus subtilis bacterium composite fertilizer |
-
2021
- 2021-06-30 CN CN202110743497.6A patent/CN113373092B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1565197A (en) * | 2003-07-09 | 2005-01-19 | 中国科学院沈阳应用生态研究所 | Plant rhizosphere growth promoting bacteria agent production method using streptomycete |
| US20150135371A1 (en) * | 2013-11-08 | 2015-05-14 | The Regents Of The University Of California | Synergy-based biocontrol of plant pathogens |
| CN103773711A (en) * | 2013-11-28 | 2014-05-07 | 中国农业科学院农业资源与农业区划研究所 | Bacillus amyloliquefaciens and microbial agent and uses thereof |
| CN103725630A (en) * | 2013-12-06 | 2014-04-16 | 大连三科生物工程有限公司 | Microbial agent with antagonism to potato common scab |
| CN104686582A (en) * | 2013-12-06 | 2015-06-10 | 大连三科生物工程有限公司 | Preparation method of microorganism bacterium agent for resisting potato scab |
| CN104498369A (en) * | 2014-12-05 | 2015-04-08 | 中国农业科学院植物保护研究所 | Trichoderma koningii and bacterial agent containing same and application thereof in prevention of cylindrocarpon destructans |
| CN107382553A (en) * | 2017-09-14 | 2017-11-24 | 海南金雨丰生物工程有限公司 | A kind of marine alga, Trichoderma harzianum and bacillus subtilis bacterium composite fertilizer |
Non-Patent Citations (11)
| Title |
|---|
| PARK, HJ等: "Isolation and antifungal and antioomycete activities of staurosporine from Streptomyces roseoflavus strain LS-A24", 《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》 * |
| PARK, HJ等: "Isolation and antifungal and antioomycete activities of staurosporine from Streptomyces roseoflavus strain LS-A24", 《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》, vol. 54, no. 8, 9 April 2006 (2006-04-09), pages 3041 - 3046 * |
| 周登博等: "抗香蕉枯萎病甲基营养型芽孢杆菌的鉴定及定殖", 《中国农学通报》 * |
| 周登博等: "抗香蕉枯萎病甲基营养型芽孢杆菌的鉴定及定殖", 《中国农学通报》, no. 36, 25 December 2017 (2017-12-25), pages 151 - 157 * |
| 毕扬等: "防治西葫芦和黄瓜白粉病的生物制剂的筛选", 《植物保护》 * |
| 毕扬等: "防治西葫芦和黄瓜白粉病的生物制剂的筛选", 《植物保护》, no. 05, 31 October 2016 (2016-10-31), pages 239 - 242 * |
| 毕扬等: "防治西葫芦和黄瓜白粉病的生物制剂的筛选", 植物保护, no. 5 * |
| 甄丹妹等: "玫瑰黄链霉菌活性代谢产物RM诱导黄瓜抗病性的信号转导途径", 《植物保护学报》 * |
| 甄丹妹等: "玫瑰黄链霉菌活性代谢产物RM诱导黄瓜抗病性的信号转导途径", 《植物保护学报》, no. 02, 15 April 2020 (2020-04-15), pages 112 - 118 * |
| 秦士娇等: "黄瓜PGPR菌株S160的鉴定及其对重金属铅的吸附研究", 《中国生物防治学报》 * |
| 秦士娇等: "黄瓜PGPR菌株S160的鉴定及其对重金属铅的吸附研究", 《中国生物防治学报》, no. 01, 28 February 2016 (2016-02-28), pages 131 - 136 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113373092B (en) | 2023-09-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US6569425B2 (en) | Bacillus licheniformis biofungicide | |
| JP2017534671A (en) | Microbial composition and method of use thereof | |
| CN109182137A (en) | The African Trichoderma harzianum of one plant of disease prevention growth-promoting and its application | |
| CN110734871B (en) | Bacillus amyloliquefaciens and application thereof in agricultural production | |
| CN110724648B (en) | Bacillus amyloliquefaciens strain and application thereof | |
| CN102067885A (en) | Trichoderma spore powder as well as preparation method and application thereof | |
| CN111690578B (en) | Salt and alkali resistant Siamese bacillus and production method and application of viable bacteria preparation thereof | |
| CN105801258A (en) | Bacillus subtilis fertilizer and preparation method and application thereof | |
| CN111040976A (en) | Bacillus amyloliquefaciens and application thereof | |
| CN113755406A (en) | Biocontrol bacterium preparation and application thereof | |
| Nofal et al. | Ecofriendly method for suppressing damping-off disease caused by Rhizoctonia solani using compost tea | |
| DE602004002293T2 (en) | A SYNERGISTIC FERMENTED PLANT GROWTH PROMOTING, BIO CONTROL COMPOSITION | |
| WO2011099878A2 (en) | Organic pesticide | |
| CN108220211B (en) | Acinetobacter oleophilic NMB17 and application thereof in plant disease control | |
| CN112574906B (en) | Bacterial strain for preventing and treating common continuous cropping diseases of greenhouse tomatoes and compound microbial agent thereof | |
| CN110042064A (en) | A kind of Xianggu mushroom strain and its insecticide and preparation method thereof using and derived from the bacterial strain | |
| Gilardi et al. | Biocontrol agents and resistance inducers reduce Phytophthora crown rot (Phytophthora capsici) of sweet pepper in closed soilless culture | |
| CN107058154B (en) | Rhodotorula orientalis strain, biocontrol microbial inoculum, biocontrol fermentation liquor as well as preparation method and application of biocontrol fermentation liquor | |
| CN106804627A (en) | A kind of complex microorganism Wettable pesticide powder and preparation method thereof and purposes | |
| CN113373092B (en) | Composite microbial preparation and preparation method thereof | |
| CN113136352B (en) | Biocontrol strain and application thereof in preventing and treating rice sheath blight and promoting rice growth | |
| CN110731345B (en) | Pesticide composition and application thereof in preventing and treating potato scab | |
| AL-TAMIMI et al. | The Efficacy Test of Nano Chitosan and Phylex in Resistance Early Blight Disease in Tomato Caused by Alternaria Solani Fungus. | |
| CN105967910A (en) | Microbial organic fertilizer capable of preventing and treating bacterial wilt as well as preparation method and application thereof | |
| Ahmed et al. | Efficiency of some organic matter on suppression of soil-borne diseases of tomato plants under organic agriculture system |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |