CN114958675B - Bacillus composition, microbial agent and application - Google Patents
Bacillus composition, microbial agent and application Download PDFInfo
- Publication number
- CN114958675B CN114958675B CN202210669672.6A CN202210669672A CN114958675B CN 114958675 B CN114958675 B CN 114958675B CN 202210669672 A CN202210669672 A CN 202210669672A CN 114958675 B CN114958675 B CN 114958675B
- Authority
- CN
- China
- Prior art keywords
- bacillus
- potato
- hmb26553
- phodg36
- composition
- 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.)
- Active
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
-
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
-
- 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
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The application relates to the technical field of pesticides, in particular to a bacillus composition, a microbial agent and application. The bacillus composition consists of bacillus subtilis HMB26553 and bacillus amyloliquefaciens PHODG 36. After the bacillus subtilis HMB26553 and the bacillus amyloliquefaciens PHODG36 are combined, remarkable synergistic effect is generated in the aspect of inhibiting pathogenic bacteria such as verticillium potato bacteria and potato scab bacteria, and the potato verticillium wilt and potato scab can be effectively prevented and treated.
Description
Technical Field
The application relates to the technical field of pesticides, in particular to a bacillus composition, a microbial agent and application.
Background
Potatoes are the fourth most important food crop worldwide, next to wheat, rice and maize. At present, china is the first country for producing potatoes, the planting area accounts for 25% of the total area of the potatoes in the world, but the single-yield of the potatoes in China is far lower than that of developed countries in the world due to the influence of plant diseases and insect pests. Particularly, in recent years, with the expansion of the planting area and the extension of the continuous cropping period of the potatoes, soil-borne diseases such as verticillium wilt and scab of the potatoes are in a trend of increasing year by year, and the yield and the quality of the potatoes are seriously threatened.
Potato Verticillium, also known as "early death disease", is a soil-borne and seed-borne vascular bundle soil-borne disease caused by Verticillium sp fungi, wherein Verticillium dahliae Verticillium dahliae and Verticillium medicago V.nonnalfalfae are the main pathogenic bacteria. At present, potato verticillium wilt occurs in regions such as inner mongolia, hebei, gansu, heilongjiang, shanxi, ningxia and the like in China, the yield loss of 10% -15% is caused by light people, and the yield loss can reach 30% -50% when serious, so that the potato verticillium wilt becomes one of important factors restricting potato production.
Potato scab is a disease caused by Streptomyces sp, which mainly damages potato tubers, influences the appearance and quality of the potato tubers, reduces commodity value, commonly occurs in potato main cultivation areas, has 30% -60% of miniature potatoes in some areas, and causes great economic loss to potato production.
At present, chemical agents are commonly used for preventing and controlling the two potato soil-borne diseases, however, the chemical bactericides are easy to cause drug resistance of pathogenic bacteria after long-term use, and cause serious problems of agricultural ecological environment pollution and the like, thereby threatening the health of human beings.
The microbial bactericide is an important measure for green prevention and control of crop diseases due to the characteristics of specific prevention effect, no toxic and side residues, environmental friendliness and the like, and is widely paid attention to. At present, few microbial fungicide products are applied to control potato verticillium wilt and scab, so that development of efficient green microbial fungicide products and technical researches for controlling potato soil-borne diseases are necessary to meet the current market demands.
Disclosure of Invention
In view of the above, the invention provides a bacillus composition, a microbial agent and application thereof. The bacillus composition can effectively inhibit pathogenic bacteria such as verticillium potato wilt and potato scab, has obvious synergistic effect, and can effectively prevent and treat verticillium potato wilt and potato scab.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a bacillus composition, which consists of bacillus subtilis (Bacillus subtilis) HMB26553 and bacillus amyloliquefaciens (B.amyloliquefaciens) PHODG 36.
In the invention, the preservation number of the bacillus subtilis HMB26553 is CGMCC No.13211, and the strain is disclosed in a patent document with the application number of 201710157385.6; the bacillus amyloliquefaciens PHODG36 has a preservation number of CGMCC No.13041, and the strain is disclosed in a patent document with a application number of 201610876803.2.
Preferably, the ratio of the number of viable bacteria of the bacillus subtilis HMB26553 to the bacillus amyloliquefaciens PHODG36 is (1-100): (1-100).
Preferably, the ratio of the number of viable bacteria of the bacillus subtilis HMB26553 to the bacillus amyloliquefaciens PHODG36 is (1-10): (1-10).
More preferably, the ratio of the number of viable bacteria of bacillus subtilis HMB26553 to the number of viable bacteria of bacillus amyloliquefaciens PHODG36 is (1-5): (1-5).
In the specific embodiment provided by the invention, the ratio of the number of viable bacteria of bacillus subtilis HMB26553 to the number of viable bacteria of bacillus amyloliquefaciens PHODG36 is 1:1.
the invention also provides a bacillus composition mother drug, which comprises the bacillus composition and a carrier.
Preferably, the bacillus composition parent drug has a viable count of (1-100) x 10 of bacillus subtilis HMB26553 or bacillus amyloliquefaciens PHODG36 10 CFU/g。
Preferably, the live bacteria number of the bacillus subtilis HMB26553 or the bacillus amyloliquefaciens PHODG36 in the bacillus composition parent drug is (1-10) multiplied by 10 10 CFU/g。
In the specific embodiment provided by the invention, the viable count of the bacillus subtilis HMB26553 or the bacillus amyloliquefaciens PHODG36 in the bacillus composition parent drug is (1-5) multiplied by 10 10 CFU/g。
Preferably, the carrier is selected from one or more of kaolin, talcum powder, clay, lime powder, gypsum, bentonite, diatomite, white carbon black, phosphate, sand stone, pumice, peat and calcium silicate. Other carrier types recognized in the art are also within the scope of the present invention.
The invention also provides a preparation method of the bacillus composition mother drug, which comprises the following steps:
and (3) centrifugally concentrating the strain fermentation broth of the bacillus subtilis HMB26553 and/or the strain fermentation broth of the bacillus amyloliquefaciens PHODG36, mixing the carrier with the obtained concentrated solution, and carrying out spray drying. The fermentation liquor of the two strains can be concentrated together or separately; the two strain concentrates may be spray dried together or separately.
The invention also provides a microbial agent, which comprises the bacillus composition and/or a bacillus composition mother drug and pharmaceutically acceptable auxiliary materials.
Preferably, the microbial agent is one of wettable powder, emulsifiable concentrate, microemulsion, water emulsion, suspending agent and water dispersible granule.
In the embodiment provided by the invention, the pharmaceutically acceptable auxiliary materials comprise one or more of wetting agents, dispersing agents, ultraviolet protective agents, fillers, penetrating agents, antifreezing agents, thickening agents, synergists, solvents, emulsifying agents, antifoaming agents and pH regulators.
In the specific embodiment provided by the invention, the microbial agent is wettable powder, and the pharmaceutically acceptable auxiliary materials comprise one or more of wetting agents, dispersing agents, ultraviolet protective agents and fillers.
In the present invention, the wetting agent is a conventional wetting agent in the art, and the wetting agent may be selected from one or more of Sodium Dodecyl Benzene Sulfonate (SDBS), sodium butyl naphthalene sulfonate (nekal BX), berol 790A (sodium alkyl sulfonate), SP-2846BW, GY-W04, LT-569, sodium dodecyl sulfate, lignin sulfonate, sodium isopropyl naphthalene sulfonate, sodium alkyl aryl sulfonate, and the like. In a specific embodiment provided by the present invention, the wetting agent is LT-569.
In the present invention, the dispersant is a dispersant conventional in the art, and the dispersant may be one or more selected from methylene bis naphthalene sulfonate (NNO), polynaphthalene formaldehyde sulfonate sodium salt (MF), morwet D-425 (alkyl naphthalene sulfonate formaldehyde condensate), phosphate esters, sulfonates, alkyl naphthalene sulfonate polycondensates, carboxylate polymers, polycarboxylate polymer modified resins, alkyl sulfates, modified alkyl sulfonates, alkyl naphthalene sulfonate polycondensate sodium salts, naphthalene sulfonate polycondensates, polycarboxylate polymer dispersants, polystyrene phenyl ether phosphate dispersants, naphthalene sulfonate formaldehyde condensate, alkylphenol polyoxyethylene ether formaldehyde condensate sulfonates, lignin sulfonates, maleic acid-acrylic copolymer sodium salts, alkyl sulfosuccinates, and the like. In a specific embodiment provided by the invention, the dispersant is MF.
In the present invention, the ultraviolet protecting agent is a conventional ultraviolet protecting agent in the art, and the ultraviolet protecting agent may be one or more selected from ascorbic acid, sodium fluorescein, dextrin, folic acid, and skimmed milk powder. In a specific embodiment provided herein, the uv protectant is ascorbic acid.
In the present invention, the filler is a filler conventional in the art, and the filler may be one or more selected from kaolin, talcum powder, clay, lime powder, gypsum, bentonite, diatomaceous earth, white carbon black, phosphate, sand, pumice, peat, and calcium silicate. In a specific embodiment provided by the invention, the filler is talc.
Preferably, the wettable powder comprises the following components in parts by weight:
the invention also provides a preparation method of the microbial agent, which comprises the following steps: mixing and stirring the bacillus composition and/or the parent drug, a wetting agent, a dispersing agent, an ultraviolet protective agent and talcum powder, and sieving the mixture with a 300-500-mesh sieve after jet milling.
The invention also provides application of the bacillus composition, a bacillus composition parent drug or a microbial agent in inhibiting plant pathogenic fungi and/or preventing potato soil-borne diseases;
preferably, the phytopathogenic fungi comprise verticillium fungi and/or streptomyces bacteria;
preferably, the Verticillium fungus is Verticillium dahliae (Verticillium dahliae)
Preferably, the Streptomyces bacteria is Streptomyces scabies.
Preferably, the potato soil-borne disease comprises potato verticillium wilt and/or potato scab.
The invention provides a bacillus composition, a microbial agent and application thereof. The bacillus composition consists of bacillus subtilis (Bacillus subtilis) HMB26553 and bacillus amyloliquefaciens (b.amyloliquefaciens) PHODG 36. Compared with the prior art, the invention has the following beneficial effects:
after the bacillus subtilis HMB26553 and the bacillus amyloliquefaciens PHODG36 are used in a combined way, remarkable synergistic effect is generated in the aspect of inhibiting pathogenic bacteria such as verticillium potato bacteria and potato scab bacteria;
the bacillus wettable powder disclosed by the invention takes the bacillus subtilis HMB26553 and the bacillus amyloliquefaciens PHODG36 as active ingredients, can quickly colonize plant root surfaces after application, achieves the effects of preventing and treating potato verticillium wilt and scab by competing infection sites with pathogenic bacteria, secreting antibacterial active substances, promoting plant nutrition absorption and the like, and can improve the yield of potatoes;
the bacillus wettable powder is safe, efficient, nontoxic and residue-free, is friendly to human bodies and environment, and can reduce the use of chemical pesticides; meanwhile, the use mode is flexible, seed dressing or drip irrigation can be selected according to different conditions, and labor and use cost are saved. Meanwhile, the preparation method of the wettable powder is simple, low in cost and suitable for industrial production.
Detailed Description
The invention discloses a bacillus composition, a microbial agent and application thereof, and a person skilled in the art can properly improve process parameters by referring to the content of the bacillus composition and the microbial agent. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The invention provides bacillus wettable powder and a preparation method and application thereof, so as to realize safe and efficient prevention and treatment of verticillium wilt and scab of potatoes.
The bacillus wettable powder comprises the active ingredients of bacillus subtilis (Bacillus subtilis) HMB26553 and bacillus amyloliquefaciens PHODG36, and further comprises a wetting agent, a dispersing agent, an ultraviolet protective agent and a filler, wherein the raw material components are mixed by proper weight, and the bacillus wettable powder is finally prepared. The prepared bacillus wettable powder has the spore content of not less than 30 hundred million CFU/g, the mixed bacterial rate of not more than 3%, the pH of not more than 7.8, the fineness of not less than 98%, the drying decrement of not more than 1.6%, the suspension rate of not less than 80%, the wetting time of not more than 96s and the preparation method is stable.
The technical scheme adopted by the invention is as follows:
the bacillus wettable powder consists of the following components in parts by weight: 10-15 parts of bacillus subtilis HMB26553 parent drug, 10-15 parts of bacillus amyloliquefaciens PHODG36 parent drug, 1-3 parts of wetting agent LT-569 and dispersing agent: 1-3 parts of MF (lubricating oil), 1-3 parts of ultraviolet protective agent ascorbic acid and 61-77 parts of talcum powder.
The preparation method of the bacillus wettable powder comprises the following steps:
1. preparation of Bacillus subtilis HMB26553 and Bacillus amyloliquefaciens PHODG36 fermentation broth
Transferring the frozen HMB26553 strain and PHODG36 strain to LB solid medium, and culturing at 37 ℃ for 24 hours to obtain an activated strain; respectively picking single bacterial colonies of the two strains, inoculating the single bacterial colonies into a Ke-shi bottle (250 mL of LB solid culture medium is filled in the Ke-shi bottle), culturing for 3d-5d at 37 ℃, washing bacterial lawn in the Ke-shi bottle with 400-500mL of sterile water, inoculating the bacterial lawn in a 1000L seed fermentation tank filled with 200L of seed culture medium, and adjusting the initial pH value to 7.8-8.0, the temperature to 32 ℃ and the ventilation volume and the rotation speed in real time according to the dissolved oxygen value so as to ensure that the dissolved oxygen is highest; when 10-12 hours after inoculation, the growth of thalli is in an exponential growth medium phase, and the thalli is transferred into a 5000L fermentation tank filled with 3000L fermentation medium, the pH value is 7.8-8.0, the temperature is 32 ℃, the ventilation volume and the rotation speed are adjusted in real time according to the dissolved oxygen value, so as to ensure that the dissolved oxygen is the highest, the thalli concentration is more than or equal to 40 hundred million/mL, the spore formation rate is more than or equal to 95%, and the fermentation can be finished, thus obtaining bacillus subtilis HMB26553 fermentation liquor and bacillus amyloliquefaciens PHODG36 fermentation liquor;
wherein, the LB solid medium: 10g of peptone, 5g of yeast powder, 5g of sodium chloride, 1000mL of water, pH 7.0-7.5 and sterilizing at 121 ℃ for 30min;
wherein the seed culture medium: 30g of corn flour, 20g of bean cake powder, 4g of sodium dihydrogen phosphate, 0.3g of potassium dihydrogen phosphate, 1g of sodium carbonate, 0.5g of magnesium sulfate, 2g of calcium carbonate and 1000mL of water, wherein the pH value is 7.0-7.5; sterilizing at 121deg.C for 30min;
wherein the fermentation medium: 30g of corn flour, 20g of bean cake powder, 4g of sodium dihydrogen phosphate, 0.3g of potassium dihydrogen phosphate, 1g of sodium carbonate, 0.5g of magnesium sulfate, 2g of calcium carbonate and 1000mL of water, wherein the pH value is 7.0-7.5; sterilizing at 121deg.C for 30min;
2. preparation of Bacillus subtilis HMB26553 and Bacillus amyloliquefaciens PHODG36 parent drug
And (3) centrifugally concentrating the HMB26553 strain fermentation liquor and the PHODG36 strain fermentation liquor by adopting a disc type centrifuge, adding kaolin into the concentrated liquor according to the adding amount of 20% -30%, and performing spray drying by utilizing a spray drying tower, wherein the inlet temperature is 170-175 ℃, and the outlet temperature is 50-55 ℃, so as to obtain the HMB26553 strain and PHODG36 strain parent drug.
3. Preparation of bacillus wettable powder
10-15 parts of HMB26553 strain parent medicine, 10-15 parts of PHODG36 strain parent medicine, 1-3 parts of wetting agent LT-569, 1-3 parts of dispersing agent MF, 1-3 parts of ultraviolet protective agent ascorbic acid and 61-77 parts of talcum powder are mixed, stirred and subjected to jet milling, and then the mixture is screened by a 300-400 mesh sieve, and the spore content is detected to be not lower than 30 hundred million CFU/g, the mixed bacteria rate is not higher than 3%, the pH is not higher than 7.8, the fineness is not lower than 98%, the drying decrement is not higher than 1.6%, the suspension rate is not lower than 80%, and the wetting time is not higher than 96s, so that the bacillus wettable powder is obtained.
Wherein, the bacillus wettable powder comprises the following components in parts by weight: 10 parts of HMB26553 strain parent drug, 10 parts of PHODG36 strain parent drug, 1 part of wetting agent LT-569, 2 parts of dispersing agent MF, 1 part of ultraviolet protective agent ascorbic acid and 76 parts of filler talcum powder.
The reagents, materials, etc. used in the present invention are commercially available.
The invention is further illustrated by the following examples:
example 1 preparation of a sterilizing composition
1. Preparation of Bacillus subtilis HMB26553 and Bacillus amyloliquefaciens PHODG36 fermentation broth
Transferring the frozen HMB26553 strain and PHODG36 strain to LB solid medium, and culturing at 37 ℃ for 24 hours to obtain an activated strain; respectively picking single bacterial colonies of the two strains, inoculating the single bacterial colonies into a Ke-shi bottle (250 mL of LB solid culture medium is filled in the Ke-shi bottle), culturing for 3d-5d at 37 ℃, washing lawn in the Ke-shi bottle with 500mL of sterile water, inoculating the lawn in a 1000L seed fermentation tank filled with 200L of seed culture medium, and adjusting the initial pH value to 7.8-8.0, the temperature to 32 ℃ and the ventilation volume and the rotation speed in real time according to the dissolved oxygen value so as to ensure that the dissolved oxygen is highest; when 10-12 hours after inoculation, the bacterial growth is in an exponential growth medium phase, and the bacterial growth is transferred into a 5000L fermentation tank filled with 3000L fermentation medium, the pH value is 7.8-8.0, the temperature is 32 ℃, the ventilation and the rotation speed are adjusted in real time according to the dissolved oxygen value, so as to ensure that the dissolved oxygen is the highest, the bacterial concentration is more than or equal to 50 hundred million/mL, the spore formation rate is more than or equal to 95%, and the bacillus subtilis HMB26553 fermentation liquid and the bacillus amyloliquefaciens PHODG36 fermentation liquid are obtained;
wherein, the LB solid medium: 10g of peptone, 5g of yeast powder, 5g of sodium chloride, 1000mL of water, pH 7.0-7.5 and sterilizing at 121 ℃ for 30min;
wherein the seed culture medium: 30g of corn flour, 20g of bean cake powder, 4g of sodium dihydrogen phosphate, 0.3g of potassium dihydrogen phosphate, 1g of sodium carbonate, 0.5g of magnesium sulfate, 2g of calcium carbonate and 1000mL of water, wherein the pH value is 7.0-7.5; sterilizing at 121deg.C for 30min;
wherein the fermentation medium: 30g of corn flour, 20g of bean cake powder, 4g of sodium dihydrogen phosphate, 0.3g of potassium dihydrogen phosphate, 1g of sodium carbonate, 0.5g of magnesium sulfate, 2g of calcium carbonate and 1000mL of water, wherein the pH value is 7.0-7.5; sterilizing at 121deg.C for 30min.
2. Preparation of a fungicidal composition
And uniformly mixing the prepared HMB26553 strain fermentation liquor and PHODG36 strain fermentation liquor according to a ratio of 1:1 for later use. In the mixed solution, the thallus concentration is more than or equal to 50 hundred million/mL, and the thallus quantity ratio of the HMB26553 strain to the PHODG36 strain is about 1:1.
test example 1 test of inhibition of germ by sterilizing composition
Test of inhibition of Fungicide composition on Potato verticillium
1. The verticillium potato virus (Verticillium dahaliae) VD-P6 to be tested is separated and stored in a plant disease biological control laboratory of the national academy of sciences of agriculture and forestry in Hebei province.
2. The test method comprises the following steps:
preparation of test strain fermentation broth:
(1) HMB26553 strain broth: taking the fermentation broth prepared in example 1 for later use;
(2) PHODG36 strain fermentation broth: taking the fermentation broth prepared in example 1 for later use;
(3) HMB26553 mixed solution with PHODG 36: the HMB26553 strain fermentation broth prepared in example 1 and the PHODG36 strain fermentation broth were mixed uniformly in a ratio of 1:1 for use.
And (3) determining the inhibition effect of the liquid to be tested on verticillium dahliae by adopting a flat plate counter method. Firstly, activating and culturing verticillium potato on a PDA plate for 10-14 days, then, taking a bacterial disc at the edge of a bacterial colony by using a sterile puncher (with the diameter of 6 mm), transferring to the center of another PDA plate, and then, respectively sucking the above several liquids to be tested by using a pipette gun, and inoculating at a position 2.0 cm away from the bacterial disc, wherein each drop is 20 mu L. The plate without inoculating the liquid to be tested and only inoculating pathogenic bacteria is used as a blank control, and each treatment is repeated for 3 times. Culturing at 25deg.C, measuring control pathogenic bacteria diameter and processed pathogenic bacteria diameter when blank control pathogenic bacteria grow to the edge of culture dish, and calculating antibacterial rate.
Antibacterial ratio (%) = (control pathogen diameter-treated pathogen diameter)/control pathogen diameter x 100.
The results show that the bacillus subtilis HMB26553 and the bacillus amyloliquefaciens PHODG36 have strong inhibition effects on verticillium potato viruses, the inhibition rate of the HMB26553 strain on verticillium potato viruses reaches 72.7%, the inhibition rate of the PHODG36 strain on verticillium potato viruses is 70.9%, and the inhibition rate of the mixed solution of the two strains on verticillium potato viruses is higher than that of the single strain and reaches 78.4% (shown in Table 1). The results show that after the bacillus subtilis HMB26553 and the bacillus amyloliquefaciens PHODG36 are combined, remarkable synergistic effect is generated in bacteriostasis.
TABLE 1 inhibition results of Bacillus HMB26553 and PHODG36 on Verticillium Solani
Test of inhibition of the sterilizing composition against Potato scab pathogen
1. The potato scab germ (Streptomyces scabies) is isolated and stored in a plant disease biological control laboratory of the national academy of sciences of agriculture and forestry in Hebei province.
2. The test method comprises the following steps:
preparation of test strain fermentation broth: as above.
Inoculating pathogenic bacteria of potato scab on oat agar medium, culturing at 28deg.C for 7-10d, washing spores with sterile water, and adjusting concentration to 10 6 And (3) CFU/mL, coating 100 mu L of spore suspension on a Gao's I culture medium plate, respectively sucking 20 mu L of the above several liquids to be tested by a pipetting gun, inoculating the liquids to the center of the plate, culturing at 28 ℃ for 7d, measuring the diameter of a bacteriostasis ring, and judging the bacteriostasis effect by the size of the bacteriostasis ring. Each treatment was repeated 5 times.
The results show that the two bacteria of the bacillus subtilis HMB26553 and the bacillus amyloliquefaciens PHODG36 have strong inhibition effect on potato scab germs, the diameter of the inhibition zone of the HMB26553 strain on potato scab germs is 10.8mm, the diameter of the inhibition zone of the PHODG36 strain on potato scab germs is 11.5mm, and the diameter of the inhibition zone of the mixed solution of the two bacteria on potato scab germs is higher than that of the single strain and reaches 13.3mm (table 2). The results show that after the bacillus subtilis HMB26553 and the bacillus amyloliquefaciens PHODG36 are combined, remarkable synergistic effect is generated in bacteriostasis.
TABLE 2 inhibition results of Bacillus HMB26553 and PHODG36 on potato scab pathogen
| Treatment of | Diameter of inhibition zone (mm) |
| HMB26553 | 10.8±0.2 |
| PHODG36 | 11.5±0.3 |
| HMB26553+PHODG36(1：1) | 13.3±0.2 |
EXAMPLE 2 preparation of Bacillus wettable powder
The bacillus wettable powder of the embodiment consists of the following components in parts by weight:
10 parts of bacillus subtilis HMB26553 parent drug, 10 parts of bacillus amyloliquefaciens PHODG36 parent drug, 1 part of wetting agent LT-569, 2 parts of dispersing agent MF, 1 part of ultraviolet protective agent ascorbic acid and 76 parts of filler talcum powder.
The preparation method of the bacillus wettable powder comprises the following steps:
1. preparation of Bacillus subtilis HMB26553 and Bacillus amyloliquefaciens PHODG36 fermentation broth
The preparation method is the same as in example 1.
2. Preparation of Bacillus subtilis HMB26553 and Bacillus amyloliquefaciens PHODG36 parent drug
And (3) centrifugally concentrating the HMB26553 strain fermentation liquor and the PHODG36 strain fermentation liquor by adopting a disc type centrifuge, adding kaolin into the concentrated liquor according to the adding amount of 30%, and performing spray drying by utilizing a spray drying tower, wherein the inlet temperature is 172 ℃, and the outlet temperature is 55 ℃, so as to obtain the HMB26553 strain and PHODG36 strain parent medicine. The spore content was measured to be 3.24X10 respectively 10 CFU/g and 3.36×10 10 CFU/g, and kaolin was added to adjust the spore content to 200 hundred million CFU/g, respectively.
3. Preparation of bacillus wettable powder
10 parts of HMB26553 strain parent drug, 10 parts of PHODG36 strain parent drug, 1 part of wetting agent LT-569, 2 parts of dispersing agent MF, 1 part of ultraviolet protective agent ascorbic acid and 76 parts of talcum powder are mixed, stirred and subjected to jet milling, and then pass through a 400-mesh sieve, and the bacillus wettable powder is obtained after detection that the total spore content is 37.6 hundred million CFU/g, the mixed bacterial rate is 2.8%, the pH is 7.8, the fineness is 98.2%, the drying decrement is 1.6%, the wetting time is 95.7s and the suspension rate is 80.7%.
Test example 2 Effect of Bacillus wettable powder on potato growth
The effect of the different amounts of the bacillus wettable powder obtained in example 2 on potato growth was evaluated by a potting test, which was carried out in a greenhouse of the institute of plant protection, academy of sciences of agriculture and forestry, hebei province. Cutting healthy potato seeds into about 35-50 g potato blocks (1-2 bud eyes/blocks), placing the potato blocks on a plastic film, scattering bacillus wettable powder on the surfaces of the potato blocks, stirring to uniformly adhere the surfaces of the potato blocks, planting the potato blocks in a flowerpot (with the diameter of 16cm and the depth of 13 cm) filled with seedling culture matrixes, and culturing in a greenhouse. Test set up 3 concentration treatments: wettable powder 1kg/667m 2 、2kg/667m 2 And 3kg/667m 2 Seed dressing, wherein each treatment is repeated for 3 times, each time 6 pots are planted, and each pot is provided with 2 seed potatoes. The potato variety is Fei Wu Rui Tao (Netherlands 15), and the pot experiment dosage is calculated according to the seed potato dosage of 150kg per mu. A blank (CK) was prepared by a 3kg seed dressing treatment with talc. After 30d sowing, the quantity of seedlings in each treatment is investigated, and the rate of seedlings is calculatedThe method comprises the steps of carrying out a first treatment on the surface of the After 40d the potato plant height was investigated and the effect of different dose treatments on potato growth was observed.
Emergence rate (%) = (emergence number/12) ×100
Corrected emergence rate (%) = (treated emergence rate/emergence rate of blank control) ×100
As can be seen from table 3, at 30d sowing, the corrected emergence rate of the wettable powder treatment was not significantly different from that of the blank control; at the time of sowing 40d, the difference between the heights of the treated plants was not significant (Table 3). The result shows that 30 hundred million CFU/g bacillus wettable powder is applied to 1-3 kg/667m 2 The seed dressing treatment is safe for emergence and growth of potatoes.
TABLE 3 influence of Bacillus wettable powder on potato emergence and plant height
| Treatment of | Correction of emergence rate (%) | Height of plant (cm) |
| CK | 100.0±0.0a | 32.9±3.0a |
| 1kg/667m 2 | 100.0±0.0a | 29.0±1.8a |
| 2kg/667m 2 | 97.2±2.8a | 29.5±3.1a |
| 3kg/667m 2 | 94.4±2.8a | 31.6±1.7a |
Test example 3 control Effect of Bacillus wettable powder on potato verticillium wilt
The root injury inoculation method is adopted to artificially inoculate verticillium dahliae, and the control effect of the bacillus wettable powder on the verticillium dahliae is evaluated. The test was carried out in a greenhouse of the institute of plant protection, academy of sciences of agriculture and forestry, hebei province. After the potato plant height was investigated in test example 2, a round of the potato plant was cut with a wallpaper knife at about 2cm from the bottom of the pot where the potato was planted, and placed in a pot containing a suspension of verticillium potato spores (10 7 Individual spores/mL) were immersed in the box for 30min and then taken out for further culture. The occurrence of verticillium in potato was investigated 21d after inoculation. Disease grade classification was according to the 5-grade classification standard:
grade 0, healthy plants, leaves asymptomatic;
1 grade, less than 25% of plants with leaf disease, and the leaf presents yellowish or irregular yellow disease spots;
2, 26-50% of plants with leaf disease, most of the disease spots become yellow or yellow brown, and the edges of the leaves are rolled up slightly and dried up;
3, plants with 51-75% of leaf disease, and few leaves wither;
4, plants with more than 76% of leaves suffering from diseases, and the leaves fall off or the plants die.
And the disease index and the control effect are calculated according to the following formula.
Disease index = Σ (disease grade x number of corresponding disease grade strains)/(4 x total number of plants) ×100
Control (%) = (control disease index-treated disease index)/control disease index x 100
Test results show that the bacillus wettable powder has better control effect on potato verticillium wilt, and the seed potato wettable powder is 1-3 kg/667m 2 After seed dressing treatment, the disease index of potato verticillium is obviously lowIn the blank (33.1), the control effect was 45.1% -47.4% (Table 4).
TABLE 4 control effect of bacillus wettable powder on potato verticillium wilt
| Treatment of | Index of disease condition | Control effect (%) |
| Blank control CK | 33.1±6.7a | - |
| 1kg/667m 2 | 18.2±4.1b | 45.1 |
| 2kg/667m 2 | 17.4±3.7b | 47.4 |
| 3kg/667m 2 | 18.0±3.8b | 45.6 |
Test example 4 field plot test for evaluating the Effect of Bacillus wettable powder on Potato
A field district test is carried out on peach villages in county of full Mongolia of the surrounding field of the Maitake City of Hebei province, and the bacillus wettable powder of 1kg/667m is evaluated 2 、2kg/667m 2 And 3kg/667m 2 The application effect of seed dressing on potatoes. The test field soil is sandy loam, the land topography is smooth, and the water and fertilizer conditions are good. The potato variety is fei wu rui ta (netherlands 15). 0.1kg of 70% thiophanate methyl wettable powder, 0.1kg of 3% zhongshengmycin wettable powder and 2.8kg of talcum powder are uniformly mixed, and then seed dressing treatment is used as a chemical agent control, and 3kg of talcum powder seed dressing treatment is used as a blank Control (CK). And calculating the dosage of the field cell test according to 150kg of seed potato dosage per mu, repeating each treatment for 4 times, and randomly arranging 20 cells. The cell area is 20m 2 (1 m.times.20m). The occurrence of verticillium wilt of 30 potato plants was continuously investigated in each cell, and the disease index and the control effect were calculated (same as in test example 3). During the potato harvest period, spot sampling was performed to measure yield and investigate the onset of tuber scab. At the time of measuring the yield, the method selects 2m according to each cell 2 The potato plants with the area are taken as sampling points, all the plants are dug out, the total weight of potato blocks is recorded, and the acre yield, the yield increase rate, the scab incidence and the prevention effect are calculated according to the following formula.
Mu yield (kg/667 m) 2 ) =measurement of yield of sweet potato block at sample point×333.5
Yield increase (%) = (treated acre yield-acre yield of the blank)/acre yield of the blank x 100
Incidence (%) = number of diseased potato tubers/number of total potato tubers harvested x 100
Control (%) = (control morbidity-treated morbidity)/control morbidity x 100
Through investigation, potato verticillium wilt does not occur in the peach mountain village plot, but potato scab occurs. After seed dressing treatment by bacillus wettable powder at different doses, the incidence rate of potato scab is obviously lower than that of a blank control. 1kg/667m 2 With 3kg/667m 2 After seed dressing treatment, the incidence rate of potato scab is not obvious from the contrast of chemical agents, and the prevention effect is respectively 56.8% and 52.5%;2kg/667m 2 After seed dressing treatment, the incidence rate (7.2%) of potato scab is obviously lower than that of chemical agent control (11.1%), and the prevention effect reaches 72.0%. In terms of yield, 1 to 3kg/667m 2 The acre yield after seed dressing treatment is higher than that of blank pairsPhoto zone (3431.8 kg/667 m) 2 ) The yield is increased by 6.1 to 15.5 percent, wherein the yield is 2kg/667m 2 The highest yield of seed dressing treatment reached 15.5% (Table 5). Thus selecting 2kg/667m wettable powder 2 Seed dressing is used as field demonstration dosage.
Table 5 effects of application of bacillus wettable powder to potatoes in field plot experiments
Test example 5 field application demonstration evaluation of the application Effect of Bacillus wettable powder on Potato
Developing field application demonstration in the village of the full-family Mongolian county of the Guangdong nationality in the North China, the village of nineteen numbers, the village on which the village is pulled and the birch forest village of the Yu-crossing, and evaluating the disease prevention and yield increase effects of the bacillus wettable powder seed dressing treatment on the potatoes. The potato variety is Xingjia No. 2. The example is provided with 2 treatments, wherein the treatment 1 is wettable powder 2kg/667m 2 Seed dressing treatment, treatment 2 was a chemical agent control treatment (same as test example 4). An exemplary area per treatment is 150 mu. Investigation of verticillium wilt disease conditions at the peak of verticillium wilt occurrence and calculation of disease index (same as test example 3); during the potato harvest period, the occurrence of potato scab was measured and investigated, and the incidence, disease preventing effect and yield increase were calculated (same as in test example 4).
Demonstration results in peach mountain village and pull-up village show that 30 hundred million CFU/g bacillus wettable powder is 2kg/667m compared with chemical agent control 2 The seed dressing can obviously reduce the disease index of the verticillium wilt of the potatoes, and the prevention effect respectively reaches 62.1% and 50.8%; in terms of yield, the wettable powder treatment of the peach mountain village is obviously higher than that of a chemical agent control, and the yield increase rate reaches 28.1%; the wettable powder treatment on the dial village had no significant difference from the chemical control, but the yield was still significantly higher than the chemical control with an increase of 14.3% (table 6). Exemplary results in nineteen villages and birch Lin Cun showed that wettable powders were 2kg/667m compared to the chemical controls 2 The seed dressing treatment can obviously reduce the bellsThe incidence rate of potato scab reaches 63.9% and 65.7% respectively. In terms of yield, both wettable powder treatments were significantly higher than the chemical controls with yields of 29.4% and 16.7%, respectively (table 7).
TABLE 6 influence of Bacillus wettable powder on potato verticillium wilt and yield in large area demonstration
Table 7 influence of 30 hundred million CFU/g Bacillus wettable powder on potato scab and yield in the area demonstration
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A bacillus composition is characterized by comprising bacillus subtilisBacillus subtilis) HMB26553 and Bacillus amyloliquefaciensB.amyloliquefaciens) PHODG 36;
the preservation number of the bacillus subtilis HMB26553 is CGMCC No.13211, and the preservation number of the bacillus amyloliquefaciens PHODG36 is CGMCC No.13041.
2. The bacillus composition of claim 1, wherein the ratio of viable count of bacillus subtilis HMB26553 to bacillus amyloliquefaciens PHODG36 is (1-100): (1-100).
3. The bacillus composition of claim 1 or 2, wherein the ratio of viable count of bacillus subtilis HMB26553 to bacillus amyloliquefaciens PHODG36 is (1-10): (1-10).
4. A bacillus composition mother liquor comprising the bacillus composition of any one of claims 1 to 3 and a carrier.
5. The bacillus composition mother liquor according to claim 4, wherein the number of viable bacteria of the bacillus subtilis HMB26553 or the bacillus amyloliquefaciens PHODG36 is (1-100) x 10 10 CFU/g。
6. The bacillus composition mother liquor according to claim 4 or 5, wherein the carrier is one or more selected from the group consisting of kaolin, talc, clay, lime powder, gypsum, bentonite, diatomaceous earth, white carbon, phosphate, sand, pumice, peat, and calcium silicate.
7. A microbial agent comprising the bacillus composition of any one of claims 1 to 3 and/or the bacillus composition mother drug of any one of claims 4 to 6, and an agropharmaceutically acceptable adjuvant.
8. The microbial agent according to claim 7, wherein the microbial agent is one of wettable powder, emulsifiable concentrate, microemulsion, water emulsion, suspending agent and water dispersible granule.
9. The microbial agent according to claim 7 or 8, wherein the agropharmaceutically acceptable auxiliary materials comprise one or more of wetting agents, dispersing agents, ultraviolet protection agents, fillers, penetrating agents, antifreezing agents, thickening agents, synergists, solvents, emulsifying agents, antifoaming agents and pH adjusting agents.
10. Use of the bacillus composition of any one of claims 1-3, the bacillus composition mother liquor of any one of claims 4-6 or the microbial inoculant of any one of claims 7-9 for inhibiting phytopathogenic fungi and/or for controlling potato soil-borne diseases;
the plant pathogenic fungi is Verticillium dahliaeVerticillium dahliae) And/or Streptomyces scabiesStreptomyces scabies)；
The potato soil-borne disease is potato verticillium wilt and/or potato scab.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210669672.6A CN114958675B (en) | 2022-06-14 | 2022-06-14 | Bacillus composition, microbial agent and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210669672.6A CN114958675B (en) | 2022-06-14 | 2022-06-14 | Bacillus composition, microbial agent and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114958675A CN114958675A (en) | 2022-08-30 |
| CN114958675B true CN114958675B (en) | 2023-07-07 |
Family
ID=82962027
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210669672.6A Active CN114958675B (en) | 2022-06-14 | 2022-06-14 | Bacillus composition, microbial agent and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114958675B (en) |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104593283A (en) * | 2014-09-11 | 2015-05-06 | 北京理工大学 | Bacillus amyloliquefaciens DPPG-74 and its use in disease prevention and growth promotion |
| CN106399177B (en) * | 2016-10-08 | 2019-10-22 | 河北省农林科学院植物保护研究所 | Bacillus amyloliquefaciens and its microbial inoculum with degradation Phos and bacteriostasis |
| CN106939290B (en) * | 2017-03-16 | 2020-02-11 | 河北省农林科学院植物保护研究所 | Bacillus subtilis HMB26553 and application thereof |
| CN109673671A (en) * | 2019-01-22 | 2019-04-26 | 河北省农林科学院植物保护研究所 | A kind of microorganism seed inorganic agent and preparation method for preventing and treating potato verticillium wilt |
| CN110477021A (en) * | 2019-09-04 | 2019-11-22 | 河北省农林科学院植物保护研究所 | The solid microbial and preparation method of a kind of pair of potato disease-preventing and yield-increasing |
| CN115725471B (en) * | 2022-11-29 | 2024-04-02 | 云南省烟草农业科学研究院 | Bacillus saxifrage strain 05-2101 and application, product and method thereof |
-
2022
- 2022-06-14 CN CN202210669672.6A patent/CN114958675B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN114958675A (en) | 2022-08-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kumar et al. | Efficacy of Bacillus subtilis MBI 600 against sheath blight caused by Rhizoctonia solani and on growth and yield of rice | |
| CN101473853B (en) | Application of Bacillus cereus Bacillus cereusCMCC63305 in agriculture field | |
| KR100800566B1 (en) | Klebsiella oxytoca c1036 and plant growth promotion, diseases control and environmental stress reduction method using the same | |
| CN110616172B (en) | Bacillus with crucifer clubroot prevention and treatment function and preparation method and application thereof | |
| CN108409479B (en) | Nematode-killing microbial organic fertilizer and preparation method and application thereof | |
| CN107509744B (en) | Compound biopesticide for preventing and treating wheat stem basal rot and preparation method and application thereof | |
| CN110669701A (en) | Microbial inoculum for preventing and treating strawberry root rot and cucumber fusarium wilt and application thereof | |
| CN108277172B (en) | Paenibacillus polymyxa GY40 and preparation and application of wettable powder thereof | |
| JPH10146187A (en) | Liquid compound fertilizer inhibiting generation of plant disease | |
| CN108294014A (en) | A kind of seed coat agent and the preparation method and application thereof of prevention root rot | |
| CN106396836B (en) | Soybean phytophthora root rot prevents and treats biological seed coating agent and preparation method thereof, application method | |
| CN105112304A (en) | Bjerkandera sp. Gause 15 for controlling vegetable root diseases and preparation thereof | |
| CN114958675B (en) | Bacillus composition, microbial agent and application | |
| CN102302005B (en) | Application of JS399-19 (2-cyano-3-amino-3-phenylacrylic acetate) in preventing and controlling wheat take-all diease | |
| CN112574906A (en) | Bacterial strain for preventing and treating common continuous cropping diseases of greenhouse tomatoes and compound microbial agent thereof | |
| KR101287141B1 (en) | Streptomyces griseus BIG105 for controlling plant diseases and uses thereof | |
| CN115152773B (en) | Composition of prothioconazole and fludioxonil for preventing and treating wheat stem rot | |
| CN116918832A (en) | Composite biocontrol microbial agent, preparation method, application and application method | |
| CN114617127B (en) | Pesticide sterilization composition and application thereof | |
| CN110760450A (en) | Culture method of trichoderma longibrachiatum spores, trichoderma longibrachiatum water dispersible granule and preparation method thereof | |
| CN106588508B (en) | Special fertilizer for preventing and treating clubroot of cruciferous crops as well as preparation method and application thereof | |
| CN109673671A (en) | A kind of microorganism seed inorganic agent and preparation method for preventing and treating potato verticillium wilt | |
| JP2004307342A (en) | Growth promoting and disease injury suppressing material for plant, and culture soil for raising seedling comprising the same material | |
| CN115948301B (en) | Oligosaccharin composite microbial inoculant for preventing and controlling banana vascular wilt and application thereof | |
| CN103798262A (en) | Sterilization composition containing thiophanate-methyl and resin acid copper salt |
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 |