CN111607537A - Preparation method and application of Burkholderia MEL01 for efficiently antagonizing fusarium graminearum - Google Patents
Preparation method and application of Burkholderia MEL01 for efficiently antagonizing fusarium graminearum Download PDFInfo
- Publication number
- CN111607537A CN111607537A CN202010411780.4A CN202010411780A CN111607537A CN 111607537 A CN111607537 A CN 111607537A CN 202010411780 A CN202010411780 A CN 202010411780A CN 111607537 A CN111607537 A CN 111607537A
- Authority
- CN
- China
- Prior art keywords
- burkholderia
- mel01
- fusarium graminearum
- fermentation
- culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
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
-
- 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
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Environmental Sciences (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Pest Control & Pesticides (AREA)
- Agronomy & Crop Science (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention relates to a preparation method and application of Burkholderia MEL01 for efficiently antagonizing fusarium graminearum, and belongs to the technical field of microorganisms. The method adopts the steps of carrying out shake-flask culture on Burkholderia MEL01 for 2 days, then carrying out standing culture for 3 days, or continuing the shake-flask culture for 3 days after carrying out the shake-flask culture for 2 days, adding fusarium graminearum hyphae every day, and continuously adding for 3 times, wherein the inhibition activity of the supernatant obtained by the fermentation method on the fusarium graminearum hyphae is obviously improved, the supernatant is used at the early stage of wheat flowering, the disease index and the synthesis of DON can be obviously reduced, the method is simple and easy to operate, and the Burkholderia MEL01 (MEL 01) can be obviously improvedBurkholderiasp.) inhibition of fermentation supernatantsAnd (4) bacterial activity.
Description
Technical Field
The invention relates to the technical field of microorganisms, and particularly relates to a preparation method and application of Burkholderia MEL01 for efficiently antagonizing fusarium graminearum.
Background
Fusarium graminearum (fusarium graminearum) is one of the important pathogenic bacteria that can cause scab in various gramineous crops such as wheat, barley, rice and corn. The ears of the crops are diseased, the grains are shriveled, and the yield of the crops is reduced. More importantly, fusarium graminearum can generate toxic metabolites, Deoxynivalenol (DON), during the process of infecting crops, and the deoxynivalenol has stable properties, heat resistance, pressure resistance, weak acid resistance and storage resistance, the structure of common food cannot be damaged in the processing process, and partial toxin can be damaged only by adding alkali or high-pressure treatment. When a person ingests food made of grains contaminated with DON, the person may suffer from toxicosis such as vomiting, diarrhea, headache, dizziness and the like, which are mainly symptoms of the digestive system and the nervous system. The DON limit standard was first promulgated by the International food code Committee (CAC) in 2015, and the DON limit in unprocessed grains was regulated to 2000. mu.g/kg, the grain product limit was regulated to 1000. mu.g/kg, and the infant grain product limit was regulated to 200. mu.g/kg. At present, chemical agents are mostly used for preventing and treating the gibberellic disease, but the chemical agents cause serious harm to the health and the environment of human beings and also cause the resistance development of fusarium graminearum. The biological control realized by utilizing microorganisms has the advantages of high efficiency, low toxicity, safety, environmental protection and the like, and is gradually one of new effective ways for controlling plant diseases.
To date, there have been some studies on the control of plant diseases by burkholderia. In 2010, Nijiahong et al found that the Pythium species JK-SH007 has strong inhibition effect on 3 main pathogenic bacteria of the poplar canker (Phomopsis acrospore), the chrysosporium (Cytosporachrysosperma) and the Fusarium aesculosum (Fusicoccumae sculi), and can effectively prevent and treat the poplar canker. In 2016, Bi Ke et al found that a burkholderia species had a significant inhibitory effect on phytopathogenic fungi such as sclerotinia sclerotiorum (Sclerotinia sclerotiorum), Blackia tabashiora (Pestalotiopsis sp.), banana vascular wilt (Fusarium oxysporum f.sp.cubense), Iris alba anthracnose (Colletotrichum sp.) and Canavana americana pestis (Pyrococcaceae hashimooka).
However, it has been rarely reported that B.burkholderia can inhibit Fusarium graminearum. Zhang Shi bin et al report that a wild rice endophytic bacterium in east county is gladiolus burkholderia Fse32 and Jianren et al report that a pine endophytic bacterium, burkholderia cepacia NSM-05, can inhibit wheat scab. However, there are no reports on fermentation methods for improving the fusarium graminearum inhibitory activity of burkholderia and improving the bacteriostatic activity of burkholderia. In addition, the research finds that the fermentation liquor of the burkholderia can not inhibit the fusarium graminearum under the general shake flask fermentation condition.
Therefore, the invention provides a natural biocontrol bacterium Burkholderia (Burkholderia sp.) MEL01 from field soil in rice and wheat crop rotation areas in the province of Bell Hubei, and an application and culture method thereof in preventing and treating wheat head blight. The simple and efficient fermentation culture method in the application can obviously improve the bacteriostatic activity of the burkholderia.
Disclosure of Invention
Aiming at the defects of the prior art, the application aims to provide a preparation method and application of burkholderia for efficiently antagonizing fusarium graminearum, and the fermentation method can obviously improve the inhibition activity of the burkholderia (burkholderia sp.) MEL01 on the fusarium graminearum and inhibit the synthesis of DON toxin.
Through diligent efforts and a great deal of experiments of the inventor, a simple preparation method of Burkholderia MEL01 for efficiently antagonizing fusarium graminearum is finally obtained, and the method comprises the following steps:
(1) selecting MEL01 single colony from the plate, inoculating into sterile LB liquid culture medium, culturing at 28 deg.C and 180r/min for 24 hr, and activating;
(2) transferring the activated bacteria liquid obtained in the step 1) into 500mL of sterile PDB fermentation medium according to the inoculation amount of 5%, and culturing for 2d under the conditions of 28 ℃ and 180 r/min;
(3) standing and culturing the fermentation liquor obtained by shaking the flask in the step 2) for 3d at the temperature of 28 ℃ to obtain MEL01 fermentation liquor;
or:
and (3) continuing to culture for 3 days in a shaking way after 2 days in a shaking way, adding fusarium graminearum hyphae every day, and continuously adding for 3 times to obtain the fermentation liquor of the burkholderia.
In the preparation method of the burkholderia for efficiently antagonizing fusarium graminearum, the burkholderia is burkholderia MEL01, which is deposited in the chinese typical culture collection with the collection number: cctccno. mk778, preservation date: 12 and 25 days 2015.
The fermentation liquid can inhibit the hypha growth and spore germination of the fusarium graminearum and inhibit the synthesis of DON.
The application of the burkholderia for efficiently antagonizing fusarium graminearum can effectively prevent and treat wheat scab by uniformly spraying the fermentation liquid on wheat ears at the early stage of flowering.
Compared with the prior art, the invention has the following beneficial effects:
(1) the inhibition rate of the Burkholderia (Burkholderia sp.) MEL01 on fusarium graminearum reaches 67.74 +/-1.34% during plate culture, the inhibition rate of MEL01 fermentation liquid on the fusarium graminearum is effectively improved to 97.8% after the fermentation culture method provided by the invention is used, and the fusarium graminearum can be inhibited from synthesizing DON.5.0 × 105The suspension of MEL01 with the bacterial concentration of CFU/mL can completely inhibit the germination of fusarium graminearum spores to form hyphae.
(2) In the Burkholderia (Burkholderia sp.) MEL01 fermentation liquor provided by the invention, in a prevention and treatment experiment of wheat scab in a field, after MEL01 bacterial suspension is sprayed, compared with a group without pesticide spraying, the disease index of wheat is reduced by 48.98%, and the content of wheat DON in the Burkholderia fermentation liquor sprayed group is not detected.
(3) The burkholderia provided by the invention is used as a biological control material for wheat scab, and has good application prospect no matter in the aspect of developing new biological pesticides or new biological control microbial agents.
Description of the drawings:
FIG. 1 shows the inhibitory activity of MEL01 on Fusarium graminearum and other fungi on PDA plates, wherein A is Fusarium graminearum, B is Geotrichum arachidicola, C is Chloromyces virescens, D is Botrytis cinerea, E is Sclerotinia sclerotiorum, F is Magnaporthe grisea, G is Botrytis cinerea, H is Ustilago virens, I is Rhizoctonia solani, and J is Pectinophora pseudopekinensis.
FIG. 2 is a graph of the effect of different MEL01 concentrations on Fusarium graminearum spore germination.
FIG. 3 shows thin layer chromatography analysis of MEL01 fermentation broth obtained by different culturing methods (spreading agent: chloroform: methanol: 9:1 v/v).
FIG. 4 shows the inhibition effect of MEL01 fermentation broth on Fusarium graminearum obtained by different culture methods, wherein a is fermentation supernatant of MEL01 shake flask 5d, b is fermentation supernatant of MEL01 shake flask after 2 days of standing for 3 days, and c is fermentation supernatant of MEL01 shake flask after 2 days of adding Fusarium graminearum hyphae and shaking for 3 days.
FIG. 5 is a graph showing the effect of MEL01 broth (2 d shake flask followed by 3d stationary culture) on Fusarium graminearum hyphae.
FIG. 6 is a graph of the effect of MEL01 broth (2 d shake flask followed by 3d stationary culture) on germination of Fusarium graminearum spores.
FIG. 7 is a graph showing the effect of MEL01 fermentation broth (2 d in shake flask followed by 3d in stationary culture) on Fusarium graminearum infection of wheat ears.
Detailed Description
The following are specific embodiments of the present invention and further description of the technical solutions of the present invention, but the present invention is not limited to the scope of the embodiments, and all changes or equivalent substitutions that do not depart from the spirit of the present invention are included in the scope of the present invention.
The media and reagent components involved in the examples:
LB culture medium: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of NaCl and 1.0L of distilled water to constant volume; the pH was 7.0.
PDA culture medium: 200g/L of potato, 20g/L of glucose, 20g/L of agar, natural PH and the constant volume of distilled water of 1.0L; sterilizing at 115 deg.C for 20 min.
PDB culture medium: 200g/L of culture medium potato, 20g/L of glucose, natural PH and distilled water to constant volume of 1.0L; sterilizing at 115 deg.C for 20 min.
CMC culture medium: 20g of CMC-Na; na (Na)2HPO42.5g；KH2PO41.5 g; peptone 2.5 g; distilled water is added to a constant volume of 1.0L; the pH value is 7.0-7.5.
Test fusarium graminearum strains:
fusarium graminearum (Fusarium graminearum) strains used for the experiments were purchased from the chinese industrial microbial cultures collection management center with the collection numbers: CICC 2697.
Example 1MEL01 plate Activity against fungi such as Fusarium graminearum
The fungi are inoculated on the center of a PDA culture medium plate, after the fungi are activated, a punch with the diameter of 7mm is used for punching a fungus cake at the edge of a colony, the fungus cake is inoculated on one side of the center of the plate, meanwhile, MEL01 is symmetrically scribed on the other side of the center of the plate, and the distance between the fungus cake and the antagonistic bacteria MEL01 is 40 mm. After the control strain is overgrown, the width of the inhibition zone (the distance from the edge of the colony of the antagonistic bacterium to the edge of the colony of the fusarium graminearum) is used as a control without inoculating the antagonistic bacterium strain Burkholderia sp.MEL01. Each treatment was repeated 3 times.
The bacteriostatic ratio (%) - (control colony diameter-treated colony diameter)/control colony diameter × 100%.
MEL01 has plate confronting activity on Fusarium graminearum and other fungi as shown in figure 1, MEL01 has obvious inhibiting effect on Fusarium graminearum, peanut leaf mold, green mold, strawberry gray mold, Sclerotinia sclerotiorum, Magnaporthe grisea, Botrytis cinerea, Ustilago virens, Rhizoctonia solani and Pestalotiopsis pseudopekinensis, wherein the inhibiting rate on Fusarium graminearum reaches 67.74 + -1.34%, and the inhibiting rate on Rhizoctonia graminearum reaches 70.01 + -2.12%
Example 2 Effect of different bacteria concentrations MEL01 on Fusarium graminearum spore germination
The concentration of the strain is 5.0 × 101～5.0×108CFU/mL MEL01 bacterial suspension and 2.0 × 105Fully mixing the CFU/mL fusarium graminearum spores, co-culturing for 6h at 25 ℃, transferring to a PDA (personal digital Assistant) plate culture for 2d, and observing whether the fusarium graminearum spores germinate to form hyphae or not, as shown in figure 2, 5.0 × 105The suspension of MEL01 with the bacterial concentration of CFU/mL can completely inhibit the germination of fusarium graminearum spores to form hyphae.
EXAMPLE 3 Effect of different culture regimes on inhibition of Fusarium graminearum Activity in MEL01 fermentation supernatants
Melt sp. mel01 single colonies were picked into sterilized LB medium and activated overnight. Activated MEL01 was washed twice with sterile water at 8000rpm for 10min and inoculated into 500mL of sterile PDB fermentation medium at 5% (5% being the volume percent of the fermentation medium).
Shaking and culturing the group A for 5d at the temperature of 28 ℃ and the speed of 180 rpm;
performing shake culture on the group B at 28 ℃ and 180rpm for 2d, and performing static culture at 28 ℃ for 3 d;
and C, performing shake culture at 28 ℃ and 180rpm for 2d, adding a proper amount of fusarium graminearum hyphae every other day for induction, and adding 1 plate hyphae with the thickness of 90mm each time.
Centrifuging the three groups of fermentation liquor at 12000rpm for 15min, performing rotary evaporation at 50 deg.C, concentrating to volume of about 50ml, centrifuging, filtering with 0.22 μm filter membrane, extracting with twice volume of ethyl acetate twice, extracting with shaking at 180rpm for 30min, standing for 1h, extracting twice, mixing ethyl acetate layers, evaporating ethyl acetate at 50 deg.C, and dissolving with 2ml HPLC water. The obtained aqueous solution was sterilized by filtration through a 0.22 μm filter, 500. mu.L of the aqueous solution was added to 15ml of PDA solid medium, and activated Fusarium graminearum was inoculated on the center of the PDA medium plate. The culture was carried out for 5 days in an incubator at 28 ℃ and the results were observed and recorded.
TLC analysis As shown in FIG. 3, addition of appropriate amounts of Fusarium graminearum hypha induced MEL01 fermentation supernatant every other day produced at least 3 new metabolites, the yellow color of which was identified as toxoflavin by LC-MS, after 2 days of shake flask culture, 3 days of standing culture at 28 ℃ and 2 days of shake flask culture, compared to 5 days of shake flask culture. The results of fungus inhibition experiments by toxoflavin show that the inhibition rate of 256 mug/mL toxoflavin on fusarium graminearum reaches 53.15 +/-2.25%, and the inhibition rate of 64 mug/mL toxoflavin on rhizoctonia cerealis reaches 100%.
Results for inhibition of fusarium graminearum as shown in fig. 4, the fermentation supernatant of MEL01 after 5 days in shake flask was unable to inhibit fusarium graminearum. However, after 2d of shaking, the inhibition efficiency of MEL01 fermentation supernatant obtained by standing and culturing 3d on fusarium graminearum reaches 97.8%; and after 2d of shake flask culture, fusarium graminearum hyphae are added every other day for induction, and the inhibition efficiency of the fermentation supernatant on fusarium graminearum reaches 70.2%. Therefore, after 2d of shaking, 3d of static culture and 2d of shaking culture, fusarium graminearum hyphae are added for induction, MEL01 is facilitated to secrete substances for inhibiting the growth of fusarium graminearum, and after 2d of shaking culture, the culture mode of 3d of static culture is more beneficial to improving the activity of fusarium graminearum of MEL01 fermentation supernatant. In addition, the biofilm detection result shows that the amount of MEL01 produced biofilm is obviously increased and is 3.2 times of that of shake flask culture for 5d in the shake flask culture mode of 2d standing for 3 d.
Example 4 Effect of MEL01 fermentation broth on Fusarium graminearum hypha and spore germination
Inoculating appropriate amount of Fusarium graminearum mycelium into 50mL PDB culture solution, and shaking at 28 deg.C and 180rpm for 24 hr to obtain uniform mycelium suspension. MEL01 broth (after shaking for 2d, stationary culture for 3d) was added, with sterile water as control. After culturing at 28 ℃ for 48 hours, morphological changes of the mycelium were observed under an inverted microscope.
The experimental results are shown in fig. 5, the surface of the hyphae of the control group is smooth, the protoplasts are uniform, and the protoplasts of the Fg hyphae added with the MEL01 fermentation liquid (after shaking for 2d, standing for 3d) are shriveled and vacuolated.
Fusarium graminearum was inoculated into a medium containing sterile 100ml CMC (20g CMC-Na, 2.5g Na)2HPO4，1.5gKH2PO42.5g peptone and 1000ml H2O, pH7.0-7.5), at 180rpm 21 ℃ -25 ℃, shaking for 3 to 5 days, filtering the culture with two layers of sterile gauze to remove the mycelia to prepare a macroconidium suspension, centrifuging at 4 ℃, 10000rpm for 20min, collecting the spores, washing the spores twice with distilled water, and adjusting the concentration of the suspension to 2 × 10 in 0.1% (v/v) Tween 20 solution5Conidia/ml.
Mixing fusarium graminearum spore suspension and MEL01 fermentation broth (shaking for 2d, standing for 3d), and observing the germination rate of fusarium graminearum spores after 12h at 28 ℃ and 180 rpm. The spore suspension and PDB medium were mixed as a control, and each treatment was repeated 3 times. Spore germination was observed microscopically and each treatment was examined microscopically at magnification x 400 and germination was scored as the length of the spore tube exceeding the diameter of the spore 1/2.
The experimental result is shown in fig. 6, the spore germination rate of fusarium graminearum spores is only 6% after passing through MEL01 fermentation liquid (after 2d of shaking bottle and 3d of static culture). The germination rate of fusarium graminearum spores in the control group is 100%. The germination rate of fusarium graminearum spores is obviously reduced after MEL01 fermentation liquid is added (after 2d of shaking bottle and 3d of static culture).
Example 5 field experiment of preventing and treating effects of MEL01 fermentation broth on wheat scab
Preparation of MEL01 bacterial suspension: inoculating MEL01 into sterilized PDB culture medium at 5%, culturing at 28 deg.C and 180rpm for 2d, standing for 3d, adding sterile phosphate buffer solution, and diluting to final thallus concentration of 108CFU/ml, and finally Tween 20 was added to a concentration of 0.1% (v/v).
Preparation of fusarium graminearum spore suspension: see example 3.
The pesticide spraying method and the spore infection method and time are as follows: at the beginning of the wheat populus (about 10% of the wheat ear is raised), a polyethylene compressed air sprayer is used at a rate of 40mL/m2Uniformly spraying MEL01 fermentation liquid suspension (or carbendazim wettable powder) on wheatear, covering with plastic film, and keeping moisture for 24 hr. After 24h, the plastic film was removed and the same polyethylene compressed air sprayer was used at 40mL/m2The concentration is 2 × 105The Fg conidia are uniformly sprayed on the wheat ears, and a plastic film is covered on the wheat ears to keep moisture for 24 hours.
Assessment of MEL01 resistance to wheat scab: after 16d (milk stage), the disease severity of the wheat is investigated and counted according to the disease grading standard of wheat scab resistance evaluation technical specification (NY/T1443.4-2007).
TABLE 1 grading of wheat scab
DI (disease index) ═ Σ (number of disease stages × number of plants of the disease stage)/(number of highest disease stages × total number of plants)
Biocontrol effect (%) (control group disease severity-treatment group disease severity)/control group disease severity × 100%
The method for measuring the DON content of the wheat grains comprises the following steps: reference is made to GBT 23503-2009' determination of deoxynivalenol in food by immunoaffinity chromatography and purification high performance liquid chromatography
FIG. 7 shows that Fusarium graminearum infection of wheat ears was completely inhibited after spraying MEL01 broth under laboratory conditions. The results of field experiments show that compared with the control without pesticide spraying, after the MEL01 fermentation liquor is sprayed, the disease index of wheat is reduced by 48.98%, and the DON content of wheat is reduced by 100%. Therefore, after the MEL01 fermentation liquid is sprayed, the growth of fusarium graminearum can be effectively inhibited, the occurrence of gibberellic disease is prevented and the use of pesticides is reduced. Meanwhile, the DON content in the wheat grains is obviously reduced, so that the quality of the wheat is improved.
TABLE 2 biological control effect of MEL01 fermentation broth on wheat scab (field experiment result)
Claims (4)
1. The preparation method of Burkholderia MEL01 for efficiently antagonizing fusarium graminearum is characterized by comprising the following steps of: the method comprises the following steps:
(1) picking single bacterial colony of Burkholderia from a flat plate, inoculating the single bacterial colony into a liquid culture medium, culturing for 24 hours at the temperature of 28 ℃ and under the condition of 180r/min, and activating;
(2) inoculating the activated bacteria obtained in the step 1) into a sterilized glucose potato shake flask fermentation medium according to the inoculation amount of 5%, and culturing for 2d under the conditions of 28 ℃ and 180r/min to obtain a fermentation seed solution;
(3) culturing the seed liquid fermented in the shake flask in the step 2) for 3d under the condition of standing at 28 ℃ to obtain a Burkholderia fermentation liquid;
or:
and (3) continuing to culture for 3 days in a shaking way after 2 days in a shaking way, adding fusarium graminearum hyphae every day, and continuously adding for 3 times to obtain the fermentation liquor of the burkholderia.
2. The method for preparing Burkholderia MEL01 for efficiently antagonizing Fusarium graminearum as claimed in claim 1, wherein the method comprises the following steps: the birkThe genus Holdersonia is Burkholderia (B) ((B))Burkholderiasp.) MEL01, which has been deposited in China Center for Type Culture Collection (CCTCC) at 12/25/2015 with the collection number of CCTCC No. M2015778.
3. The use of a burkholderia MEL01 strain for efficiently antagonizing fusarium graminearum as claimed in claim 1, wherein: the fermentation liquid can inhibit the hypha growth, spore germination and DON synthesis of fusarium graminearum.
4. The use of a burkholderia MEL01 strain for efficiently antagonizing fusarium graminearum as claimed in claim 3, wherein: the MEL01 fermentation liquid is uniformly sprayed on wheat ears at the early stage of flowering to prevent and control wheat scab.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010411780.4A CN111607537B (en) | 2020-05-15 | 2020-05-15 | Preparation method and application of Burkholderia MEL01 for efficiently antagonizing fusarium graminearum |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010411780.4A CN111607537B (en) | 2020-05-15 | 2020-05-15 | Preparation method and application of Burkholderia MEL01 for efficiently antagonizing fusarium graminearum |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111607537A true CN111607537A (en) | 2020-09-01 |
| CN111607537B CN111607537B (en) | 2022-01-04 |
Family
ID=72198291
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010411780.4A Active CN111607537B (en) | 2020-05-15 | 2020-05-15 | Preparation method and application of Burkholderia MEL01 for efficiently antagonizing fusarium graminearum |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111607537B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111172049A (en) * | 2018-11-09 | 2020-05-19 | 江苏省中国科学院植物研究所 | Toxoflavin high-yield strain and application thereof |
| CN112899186A (en) * | 2021-01-29 | 2021-06-04 | 山东省花生研究所 | Burkholderia and application thereof in biological control of fusarium graminearum |
| CN115505541A (en) * | 2022-07-11 | 2022-12-23 | 安徽农业大学 | Biocontrol bacterium burkholderia pyrrocinia suitable for wheat fungal diseases and application thereof |
| CN115851495A (en) * | 2022-09-07 | 2023-03-28 | 西北农林科技大学 | Biocontrol bacterium for preventing and treating wheat scab, application of biocontrol bacterium and biocontrol bacterium agent |
| CN116676218B (en) * | 2023-05-16 | 2024-02-27 | 上海市农业科学院 | Burkholderia gladioli and application thereof in preventing and controlling strawberry diseases |
| CN117898307A (en) * | 2024-01-15 | 2024-04-19 | 安徽农业大学 | Application of Burkholderia pyrrocinia in inhibiting wheat scab mycotoxin synthesis |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106011026A (en) * | 2016-07-15 | 2016-10-12 | 湖北大学 | Chitosanase-producing and fungus-restraining Burkholderia sp. and application thereof |
| CN107099478A (en) * | 2017-06-06 | 2017-08-29 | 江苏省中国科学院植物研究所 | One plant of Lycoris aurea endogenetic bacteria bulkholderia cepasea HDXY 02 and its application |
| CN110656068A (en) * | 2019-10-31 | 2020-01-07 | 南京林业大学 | Method for forming biological membrane by burkholderia pyrrocinia |
| CN110669686A (en) * | 2019-03-07 | 2020-01-10 | 慕恩（广州）生物科技有限公司 | Burkholderia and application thereof |
-
2020
- 2020-05-15 CN CN202010411780.4A patent/CN111607537B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106011026A (en) * | 2016-07-15 | 2016-10-12 | 湖北大学 | Chitosanase-producing and fungus-restraining Burkholderia sp. and application thereof |
| CN107099478A (en) * | 2017-06-06 | 2017-08-29 | 江苏省中国科学院植物研究所 | One plant of Lycoris aurea endogenetic bacteria bulkholderia cepasea HDXY 02 and its application |
| CN110669686A (en) * | 2019-03-07 | 2020-01-10 | 慕恩（广州）生物科技有限公司 | Burkholderia and application thereof |
| CN110656068A (en) * | 2019-10-31 | 2020-01-07 | 南京林业大学 | Method for forming biological membrane by burkholderia pyrrocinia |
Non-Patent Citations (5)
| Title |
|---|
| BOKNAM JUNG 等: "Development of a Selective Medium for the Fungal Pathogen Fusarium graminearum Using Toxoflavin Produced by the Bacterial Pathogen Burkholderia glumae", 《THE PLANT PATHOLOGY JOURNAL》 * |
| SETU BAZIE TAGELE 等: "Effectiveness of multi-trait Burkholderia contaminans KNU17BI1 in growth promotion and management of banded leaf and sheath blight in maize seedling", 《MICROBIOLOGICAL RESEARCH》 * |
| SETU BAZIE TAGELE 等: "Potential of Novel Sequence Type of Burkholderia cenocepacia for Biological Control of Root Rot of Maize ( Zea mays L.) Caused by Fusarium temperatum", 《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》 * |
| 张婧婷 等: "洋葱伯克氏菌Burkholderia contaminans抗菌蛋白分离纯化及生物学特性分析", 《食品科学》 * |
| 张慧雯 等: "不同条件下植物病原真菌对链霉菌702的敏感性研究", 《安徽农业科学》 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111172049A (en) * | 2018-11-09 | 2020-05-19 | 江苏省中国科学院植物研究所 | Toxoflavin high-yield strain and application thereof |
| CN111172049B (en) * | 2018-11-09 | 2023-05-26 | 江苏省中国科学院植物研究所 | Strain Huang Sugao producing strain and application thereof |
| CN112899186A (en) * | 2021-01-29 | 2021-06-04 | 山东省花生研究所 | Burkholderia and application thereof in biological control of fusarium graminearum |
| CN112899186B (en) * | 2021-01-29 | 2022-08-02 | 山东省花生研究所 | Burkholderia and application thereof in biological control of fusarium graminearum |
| CN115505541A (en) * | 2022-07-11 | 2022-12-23 | 安徽农业大学 | Biocontrol bacterium burkholderia pyrrocinia suitable for wheat fungal diseases and application thereof |
| CN115505541B (en) * | 2022-07-11 | 2023-07-21 | 安徽农业大学 | Biocontrol bacterium Burkholderia pyrrocinia suitable for wheat fungal diseases and application thereof |
| CN115851495A (en) * | 2022-09-07 | 2023-03-28 | 西北农林科技大学 | Biocontrol bacterium for preventing and treating wheat scab, application of biocontrol bacterium and biocontrol bacterium agent |
| CN115851495B (en) * | 2022-09-07 | 2024-05-24 | 西北农林科技大学 | Biocontrol bacterium for preventing and treating wheat scab, application thereof and biocontrol bacterium agent |
| CN116676218B (en) * | 2023-05-16 | 2024-02-27 | 上海市农业科学院 | Burkholderia gladioli and application thereof in preventing and controlling strawberry diseases |
| CN117898307A (en) * | 2024-01-15 | 2024-04-19 | 安徽农业大学 | Application of Burkholderia pyrrocinia in inhibiting wheat scab mycotoxin synthesis |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111607537B (en) | 2022-01-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111607537B (en) | Preparation method and application of Burkholderia MEL01 for efficiently antagonizing fusarium graminearum | |
| CN111534460B (en) | Preparation method and application of bacillus beiLeisi capable of efficiently antagonizing fusarium graminearum | |
| CN111154688B (en) | Biocontrol bacillus beleisi SF259 and application thereof | |
| CN110257290B (en) | Plant pathogenic bacteria inhibitor, and strain and application thereof | |
| CN107736379B (en) | Application of bacillus amyloliquefaciens in preventing and treating plant fungal diseases | |
| CN113969247B (en) | Bacterium for inhibiting tobacco disease pathogenic bacteria and application thereof | |
| CN110713951B (en) | Pantoea agglomerans strain and application thereof | |
| CN107974427B (en) | Marine streptomyces with bacteriostatic activity | |
| CN104403970B (en) | A kind of bacillus subtilis and the application in control of plant disease thereof | |
| CN111560327B (en) | Preparation and application of alcaligenes faecalis capable of antagonizing fusarium graminearum and efficiently degrading DON (Don) | |
| CN111394285A (en) | Bacillus belgii and application thereof in degrading deoxynivalenol | |
| CN114854618A (en) | Bacillus belgii SF327 and application thereof | |
| CN114736821B (en) | Bacillus belgii SF305 with antagonistic effect on rubber tree red root pathogen and application thereof | |
| CN115058358B (en) | Salt-tolerant bacillus and application thereof | |
| CN110184224B (en) | Staphylococcus epidermidis and application thereof | |
| CN100467588C (en) | One strain of streptonmyces lydicus and its application in treatment and prevention of plant's disease | |
| CN114940958A (en) | Bacillus siamensis, prepared microbial agent and application thereof | |
| CN107629985B (en) | Plant endophytic bacterium with antagonistic effect on plant pathogenic fungi | |
| CN110628661B (en) | Bacillus mojavensis CJX-61 and application thereof | |
| CN111394270B (en) | Nocardia gamboge and application thereof | |
| CN107012110A (en) | A kind of entomopathogenic nematode symbiotic bacteria and its application with inhibition of potato late blight | |
| CN116716225A (en) | Radiation-resistant streptomycete and application thereof | |
| CN114946885B (en) | Microbial pesticide for resisting leaf spot disease of polygonatum odoratum | |
| CN116042436A (en) | Bacillus bailii SF334 and application thereof | |
| CN114231435A (en) | Actinomycetes and application thereof |
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 | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230411 Address after: 441300 Liu Dian Village, Sanligang Town, Suixian County, Suizhou City, Hubei Province Patentee after: Suizhou Chutian Gardening Co.,Ltd. Address before: 430062 368 Friendship Avenue, Wuchang District, Wuhan, Hubei. Patentee before: Hubei University Patentee before: Nanyang Normal University Patentee before: INSTITUTE FOR PLANT PROTECTION AND SOIL SCIENCE, HUBEI ACADEMY OF AGRICULTURAL SCIENCES |