CN116496922A - Bacillus amyloliquefaciens and application thereof - Google Patents
Bacillus amyloliquefaciens and application thereof Download PDFInfo
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- CN116496922A CN116496922A CN202211511385.9A CN202211511385A CN116496922A CN 116496922 A CN116496922 A CN 116496922A CN 202211511385 A CN202211511385 A CN 202211511385A CN 116496922 A CN116496922 A CN 116496922A
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- bacillus amyloliquefaciens
- grape
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- fermentation
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Classifications
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- 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
- C12N1/205—Bacterial isolates
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- 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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P21/00—Plant growth regulators
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
- C05G3/60—Biocides or preservatives, e.g. disinfectants, pesticides or herbicides; Pest repellants or attractants
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- 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
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- 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
Abstract
The invention provides bacillus amyloliquefaciens and application thereof. The name of the bacillus amyloliquefaciens is BJ-10, and the strain is preserved in China general microbiological culture Collection center (CGMCC) No.22613. The strain has the functions of preventing diseases and promoting growth, has obvious antagonism on powdery mildew, grape canker pathogen, grape anthracnose pathogen, grape black root pathogen, grape vine blight pathogen, tomato early blight pathogen, tomato gray mold pathogen, phytophthora capsici, pepper wilt pathogen, rhizoctonia solani, strawberry phytophthora capsici and the like, and the fermentation liquor can obviously promote the growth of seedlings of grapes, cucumbers and the like and improve the yield.
Description
Technical Field
The invention belongs to the field of microorganism application, and in particular relates to a bacillus amyloliquefaciens strain capable of preventing diseases and promoting growth and application thereof in the technical field of biocontrol bactericides.
Background
The frequency of fungal diseases is one of the main factors affecting the production of fruit and vegetable crops, chemical control is a main control measure, but the problems of pesticide residues, ecological environment pollution and pathogen resistance are easily caused. The biological control has the characteristics of environmental friendliness, ecological safety, difficult generation of drug resistance by pathogenic bacteria and the like, and is gradually popularized and applied in agricultural production. Biological control of diseases includes the use of microbial agents, plant inducers and the like, wherein the microbial agents mainly comprise bacteria, fungi and actinomycetes, bacillus spp is a gram positive bacterium of endophytic spores, and is a type which is more studied in the current biocontrol bacteria, and mainly comprises bacillus amyloliquefaciens, bacillus cereus, bacillus subtilis, bacillus polymyxa and the like. The bacillus amyloliquefaciens has the characteristics of fast growth, long storage period, easiness in generation of stress-resistant spores and the like, can colonize and multiply on the surface of a host after being used, forms a protective layer and blocks infection of various pathogenic bacteria, so that the bacillus amyloliquefaciens is a research hot spot for current biological control.
The biological control mechanism of bacillus amyloliquefaciens mainly comprises two types, namely a first type for promoting plant growth and a second type for producing secondary metabolites, FZB42 is the most studied 1 strain in the genus, and has been used as a biological fertilizer for commercial production by ABiTEP GmbH company. At present, microorganisms with high antagonistic activity are screened, and the novel microorganism product which can gradually replace chemical pesticides is developed, so that the method has important significance for disease prevention and control of fruit and vegetable crops.
Disclosure of Invention
The invention relates to bacillus amyloliquefaciens with disease prevention and growth promotion functions and application thereof.
The bacillus amyloliquefaciens (Bacilus amyloliquefaciens) BJ-10 strain provided by the invention is preserved in China general microbiological culture Collection center (No. 1 and No. 3 of North West road in the Korean area of Beijing) in the 5 th month 26 days 2021, and the preservation number is CGMCC No.22613. The bacillus amyloliquefaciens (Bacilus amyloliquefaciens) BJ-10 provided by the invention is separated from soil. The bacterial colony of the bacillus amyloliquefaciens BJ-10 is light yellow, opaque, and has raised surface with folds and irregular edges.
The invention provides a method for knowing the inhibition effect of bacillus amyloliquefaciens (Bacilus amyloliquefaciens) BJ-10 on plant pathogenic fungi such as powdery mildew, grape canker pathogen, grape anthracnose pathogen, grape black root pathogen, vine gummy stem pathogen, tomato early blight pathogen, tomato gray mold pathogen, phytophthora capsici, pepper fusarium wilt pathogen, strawberry rhizoctonia solani, strawberry phytophthora capsici and the like.
The invention provides a preparation method of bacillus amyloliquefaciens (Bacilus amyloliquefaciens) BJ-10 fermentation liquor and application thereof in preventing and treating grape powdery mildew and grape gray mold.
The invention provides application of bacillus amyloliquefaciens (Bacilus amyloliquefaciens) BJ-10 in promoting cucumber and grape growth.
The invention provides acquisition and identification of bacillus amyloliquefaciens BJ-10, wherein the acquisition method comprises the following steps of:
1) Grinding a soil sample collected from a Beijing Changpin vegetable garden;
2) Placing the ground soil sample into a centrifuge tube filled with sterile water, and vibrating to obtain soil suspension;
3) Gradient diluting the soil suspension to obtain soil suspension with different concentrations;
4) Selecting concentration of 10 5 、10 6 And 10 7 Adding the soil suspension into an NA culture medium flat plate for coating treatment, and culturing in a 28 ℃ incubator to obtain a bacterial colony;
5) Selecting single colonies with different forms, and performing streak culture for 24 hours to obtain corresponding colonies, wherein the vigorous growth person is named BJ-10;
the formula of the culture medium is as follows: beef extract 5g, peptone 10g, sodium chloride 10g, agar 20g and ultra-pure water 1000mL.
The isolated strain BJ-10 was subjected to morphological characterization by observation of biological characteristics, as follows: the bacterial colony is observed for morphology, size, concavo-convex degree, edge, gram staining and the like, and the 16S rDNA sequence of the bacterial colony is analyzed and phylogenetic tree is constructed.
The application of the bacillus amyloliquefaciens in preparing a microbial agent or microbial fertilizer also belongs to the protection scope of the invention;
the microbial agent or microbial fertilizer has the following functions:
1) Promoting plant growth;
2) Inhibiting plant pathogenic bacteria;
3) Preventing and treating plant diseases.
The plant pathogenic bacteria are one or more of grape canker (Lasiodiplodia citricola), grape anthracnose (Colletotrichum viniferum), grape black root germ (Dactylonectria macrodidyma), grape vine blight germ (Diadorthe eres), tomato early blight germ (Alternaria solani), corn big spot germ (Exserohilum turcicum (pass.) Leonay et Suggs), pepper phytophthora capsici (Phytophora capsiciLeonia), pepper fusarium (Fusarium oxysporum f.sp.capsicum), tomato gray mold germ (Botrytis cinerea), strawberry phytophthora (Pytophthora cactorum), strawberry rhizoctonia solani (Rhizoctonia solani) and grape powdery mildew germ (erygium necator).
The promotion of plant growth is reflected in plant height, fresh weight and dry weight increase; the plant comprises cucumber and/or grape.
The invention also provides a microbial inoculum, wherein the active ingredient of the microbial inoculum is the bacillus amyloliquefaciens; the microbial inoculum is any one of the following microbial inoculum:
1) A microbial agent for promoting plant growth;
2) A microbial agent for inhibiting plant pathogenic bacteria;
3) A microbial inoculum for preventing and treating plant diseases.
The invention also provides a microbial fertilizer, wherein the active ingredient of the microbial fertilizer is the bacillus amyloliquefaciens of claim 1;
the microbial fertilizer has the following functions:
1) Promoting plant growth;
2) Inhibiting plant pathogenic bacteria;
3) Preventing and treating plant diseases.
The preparation method of the microbial inoculum taking the bacillus amyloliquefaciens as the active ingredient is characterized by comprising the following steps:
a: strain activation: inoculating the strain BJ-10 on an LB culture medium plate by a plate streaking method with an inoculating needle, and placing the strain in an incubator for culturing for 2d at a constant temperature of 28 ℃ to obtain an activated strain;
b: seed liquid culture: selecting a single colony by using a sterilized toothpick, inoculating the single colony into an LB liquid culture medium, and shaking and culturing the single colony in a full-temperature shaking incubator at 28 ℃ and 180rpm for 12 hours to obtain seed liquid;
c: fermentation: the seed liquid is poured into a triangular flask with a liquid loading amount of 100mL/500mL of fermentation medium, and shake culture is carried out for 72 hours at 28 ℃ and 180rpm, thus obtaining fermentation liquid.
8. The method according to claim 7, wherein the LB medium plate in the step A is an agar medium; the liquid culture medium in the step B is 5g of yeast extract, 10g of tryptone, 10g of sodium chloride, 1L of ultrapure water and the pH value is 6.5-8.0.
9. The process of claim 7, wherein the fermentation medium comprises 20g of maltose, 10g of peptone and 10g of CaCO 3 10g，NaHPO 3 2g，KH 2 PO 3 1g, 1L of ultrapure water.
10. The preparation method according to claim 7, wherein the viable count content of Bacillus amyloliquefaciens BJ-10 in the fermentation broth of Bacillus amyloliquefaciens BJ-10 is 3.0X10 8 ～2.0×10 9 CFU/mL。
And fermenting and culturing the bacillus amyloliquefaciens to obtain fermentation liquor. The fermentation broth is prepared by the following method:
(1) Inoculating bacillus amyloliquefaciens to an LB culture medium, and culturing for 24 hours at the temperature of 28 ℃ to obtain an activated strain; LB medium composition: 5g of yeast extract, 10g of tryptone, 10g of sodium chloride and 20g of agar.
(2) Inoculating the activated strain in the step (1) into an LB liquid culture medium, and shake culturing for 12 hours at 28 ℃ and 180r/min to obtain seed liquid; the liquid culture medium consists of: 5g of yeast extract, 10g of tryptone, 10g of sodium chloride and water as a solvent;
(3) Inoculating 10% of inoculum size of the seed liquid volume concentration obtained in the step (2) into a bran liquid fermentation medium, and shake culturing for 72h at 180r/min to obtain fermentation liquid;
the bran liquid culture medium consists of: maltose 20g, peptone 10g, bran 10g, caCO 3 10g、NaHPO 3 2g、KHPO 3 1g, water as solvent, pH7.0-7.4.
Further, the microbial agent contains 2×10 bacillus amyloliquefaciens thallus 8 ～2×10 9 CFU/mL。
Compared with the prior art, the invention has the beneficial effects that:
(1) The microbial inoculum is any one of the following microbial inoculum:
1) An amylase-producing microbial agent;
2) A protease-producing microbial agent;
3) A cellulase-producing microbial agent;
4) An amylase-producing microbial agent;
5) A microbial agent for inhibiting plant pathogenic fungi;
6) A microbial inoculum for controlling plant pathogenic fungi diseases;
7) A microbial agent for promoting plant growth;
the plant pathogenic bacteria are plant pathogenic fungi such as grape powdery mildew, grape canker, grape anthracnose, grape black root, grape vine blight, tomato early blight, tomato gray mold, phytophthora capsici, pepper wilt, strawberry rhizoctonia solani, strawberry phytophthora capsici and the like, and the plant fungal disease prevention and control is grape powdery mildew and grape gray mold.
The plant growth promotion is reflected in plant height, fresh weight and dry weight increase; the plants include, but are not limited to, cucumber, grape.
Indoor potted plant experiment shows that the concentration is 1X 10 7 The prevention and treatment effect of the CFU/mL BJ-10 fermentation liquor on grape powdery mildew after 3 times of application reaches 78.06 percent. The field test shows that the control effect on grape powdery mildew after 3 times of application is up to 72.81%. The field test shows that the control effect on grape gray mold after 2 times of application is up to 73.06 percent
The bacillus amyloliquefaciens BJ-10 has the functions of preventing diseases and promoting growth, has obvious antagonism on powdery mildew, canker pathogen, anthracnose pathogen, black root pathogen, gummy stem blight, early blight pathogen, gray mold pathogen, phytophthora capsici, fusarium oxysporum, rhizoctonia solani, phytophthora strawberry and the like, and the fermentation liquor can obviously promote the growth of seedlings of grapes, cucumbers and the like and improve the yield. In addition, the bacillus amyloliquefaciens BJ-10 has a good control effect on grape powdery mildew, the actual control effect of the field is 69.34% -72.81%, and the control effect of the bacillus amyloliquefaciens BJ-10 is not remarkably different from that of 4% pyrimidine nucleoside antibiotics on the market; meanwhile, the bacillus amyloliquefaciens BJ-10 has a good disease prevention effect on grape gray mold, the actual field prevention effect is 71.65% -73.06%, the control effect of the bacillus amyloliquefaciens BJ-10 is electrodeless and obviously different from that of a control medicament 50% fludioxonil wettable powder, and the bacillus amyloliquefaciens BJ-10 can be developed into a bacterial fertilizer and microbial pesticide preparation integrating disease prevention and growth promotion.
Drawings
FIG. 1 shows the colony morphology of Bacillus amyloliquefaciens BJ-10.
FIG. 2 shows a Bacillus amyloliquefaciens BJ-10 phylogenetic tree constructed based on the 16S rDNA sequence.
FIG. 3 shows the results of BJ-10 amylase assay.
FIG. 4 shows the results of BJ-10 protease assay.
FIG. 5 shows the results of BJ-10 cellulase detection.
FIG. 6 shows the results of BJ-10 lipase detection, with E.coli on the left and BJ-10 on the right.
FIG. 7 shows the BJ-10 methyl red test results.
FIG. 8 shows the results of glucose fermentation test.
FIG. 9 shows the results of sucrose fermentation assay.
FIG. 10 shows lactose fermentation test results.
FIG. 11 shows mannitol fermentation test results.
FIG. 12 shows inositol fermentation assay results.
FIG. 13 shows the effect of Bacillus amyloliquefaciens on controlling powdery mildew of greenhouse potted grapes.
FIG. 14 shows a graph of B.amyloliquefaciens versus cucumber promoter effect.
FIG. 15 shows a graph of B.amyloliquefaciens versus grape pro-effect.
The strain preservation description of the invention is as follows:
strain name: bacillus amyloliquefaciens
Classification name: bacilus amyloliquefaciens
Preservation mechanism: china academy of sciences microbiological institute
The preservation organization is abbreviated as: CGMCC
Address: beijing city, chaoyang area, north Chenxi Lu No. 1 and 3
Preservation date: 2021, 05, 26
Accession numbers of the preservation center: CGMCC No.22613
Detailed Description
The invention will be further described with reference to specific examples, which are given by way of illustration only, and the scope of the invention is not limited thereto.
The experimental methods used in the following experiments are conventional methods unless otherwise specified.
Materials, reagents and the like used in the following experiments are commercially available unless otherwise specified.
The bacillus is bacillus amyloliquefaciens (Bacilus amyloliquefaciens) BJ-10, and the preservation unit is as follows: china general microbiological culture Collection center (CGMCC), the collection date: 2021, 26 months 05, deposit number: CGMCC No.22613. The Bacillus amyloliquefaciens belongs to the bacteria kingdom, the phylum of the back wall fungus, the class of the bacillus, the order of the bacillus, the family of the bacillus and the genus of the bacillus.
The preparation method of the microbial inoculum taking bacillus as an active ingredient comprises the following steps:
(1) Inoculating bacillus amyloliquefaciens to an LB culture medium, and culturing for 24 hours at the temperature of 28 ℃ to obtain an activated strain; LB medium composition: 5g of yeast extract, 10g of tryptone, 10g of sodium chloride and 20g of agar.
(2) Inoculating the activated strain in the step (1) into an LB liquid culture medium, and shake culturing for 12 hours at 28 ℃ and 180r/min to obtain seed liquid; the liquid culture medium consists of: 5g of yeast extract, 10g of tryptone, 10g of sodium chloride and water as a solvent;
(3) Inoculating 10% of inoculum size of the seed liquid volume concentration obtained in the step (2) into a bran liquid fermentation medium, and shake culturing for 72h at 180r/min to obtain fermentation liquid.
The bran liquid culture medium consists of: maltose 20g, peptone 10g, bran 10g, caCO 3 10g、NaHPO 3 2g、KHPO 3 1g, water as solvent, pH7.0-7.4.
Test plant pathogenic bacteria: powdery mildew of grape; grape canker pathogen; grape anthracnose pathogen; black root germ of grape; vine blight germ; botrytis cinerea; tomato early blight bacteria; the corn leaf spot germ; phytophthora capsici; fusarium capsici; phytophthora strawberry; rhizoctonia solani of strawberry.
EXAMPLE 1 isolation and characterization of Bacillus amyloliquefaciens (Bacilus amyloliquefaciens) BJ-10
Isolation of strain BJ-10
Air-drying a soil sample from the Beijing Changping area at room temperature, grinding and sieving, weighing 10g of the ground and sieved soil sample, adding 90mL of sterile water, and sufficiently shaking to obtain a soil suspension, namely the soil sample 10 -1 Is then shaken uniformly and then diluted to 10 in gradient -5 、10 -6 And 10 -7 . 100. Mu.L of dilutions of different gradients were spread evenly on NA plates, each concentration gradient was repeated for 3 dishes and incubated at 28℃for 48h. Single colonies with different culture characteristics are picked from the flat plate, and are continuously streaked and purified on the NA flat plate, so that pure bacterial culture is obtained, numbered and stored.
Identification of Strain BJ-10
1. Morphological features: the morphology of BJ-10 is shown in FIG. 1, the colony is yellowish, opaque, the surface is raised with folds and the edges are irregular.
2. Physiological and biochemical characteristics:
2.1 detection of amylase
Detection medium: amylase selection medium (LB medium+1.0% soluble starch): 10g of peptone, 5g of yeast extract, 5g of NaCl, 10g of soluble starch, 20g of agar and 1000mL of distilled water, and pH7.8.
The detection method comprises the following steps: and (3) dripping 10 mu L of shaking culture strain BJ-10 to be detected and escherichia coli control strain (without starch hydrolase activity) on a detection plate, airing, culturing at the constant temperature of 28 ℃ for 24-28h, covering and dyeing with dilute iodine solution, flushing with clear water, and observing the size of a transparent ring.
Detection result: as shown in FIG. 3, it can be seen that the strain BJ-10 was surrounded by a transparent ring, indicating that it was able to produce amylase, a positive starch hydrolysis. The E.coli is not produced with transparent rings around and has no starch hydrolase activity.
2.2 protease detection
Detection plate: the method comprises heating skim milk (1 g/100mL water) with 10% of skim milk agar medium (agar powder) in a microwave oven, removing fat film if fat film exists, and adding 10mL into 100mL sterilized cold water agar medium with pipetting gun.
The detection method comprises the following steps: and (3) shaking the strain BJ-10 to be detected in an LB culture medium overnight, dripping 10 mu L of the strain on a detection plate, airing, culturing at the constant temperature of 28 ℃, and observing a protein degradation ring for 12 hours.
Detection result: as shown in FIG. 4, it can be seen that the strain BJ-10 was surrounded by a more pronounced transparent circle, indicating that it was able to produce protease, which was positive for proteolytic hydrolysis. The clear circles are also obvious around the escherichia coli, and the escherichia coli has certain proteolytic activity.
2.3 detection of cellulases
Detection plate: semi-LB medium (halved amount of carbon source, nitrogen source) plus 0.2% sodium carboxymethylcellulose.
The detection method comprises the following steps: shaking the strain BJ-10 to be detected in LB culture medium overnight, taking 10 mu L, dripping the strain onto a detection plate, airing, culturing at a constant temperature of 28 ℃ for 24 hours, washing out colonies, adding 1g/L Congo red solution for dyeing for 1 hour, soaking and washing for 2 times with 1M NaCl solution for decoloring, soaking for 30 minutes each time, and observing whether cellulose hydrolysis rings exist around the colonies.
Detection result: as shown in FIG. 5, it can be seen that a white transparent ring was produced around strain BJ-10, indicating that they all produced cellulases, which were positive for hydrolysis. The E.coli can also produce transparent circles around it and also has cellulolytic enzyme activity.
2.4 Lipase assay
Detection medium: oil culture medium: 10g of peptone, 5g of beef extract, 10g of peanut oil or sesame oil, 1mL of 1.6% neutral red water solution, 15g of agar, 1000mL of distilled water and pH7.2.
The detection method comprises the following steps: preparing a grease culture medium, and sterilizing for later use. Before pouring the plate, the grease culture medium is fully vibrated to ensure that grease is evenly distributed, and then poured into a culture dish. And (3) dropwise adding the bacteria BJ-10 to be detected on the flat plate after the flat plate is solidified, and culturing at the constant temperature of 28 ℃ for 24 hours after airing. Colonies at the bottom of the plate were observed and if red spots appeared, indicating that fatty acids were hydrolysed, a positive reaction.
Detection result: as shown in FIG. 6, it can be seen that the E.coli and strain BJ-10 showed no red spots around the colony on the bottom of the plate, and were unable to hydrolyze fatty acids, and were unable to produce lipase, and lipase hydrolysis was negative.
2.5 methyl Red detection
Detection medium: glucose peptone liquid medium: peptone 5.0g, glucose 5.0g, K 2 HPO 4 5.0g, 1000mL of distilled water, and adjusting the pH to 7.0-7.2. 4-5 mL of each tube is sterilized at 115 DEG C20min。
Methyl red indicator: 0.1g of methyl red was dissolved in 300mL of 95% ethanol, and 200mL of distilled water was added thereto.
The detection method comprises the following steps: inoculating the activated strain into glucose peptone liquid culture medium, inoculating 6 tubes of each strain, and culturing at 37 ℃ for 4-7 d by taking the 6 tubes as non-inoculating control. Taking 3 tubes (repeated) of glucose peptone liquid culture medium, dropwise adding 2-3 drops of methyl red indicator into the tubes, fully shaking uniformly, and observing color change. If the culture solution is red, the culture solution is M.R. positive reaction; such as yellow, i.e., negative.
Detection result: as shown in FIG. 7, the solution in the tube inoculated with Bacillus amyloliquefaciens BJ-10 turned yellow and was a negative reaction.
2.6 sugar alcohol fermentation experiments
Detection medium: sugar alcohol fermentation basal medium: 10g of peptone, 5g of NaCl and 1000mL of distilled water, pH7.6 is regulated, and 1.6% bromocresol purple ethanol solution is added.
The detection method comprises the following steps: the sugar alcohol fermentation culture medium is respectively added with glucose (1%), lactose (0.75%), sucrose (0.75%), mannitol (0.75%), inositol (0.75%), and after subpackaging test tubes, each tube is put into a Du's fermentation tube, the strain BJ-10 to be tested is inoculated into sugar-containing culture solution, the culture solution without inoculation is used as a control, and the culture solution is cultured for 7 days at 30 ℃, and the color change of each test tube and the generation of bubbles in the Du's fermentation tube are observed. The indicator bromocresol purple has an indication range of pH5.3 (yellow) to 6.8 (purple).
Detection result:
the results of glucose fermentation experiments are shown in FIG. 8, and the results show that the color of the test tube culture medium of BJ-10 is changed from purple to yellow, and no bubbles are generated in the Du tube, so that the BJ-10 can decompose glucose and generate acid without generating gas.
As shown in FIG. 9, the color of the BJ-10 test tube culture medium is changed from purple to yellow, and no bubbles are generated in the Du tube, so that the BJ-10 can decompose sucrose and generate acid without generating gas.
As shown in FIG. 10, the results of lactose fermentation experiments show that the color of the BJ-10 test tube medium is not changed, and no bubbles are generated in the Du tube, which indicates that BJ-10 cannot decompose lactose.
As shown in FIG. 11, the test tube culture medium of BJ-10 changes from purple to yellow, and no bubble is generated in Du's tube, which indicates that BJ-10 can decompose mannitol and produce acid without gas.
As shown in FIG. 12, the color of the BJ-10 test tube medium is changed from purple to yellow, and no bubbles are generated in the Du tube, which indicates that BJ-10 cannot decompose inositol.
(III), identifying the BJ-10 strain molecules:
1.1 16S rDNA amplification
The total DNA of the strain BJ-10 is used as a template, and bacterial 16S rDNA region universal primers 27f and 1492r are used for PCR amplification, wherein the sequences of the primers are as follows:
27f：5’-AGAGTTTGATCCTGGCTCAG-3’
1492r：5’-TACCTTGTTACGACTT-3’
the PCR reaction system was (50. Mu.L): 25 μl2× Taq PCR Master Mix,21 μl LddH 2 O, 1. Mu.L 27f, 1. Mu.L 1492r, 2. Mu.L DNA template.
PCR amplification reaction procedure: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s, annealing at 54℃for 30s and extension at 72℃for 90s,34 cycles; extending at 72 ℃ for 8min; the reaction was terminated at 12 ℃.
1.2PCR product detection
The 16S rDNA sequence of the amplified strain BJ-10 was electrophoretically detected by 1% agarose gel electrophoresis, and then the result was stored and analyzed by photographing with a gel imaging system.
1.316S rDNA sequence analysis
The PCR amplified product of the 16S rDNA gene of the strain BJ-10 is sent to Beijing engine biotechnology limited company for sequencing, the complete sequence is spliced and output, the 16S rDNA of the strain BJ-10 is subjected to BLAST homologous sequence comparison, and then a phylogenetic tree is constructed by adopting an adjacent method. The 16SrDNA sequence of the bacillus amyloliquefaciens BJ-10 is shown as a sequence 1 in a sequence table.
1.4 phylogenetic Tree construction method
The sequences of the related strains were downloaded from Genbank, the sequences of strain BJ-10 were aligned with the downloaded related strain 16S rDNA sequences using MEGA 5.2 software, and then a phylogenetic tree was constructed by the adjacency method. Bacillus amyloliquefaciens BJ-10 phylogenetic tree constructed based on the result of the 16S rDNA sequence BLAST is shown in FIG. 2.
Example 2 determination of bacteriostatic Activity
The method comprises the steps of (1) measuring the indoor biological activity of bacillus amyloliquefaciens BJ-10 on powdery mildew, eluting powdery mildew spores of the plant leaves collected in the field by deionized water, filtering, centrifuging (1000 r/min) for 5min, pouring out supernatant, adding deionized water, and centrifuging. Finally, the spores are resuspended to 1X 10 per milliliter with deionized water 6 Spores and 0.5% dextrose solution was added.
2. Under aseptic operation conditions, 50mL of the sterilized culture medium melted in advance is added into an aseptic conical flask according to test treatment, 500 mu L of liquid medicine is sequentially sucked from low concentration to high concentration, and the liquid medicine is respectively added into the conical flask and fully and uniformly shaken. Equal amount of the mixture was poured into 3 petri dishes with a diameter of 9cm, and drug-containing plates with corresponding concentrations were prepared. The corresponding concentration gradient and inhibition rate of each medicament are shown in the attached table.
3. The test was run with no drug treatment as a blank, and 3 replicates per treatment.
4. More than 3 fields were randomly observed for each treatment, the total number of spores was investigated and not less than 200 spores were counted, and the germination number and total number of spores were recorded, respectively. Spore bud tube lengths greater than the spore short radius are considered germination. The test results are shown in Table 1.
TABLE 1 determination of indoor biological Activity of Bacillus amyloliquefaciens BJ-10 against powdery mildew
| Medicament | Toxicity regression equation | Correlation coefficient | EC 50 |
| BJ-10 fermentation broth | y＝0.7492x-0.8806 | 0.9943 | 7.07×10 7 CFU/mL |
| 42.4% Azole/fluoroamide suspension | y＝1.047x+4.8964 | 0.9725 | 1.26μg/mL |
| 80% sulfur water dispersible granule | y＝2.114x+7.4888 | 0.9009 | 0.07μg/mL |
| 20% penthiopyrad suspending agent | y＝0.6716x+4.6467 | 0.9832 | 3.36μg/mL |
Antagonism of BJ-10 suspensions and pathogenic bacteria
The method adopts a counter culture method: taking 11 plant pathogenic fungi (corn big spot pathogen, tomato gray mold pathogen, tomato early blight pathogen, grape anthracnose pathogen, grape canker pathogen, grape black root pathogen, grape vine blight pathogen, strawberry rhizoctonia solani, strawberry phytophthora, pepper fusarium wilt and pepper phytophthora capsici) as targets, taking bacterial cakes with the diameter of 4mm along the edges of a PDA flat plate in dark culture for 3-7 days at 25 ℃, placing the bacterial cakes in the center of a new PDA flat plate, equidistantly inoculating sterilized filter paper sheets with the diameter of 4mm at a position 3cm away from the center, absorbing 5 mu L of cultured BJ-10 bacterial suspension to wet the filter paper sheets, taking the filter paper sheets with 5 mu L of sterile water as a control, repeating the process for 3 times, and culturing in dark at 25 ℃. When the pathogenic bacteria mycelium in the control group grows to the filter paper, the colony diameter and the antibacterial zone width are measured, and the antibacterial rate is calculated. The results are shown in Table 2.
Antibacterial ratio (%) = (control colony diameter-treated colony diameter)/control colony diameter×100%
TABLE 2 determination of the bacteriostatic Activity against phytopathogenic fungi
The antibacterial activity of BJ-10 on 11 plant pathogenic fungi is measured by a counter culture method, the measurement result is shown in a table 2, the BJ-10 has different degrees of inhibition effects on the growth of 11 pathogenic fungi hyphae, the inhibition effect on the growth of the corn big spot hyphae is strongest, the inhibition rate reaches 77.27%, the inhibition zone width is 19.32mm, the antibacterial effect on tomato gray mold bacteria, grape gummy stem blight bacteria, grape anthracnose bacteria and pepper fusarium wilt bacteria is good, the inhibition rate is 46.45% -58.29%, the inhibition zone width is 10.20-14.57mm, the inhibition effects on tomato early blight bacteria, strawberry rhizoctonia solani, pepper phytophthora capsici bacteria and strawberry phytophthora capsici bacteria are general, and the inhibition rate is below 35%.
Example 3 control Effect of Bacillus amyloliquefaciens BJ-10 fermentation broth on grape powdery mildew (greenhouse potting method)
Test Co-setting Bacillus amyloliquefaciens BJ-10 fermentation liquor 1×10 5 CFU/mL、1×10 6 CFU/mL、1×10 7 CFU/mL 3 concentration gradients, clear water as a blank control; every 6 pot grape seedlings (Ma Selan) are treated, the grape seedlings are subjected to foliar spray treatment, the foliar spray is carried out once every 7 days, the total is carried out 3 times, and the investigation is carried out 7 days after the last treatmentDisease effect. The test results are shown in Table 3.
Disease grading standard: grade 0 no disease spots; the area of the grade 1 lesion accounts for less than 5% of the whole leaf area; the area of the 3-level lesion accounts for 6-10% of the whole leaf area; the area of the 5-level lesion accounts for 11% -20% of the whole leaf area; the 7-level lesion area accounts for 21% -40% of the whole leaf area; the 9-stage disease spot area accounts for more than 40% of the whole leaf area.
The disease index calculating method comprises the following steps:
TABLE 3 control effect of Bacillus amyloliquefaciens BJ-10 on grape powdery mildew (greenhouse potting method)
The different capital and lowercase letters in the table indicate significant and extremely significant differences between treatments (DMRT method, P < 0.05, P < 0.01)
According to the test result of the greenhouse potting method, the bacillus amyloliquefaciens BJ-10 fermentation liquor has a good control effect on potted grape powdery mildew, the average control effect is 51.09% -78.06%, the field natural conditions are different from the indoor conditions, and whether the fermentation liquor can be applied in actual production needs further field test verification. The effect diagram is shown in fig. 13.
Example 4 field control Effect of Bacillus amyloliquefaciens BJ-10 fermentation broth on grape powdery mildew
The test is carried out by setting 5 treatments, and the concentration of the fermentation liquid of the bacillus amyloliquefaciens BJ-10 is 1 multiplied by 10 5 CFU/mL、1×10 6 CFU/mL、1×10 7 CFU/mL,4% pyrimidine nucleoside aqua as control agent, clear water as blank control, spray application for the first time at 2021, 09, 30, 10, 08, second, 10, 15, and third, respectively, and mixingThe medicine is applied for 3 times. The disease prevention effect was investigated for the first time on day 22 of 10 in 2021 (7 days after 3 rd administration), and for the second time on day 29 of 10 in 2021 (14 days after 3 rd administration), two surveys were taken together. The test results are shown in Table 4.
Disease grading standard: grade 0 no disease spots; the area of the grade 1 lesion accounts for less than 5% of the whole leaf area; the area of the 3-level lesion accounts for 6-10% of the whole leaf area; the area of the 5-level lesion accounts for 11% -20% of the whole leaf area; the 7-level lesion area accounts for 21% -40% of the whole leaf area; the 9-stage disease spot area accounts for more than 40% of the whole leaf area.
The disease index calculating method comprises the following steps:
TABLE 4 field control Effect of Bacillus amyloliquefaciens BJ-10 on grape powdery mildew
Note that: the different capital and lowercase letters in the table indicate significant and extremely significant differences between treatments (DMRT method, P < 0.05, P < 0.01)
The investigation result 7 days after the last application shows that the bacillus amyloliquefaciens BJ-10 fermentation liquor has better control effect on grape powdery mildew, the average control effect is 69.34% -72.81%, the control effect of 4% pyrimidine nucleoside antibiotic aqueous agent on grape powdery mildew is 73.16%, and 3 concentration gradients of the bacillus amyloliquefaciens BJ-10 fermentation liquor have no significant difference with the control medicament 4% pyrimidine nucleoside antibiotic aqueous agent; the investigation result of 14 days after the last application shows that the average control effect of the bacillus amyloliquefaciens BJ-10 fermentation liquor on the grape powdery mildew is 66.61% -70.23%, the control effect of the 4% pyrimidine nucleoside antibiotic aqueous agent on the grape powdery mildew is 70.83%, and the 3 concentration gradients of the bacillus amyloliquefaciens BJ-10 fermentation liquor are not obviously different from that of the control medicament 4% pyrimidine nucleoside antibiotic aqueous agent.
Example 5 control Effect of Bacillus amyloliquefaciens BJ-10 fermentation broth on grape gray mold
The test is carried out by setting 5 treatments, and the concentration of the fermentation liquid of the bacillus amyloliquefaciens BJ-10 is 1 multiplied by 10 6 CFU/mL、1×10 7 CFU/mL,50% fludioxonil wettable powder and 2 hundred million CFU/g trichoderma harzianum wettable powder are used as control agents, clear water is used as blank control, and the first spray application and the second spray application are respectively carried out on the 08 th of 2022 and the 18 th of 06 months for 2 times. Disease prevention effect investigation was performed at 2022, 09 and 16. The test results are shown in Table 5.
And (5) surveying all the clusters in each district, recording the damage degree of the clusters, and calculating the disease index and the prevention and treatment effect.
The method for grading the ears comprises the following steps: grade 0 no disease spots; the area of the grade 1 disease spots accounts for less than 5% of the area of the whole cluster; the area of the grade 3 disease spots accounts for 6 to 15 percent of the area of the whole cluster; the area of the 5-level disease spots accounts for 16% -25% of the area of the whole cluster; the 7-level disease spot area accounts for 26% -50% of the whole ear area; the 9-grade disease spot area accounts for more than 51% of the whole ear area.
The disease index calculating method comprises the following steps:
table 5BJ-10 investigation results of grape gray mold control experiment
Note that: the different capital and lowercase letters in the table indicate significant and extremely significant differences between treatments (DMRT method, P < 0.05, P < 0.01)
As shown by the investigation result, the fermentation liquid of the bacillus amyloliquefaciens BJ-10 is used for controlling Botrytis cinereaThe compound pesticide has better control effect, the average control effect is 71.65% -73.06%, the control effect of 2 hundred million cfu/g trichoderma harzianum wettable powder on grape gray mold is 78.41%, and the control effect of 50% fludioxonil wettable powder on grape gray mold is 74.82%. On the level of 0.05, 2 hundred million cfu/g trichoderma harzianum wettable powder has a significantly higher control effect on grape gray mold than 2 concentration gradients of bacillus amyloliquefaciens BJ-10 fermentation broth; on the level of 0.01, the 2 hundred million cfu/g trichoderma harzianum wettable powder has a significantly higher control effect on grape gray mold than the effective viable count of 10 7 CFU/mL bacillus amyloliquefaciens BJ-10 fermentation broth with effective viable count of 10 6 The CFU/mL bacillus amyloliquefaciens BJ-10 fermentation broth and the control agent 50% fludioxonil wettable powder have electrodeless significance difference.
Example 6 seed-metering of Bacillus amyloliquefaciens BJ-10 fermentation broth on cucumber and grape seed-metering
1. Preparing bacterial liquid: the glycerol bacteria of BJ-10 preserved at-80 ℃ are picked up by an inoculating loop, and streak culture is carried out on LB culture medium. Picking single colony streaked by using toothpick, placing in 1mL LB liquid medium, shake culturing at 28deg.C under 180rpm/min for 8-10 hr to obtain seed solution, transferring into 150mL fermentation medium (maltose 20g/L, peptone 10g/L, bran 10g/L, caCO3 10g/L g, naHPO) at a ratio of 10:100 3 2g/L、KHPO 3 1g/L, pH 7.0-7.4), 28 ℃,180 rpm/min, shaking culture for 72 hours to obtain fermentation broth with the concentration of 8 multiplied by 10 8 CFU/mL. The fermentation broth was centrifuged at 12,000rpm at 4℃for 10min, and the supernatant was filtered through a 0.22 μm bacterial filter to obtain a fermentation filtrate containing sterile cells.
2. Test of bacillus amyloliquefaciens on cucumber growth promotion
Respectively using clear water, 100 times dilution of fermentation filtrate and concentration of 8×10 6 The CFU/mL bacillus amyloliquefaciens fermentation broth and 100-time dilution of bran culture medium are used for root irrigation treatment of cucumber in seedling stage every 7 days, the treatment is carried out for 3 times, and physiological indexes such as chlorophyll content, plant height, dry weight, fresh weight and the like of cucumber seedlings under different conditions are detected in 14 days after the last treatment, and the results are shown in table 6.
Strong seedling index = (stem thickness/plant height) ×whole plant dry mass
TABLE 6 growth promoting effect of Bacillus amyloliquefaciens on cucumber
Note that: the different capital and lowercase letters in the table indicate significant and extremely significant differences between treatments (DMRT method, P < 0.05, P < 0.01)
The results show that: when diluted 100 times, there were significant differences in physiological index between the fresh water control, fermentation filtrate, bran medium and several treatments of the fermentation broth. Several treatment groups have obvious growth promoting effect on cucumber growth. The cucumber seedlings treated by different concentrations have obviously improved plant height, fresh weight and dry weight, wherein the concentration is 8 multiplied by 10 6 The cucumber seedling strengthening index after the root-filling treatment of the CFU/mL bacillus amyloliquefaciens fermentation liquid is obviously higher than that of other treatments, as shown in figure 14.
3. Grape growth promotion test by bacillus amyloliquefaciens
The concentrations are respectively set to be 1 multiplied by 10 6 CFU/mL、1×10 7 The CFU/mL bacillus amyloliquefaciens BJ-10 fermentation broth and clear water are used as controls, 3 treatments are performed, each treatment is performed on 6 grape seedlings (Ma Selan), the grape seedlings are subjected to foliar spray treatment, each 7 days are subjected to foliar spray treatment, the total is performed for 3 times, and physiological indexes such as grape seedling height, dry weight, fresh weight and the like are detected 14 days after the last treatment, and the results are shown in table 7.
TABLE 7 results of growth promotion of grape by Bacillus amyloliquefaciens fermentation broth
| Treatment of | Height of plant (cm) | Fresh weight (g) | Dry weight (g) |
| CK | 14.68±0.78 c | 5.01±0.16 b | 0.68±0.03 b |
| 1×10 6 CFU/mL | 28.26±2.08 b | 8.54±2.00 a | 1.28±0.09 a |
| 1×10 7 CFU/mL | 40.63±4.30 a | 10.59±0.38 a | 1.60±0.51 a |
Note that: the different capital and lowercase letters in the table represent significant and very significant differences between treatments (DMRT method, P < 0.05, P < 0.01)
The results show that: there were significant differences in physiological index between the 3 treatments. Several treatment groups have obvious growth promoting effect on cucumber growth. The grape seedlings treated by different concentrations have obviously improved plant height, fresh weight and dry weight, and are shown in figure 15.
The above examples illustrate only a few embodiments of the invention, which are described in detail, but are merely illustrative of the invention and not limiting. It will be apparent to those skilled in the art that many modifications, variations and improvements may be made therein without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. The bacillus amyloliquefaciens is characterized in that the name of the bacillus amyloliquefaciens is bacillus amyloliquefaciens BJ-10, and the bacillus amyloliquefaciens is stored in the China general microbiological culture collection center (CGMCC) No.22613.
2. The use of the bacillus amyloliquefaciens of claim 1 in the preparation of a microbial agent or microbial fertilizer;
the microbial agent or microbial fertilizer has the following functions:
1) Promoting plant growth;
2) Inhibiting plant pathogenic bacteria;
3) Preventing and treating plant diseases.
3. The use according to claim 2, wherein the phytopathogen is one or more of the group consisting of Botrytis cinerea (Lasiodiplodia citricola), colletotrichum viticola (Colletotrichum viniferum), nigella viticola (Dactylonectria macrodidyma), gummis viticola (Diaporthe eres), solarium solani (Alternaria solani), leprosy et Suggs (Exserohilum turcicum (pass.) leprosy et Suggs), phytophthora capsici (Phytophora capsiciLeonia), fusarium capsici (Fusarium oxysporum f.sp.capsicum), botrytis cinerea, phytophthora strawberry (Pytophthora cactorum), rhizoctonia solani (Rhizoctonia solani), erysiphe necator (Erysiphe necator).
4. The use according to claim 2, wherein said promotion of plant growth is manifested in plant height, fresh weight, dry weight increase; the plant comprises cucumber and/or grape.
5. A microbial agent, characterized in that the active ingredient of the microbial agent is the bacillus amyloliquefaciens of claim 1; the microbial inoculum is any one of the following microbial inoculum:
1) A microbial agent for promoting plant growth;
2) A microbial agent for inhibiting plant pathogenic bacteria;
3) A microbial inoculum for preventing and treating plant diseases.
6. A microbial fertilizer, characterized in that the active ingredient of the microbial fertilizer is the bacillus amyloliquefaciens of claim 1;
the microbial fertilizer has the following functions:
1) Promoting plant growth;
2) Inhibiting plant pathogenic bacteria;
3) Preventing and treating plant diseases.
7. A method for preparing the microbial inoculum taking the bacillus amyloliquefaciens as an active ingredient, which is characterized by comprising the following steps:
a: strain activation: inoculating bacillus amyloliquefaciens BJ-10 on an LB culture medium plate by a plate streaking method through an inoculating needle, and placing the bacillus amyloliquefaciens BJ-10 in an incubator for culturing for 2d at the constant temperature of 28 ℃ to obtain an activated strain;
b: seed liquid culture: selecting a single colony by using a sterilized toothpick, inoculating the single colony into an LB liquid culture medium, and shaking and culturing the single colony in a full-temperature shaking incubator at 28 ℃ and 180rpm for 12 hours to obtain seed liquid;
c: fermentation: pouring the seed liquid into a triangular flask with a liquid loading amount of 100mL/500mL of fermentation medium, and shake culturing at 28 ℃ and 180rpm for 72 hours to obtain fermentation liquid;
bacillus amyloliquefaciens BJ-10 is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.22613.
8. The method according to claim 7, wherein the LB medium plate in the step A is an agar medium; the liquid culture medium in the step B is 5g of yeast extract, 10g of tryptone, 10g of sodium chloride, 1L of ultrapure water and the pH value is 6.5-8.0.
9. The process of claim 7, wherein the fermentation medium comprises 20g of maltose, 10g of peptone and 10g of CaCO 3 10g，NaHPO 3 2g，KH 2 PO 3 1g, 1L of ultrapure water.
10. The preparation method according to claim 7, wherein the viable count content of Bacillus amyloliquefaciens BJ-10 in the fermentation broth of Bacillus amyloliquefaciens BJ-10 is 3.0X10 8 ～2.0×10 9 CFU/mL。
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