CN104403970A - Bacillus subtilis and application thereof in plant disease control - Google Patents
Bacillus subtilis and application thereof in plant disease control Download PDFInfo
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- CN104403970A CN104403970A CN201410699358.8A CN201410699358A CN104403970A CN 104403970 A CN104403970 A CN 104403970A CN 201410699358 A CN201410699358 A CN 201410699358A CN 104403970 A CN104403970 A CN 104403970A
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- powdery mildew
- bacillus subtilis
- plant
- subtilis
- control
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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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- 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
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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
Abstract
The invention provides a bacillus subtilis and an application thereof in plant disease control, relates to the technical field of microorganism screening, and specifically provides a bacillus subtilis FX1 screened from the soil of a vegetable greenhouse, wherein the preservation number of the bacillus subtilis FX1 is CCTCC NO.M2014563. The strain has very strong antibacterial effect and can be widely applied to the plant disease control. The cucumber powdery mildew control efficiency of the strain is up to 95.5%, the strawberry powdery mildew control efficiency of the strain is up to 94.8%, and the grape brown spot control efficiency is up to 83.2%. Besides controlling the plant powdery mildew and brown spot, the bacillus subtilis FX1 further has remarkable antagonism to plant yellow rot and fruit rot caused by fusarium and rhizoctonia solani, the control efficiency is up to more than 80%, and the application prospect is wide.
Description
Technical field
The present invention relates to microbe to screen technical field, be specifically related to a kind of subtilis and the application in control of plant disease thereof.
Background technology
Be on the increase along with floristic, the continuous variation of plant growing mode, Plant diseases is on the rise, and traditional chemical prevention and control method also exists many drawbacks, such as: a large amount of uses of chemical pesticide cause serious environmental pollution, the pesticide residue of agricultural-food are also more and more serious, and it is also more and more faster etc. a series of to people's lives injurious effects that germ resistance produces speed.Advocating now in low-carbon economy, environmental protection economical society, seek to protect human lives again fertile new technology extremely adapt to the development of society.
Biological control is a kind of method utilizing biology or its product to tackle another biology, is divided into controlling worm with worm, controlling worm, fungus treatment three major types with bacterium.Which utilizes the mutual relationship between living species, with a kind of or a class biological or its Product inhibiton is another kind of or another kind of biology.The method of biological control has a lot, and microbial control is exactly wherein a kind of.Microbial control mainly utilizes useful microorganism, by interbiotic Competition, antibiosis, parasitization, bacteriolysis and induction of resistance etc., suppress survival and the activity of some pathogen, its superiority is that aboundresources, selectivity are strong, noresidue, pollution-free, cost is low, hold concurrently and anti-doublely to control, increasing both production and income, to keep ecological balance, play long-acting effect.
Microorganism for biological control mainly comprises a lot of biotic population such as fungi, bacterium, actinomycetes and yeast, wherein fungi mainly comprises wood mould, chaetomium, Penicillium notatum, mycorrhizal fungi etc., and bacterium mainly comprises genus bacillus, pseudomonas, soil radiation bacillus etc.Wherein, the scope of restraining fungi of sporeformer is very wide, comprises root disease, trunk disease, leaf China portion's disease and and gathers in the crops rear fruit disease, as Fusarium and Verticillium wilts, damping-off etc.Because it has endogenous spore, strong stress resistance, nutritional needs is simple, and reproduction speed is fast, and is easy to surely grow at roots of plants circle, so be widely used in the biological control of Plant diseases.The genus bacillus kind being applied to biological and ecological methods to prevent plant disease, pests, and erosion at present mainly contains subtilis, bacillus cereus, bacillus megaterium, bacillus polymyxa and bacillus pumilus etc.
Antagonistic microbe controlling plant diseases is utilized to be one of very active research field of current plant pathology educational circles.Facts have proved, introduce bacterium controlling plant diseases pathogenic bacteria being had to antagonistic action, be a kind of economical and effective and the controlling way had no side effect.Abundant microbe species is had in the growing environment of plant, exploitation Useful Bacillus potentiality are very large, but Biocontrol Bacillus protection effect is sometimes unstable, its protection effect of the genus bacillus that different zones screens is widely different, so the more how useful genus bacillus of exploitation is one of emphasis of development biocontrol fungicide.
Summary of the invention
The present invention, for solving prior art problem, provides a kind of subtilis and application thereof.Described subtilis be from the soil of green house of vegetables screening and separating to, there is very strong fungistatic effect, can effective controlling plant diseases.
One aspect of the present invention relates to a kind of subtilis FX1 (Bacillus subtilis FX1), be preserved in the China typical culture collection center of Wuhan, China Wuhan University on November 12nd, 2014, deposit number is CCTCC NO:M2014563.
One aspect of the present invention relates to a kind of biological prevention and control agent, comprises above-mentioned subtilis FX1.
The invention still further relates to the application of above-mentioned biological prevention and control agent in control of plant disease.
Described Plant diseases is any one in powdery mildew of cucumber, powdery mildew of strawberry, brown spot of grape, the yellow maize ear rot of plant and fruit rot.
The application of described biological prevention and control agent in control of plant disease, concrete grammar is for diluting 0-700 doubly by biological prevention and control agent, preferred 500-700 doubly.
The present invention has screened the novel subtilis FX1 of a strain from the soil of green house of vegetables.Described bacterial strain has very strong fungistatic effect, can be widely used in the control of Plant diseases.Described bacterial strain prevents and treats efficiency up to 95.5% to powdery mildew of cucumber, prevents and treats efficiency up to 94.8% to powdery mildew of strawberry, prevents and treats efficiency up to 83.2% to brown spot of grape.Except plant powdery mildew and brown spot, described subtilis FX1 also all has obvious antagonistic action to by the yellow maize ear rot of sickle-like bacteria and the microbial plant of miliary damping-off and fruit rot, and preventing and treating efficiency can reach more than 80%, has a extensive future.
Embodiment
The present invention is set forth further below in conjunction with specific embodiment.For concrete grammar used in embodiment or material, those skilled in the art on the basis of the technology of the present invention thinking, can carry out conventional replacement according to existing technology and select, and be not limited only to the concrete record of the embodiment of the present invention.
Embodiment 1 bacterial strain screening be separated
1, the preparation of soil sample diluent:
Soil is fetched from Chengyang village, north, Haiyang green house of vegetables, take 0.5g pedotheque and be dissolved in the soil solution making 1:10 in 4.5ml sterilized water, then therefrom draw the 0.5ml soil solution and be placed in 4.5ml sterilized water, make the soil solution of 1:100, analogize in this approach, preparation 1:10
6-10
7soil dilution solution.
2, strains separation and purifying:
The diluent 0.1ml getting 3-4 suitable concentration is spread evenly across on nutrient agar; Take out after 37 DEG C of cultivation 2d, the single bacterium colony of picking continues separation and purification, until the colonial morphology solid colour that each flat board grows, is single bacterial strain.
Adopt aforesaid method, applicant filters out 12 strain bacterium altogether, respectively called after FX1, FX2, FX3 ... FX12.
3, pathogenic bacteria face-off experiment:
Monofilament shell powdery mildew (this bacterium is one of the main pathogenic fungi of powdery mildew of cucumber, is to screen in the green cucumber booth of Xi Fu town, Chengyang District, Qingdao City in June, 2013 by contriver garden is big) the bacterium cake of 5mm is about at PDA plate center place inoculation diameter; Four angles of PDA flat board are inoculated respectively bacterial strain FX1, the FX2 of equivalent, FX3 ... FX12, cultivates after 3 days for 30 DEG C, observes face-off effect.According to suppressing the size of circle to be screened, applicant obtains four strains altogether and has bacterial strain compared with high inhibition effect to monofilament shell powdery mildew, is respectively FX1, FX4, FX6 and FX10.
Embodiment 2 powdery mildew of cucumber prevention effect confirmatory experiment
1, bacterium solution preparation
Four strain bacterium FX1, FX4, FX6, the FX10 screened are inoculated in nutrient broth medium respectively, and cultivate 2-3 days for 30 DEG C, obtaining viable bacteria content is 10
8-10
9the bacterium liquid of cfu/ml.
2, experimentation
1) experimental group: use respectively same amount FX1, FX4, FX6, FX10 tetra-strain bacterium spray cucumber leaves; Every strain bacterium establishes three treatment group, and each treatment group is two ridge cucumber of Stochastic choice, totally 100, arranges protection row between each treatment group.
In the growth mid-term of cucumber, after above-mentioned bacterium liquid respectively thin up 500 times, be evenly sprayed on the cucumber leaves of each treatment group; 7 days, each sprinkling interval, is used in conjunction three times; The complete bacterium liquid of last sprinkling, after 10 days, checks the incidence of each treatment group powdery mildew of cucumber.
2) control group: use and do not spray cucumber leaves containing the nutrient broth medium of bacterium, other operations are all identical with experimental group.
3, interpretation of result
Disease scale standard: 0 grade without scab; 1 grade: lesion area accounts for less than 1/4 of leaf area; 2 grades: lesion area accounts for the 1/4-2/4 of leaf area; 3: lesion area accounts for the 2/4-3/4 of leaf area; 4 grades: lesion area accounts for more than 3/4 of leaf area.
Disease index=[∑ (sick level value × this level morbidity number of sheets)/(investigating total number of sheets × the highest sick level value)] × 100%
Prevent and treat calculation formula=[1-(disease index of the disease index/contrast of process)] × 100% of efficiency.
Analyze experimental data with SPSS statistical software, result is as shown in table 1:
Table 1 powdery mildew of cucumber controlling experiment
As can be seen from the data of table 1, the control of FX1 bacterial strain to powdery mildew of cucumber that the present invention screens is most effective, is 95.5%, can be used as the control that biological prevention and control agent is widely used in powdery mildew of cucumber.
The qualification of embodiment 3 FX1 bacterial strain
1) the physiology and chemistry characteristic of FX1 bacterial strain:
Bacterium colony is flat, and surface irregularity is opaque, dirty white, gemma size 0.6-0.9 × 0.9-1.4 μm, oval or column, middle life or near middle raw; Gram-reaction is positive, and catalase reaction is positive, V.P reacting positive, and Starch Hydrolysis is positive, and gelatine liquefication is positive, decomposes casein positive, growth temperature range 25-40 DEG C, pH scope 5-8.
2) molecular biology identification of FX1 bacterial strain:
Adopt molecular biological method to identify the FX1 bacterial strain that above-mentioned screening obtains, record its 16s rDNA sequence, and carry out blast comparison in GenBank nucleic acid database.In conjunction with biological characteristics and the 16srDNA comparison result of FX1 bacterial strain, applicant confirms that FX1 bacterial strain is subtilis (Bacillus subtilis), called after subtilis FX1 (Bacillussubtilis FX1).
Above-mentioned subtilis FX1 (Bacillus subtilis FX1) is preserved in the China typical culture collection center of Wuhan, China Wuhan University on November 12nd, 2014 by applicant, and deposit number is CCTCC NO:M2014563.
The application of embodiment 4 subtilis FX1 in control powdery mildew of strawberry
1, bacterium solution preparation
Subtilis FX1 is inoculated in nutrient broth medium, and cultivate 2 days for 30 DEG C, obtaining viable bacteria content is 10
8-10
9the bacterium liquid of cfu/ml.
2, using method
1) experimental group: three treatment group, each treatment group 10 ridge strawberry, sets up protection row by totally 1200 between each treatment group;
Come into bloom strawberry, after subtilis FX1 thin up 300,500,700 times, be evenly sprayed at From Strawberry Leaves respectively anyway on face and melon stem; 7 days, each sprinkling interval, is used in conjunction three times; The complete bacterium liquid of last sprinkling, after 10 days, checks the incidence of powdery mildew of strawberry.
2) control group: use not containing the nutrient broth medium of bacterium, other operations are all identical with experimental group.
Adopt the method identical with embodiment 2 to analyze experimental data, result is as shown in table 2:
Table 2 subtilis FX1 is to the prevention effect of powdery mildew of strawberry
As can be seen from the data of table 2, the subtilis FX1 that the present invention screens also effectively can prevent and treat powdery mildew of strawberry, and when bacterium liquid is diluted to 700 times, the prevent and treat efficiency of this bacterial strain to powdery mildew of strawberry still can higher than 92%, effect highly significant.
Embodiment 5 subtilis FX1 is preventing and treating the application in brown spot of grape
1, bacterium solution preparation
Subtilis FX1 is inoculated in nutrient broth medium, and cultivate 2 days for 30 DEG C, obtaining viable bacteria content is 10
8-10
9the bacterium liquid of cfu/ml.
2, using method
1) experimental group: three treatment group, each treatment group 2 ridge grape, sets up protection row by totally 100 between each treatment group;
Choose the growth grape seedlings of 3 years, coming into bloom, after subtilis FX1 thin up 300,500,700 times, be evenly sprayed at grape leave respectively anyway on face; 7 days, each sprinkling interval, is used in conjunction three times; The complete bacterium liquid of last sprinkling, after 10 days, checks the incidence of brown spot of grape.
2) control group: use not containing the nutrient broth medium of bacterium, other operations are all identical with experimental group.
Adopt the method identical with embodiment 2 to analyze experimental data, result is as shown in table 3:
Table 3 subtilis FX1 is to the prevention effect of brown spot of grape
As can be seen from the data of table 3, subtilis FX1 of the present invention can not only effectively prevent and treat powdery mildew of cucumber and powdery mildew of strawberry, its to the prevention effect of brown spot of grape also clearly, 300 times of diluents prevent and treat efficiency up to 83.2%.
Further, applicant is found by great many of experiments, except plant powdery mildew and brown spot, the subtilis FX1 that the present invention screens also all has obvious antagonistic action to by the yellow maize ear rot of sickle-like bacteria and the microbial plant of miliary damping-off and fruit rot, prevent and treat efficiency and all can arrive more than 80%, can controlling plant diseases be widely used in, have a extensive future.
Claims (6)
1. a subtilis, is characterized in that, deposit number is CCTCC NO:M2014563.
2. a biological prevention and control agent, is characterized in that, comprises subtilis according to claim 1.
3. the application of biological prevention and control agent according to claim 2 in control of plant disease.
4. apply as claimed in claim 3, it is characterized in that, described Plant diseases is any one in Powdery Mildew, brown spot, yellow maize ear rot and fruit rot.
5. apply as claimed in claim 3, it is characterized in that, described application biological prevention and control agent is diluted 0-700 doubly.
6. apply as claimed in claim 3, it is characterized in that, described application biological prevention and control agent is diluted 500-700 doubly.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104920478A (en) * | 2015-05-08 | 2015-09-23 | 长阳勤劳农夫农产品有限公司 | Spraying agent for controlling strawberry pathogenic bacteria |
| CN105145310A (en) * | 2015-09-23 | 2015-12-16 | 山东农业大学 | Integrated ecological cucumber cultivation method for reducing pest and disease damage and improving fruit quality |
| CN105145632A (en) * | 2015-07-20 | 2015-12-16 | 潘灿平 | Composite bacterial enzyme system with multiple effects and application thereof |
| CN105309245A (en) * | 2015-11-10 | 2016-02-10 | 江苏省农业科学院 | Method utilizing bacillus subtilis PTS-394 wettable powder for preventing and treating crop soil-borne disease |
| CN106942276A (en) * | 2017-03-16 | 2017-07-14 | 河北省农林科学院植物保护研究所 | Bacillus subtilis BAB 1 prevents and treats the application of cucurbits powdery mildew |
| CN110129235A (en) * | 2019-05-28 | 2019-08-16 | 青岛力力惠生物科技股份有限公司 | A kind of heat-resisting plant lactobacillus and its application in agricultural production |
| CN112195120A (en) * | 2020-09-27 | 2021-01-08 | 基因赛奥(大连)生物科技发展有限公司 | Biocontrol strain G2C3 for preventing and treating powdery mildew of strawberries and application thereof |
| CN113881588A (en) * | 2021-06-01 | 2022-01-04 | 河北科技师范学院 | Bacillus megaterium and application thereof |
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| CN101993836B (en) * | 2010-06-13 | 2012-05-23 | 河南省农业科学院 | Bacillus subtilis strain YB-81, fungicide and preparation method and application thereof |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104920478A (en) * | 2015-05-08 | 2015-09-23 | 长阳勤劳农夫农产品有限公司 | Spraying agent for controlling strawberry pathogenic bacteria |
| CN104920478B (en) * | 2015-05-08 | 2018-01-19 | 安徽勤劳农夫生态农业有限公司 | A kind of spray for controlling strawberry pathogenic bacteria |
| CN105145632A (en) * | 2015-07-20 | 2015-12-16 | 潘灿平 | Composite bacterial enzyme system with multiple effects and application thereof |
| CN105145632B (en) * | 2015-07-20 | 2018-03-23 | 潘灿平 | It is a kind of with the compound bacteria enzyme system of multiple effect and its application |
| CN105145310A (en) * | 2015-09-23 | 2015-12-16 | 山东农业大学 | Integrated ecological cucumber cultivation method for reducing pest and disease damage and improving fruit quality |
| CN105309245A (en) * | 2015-11-10 | 2016-02-10 | 江苏省农业科学院 | Method utilizing bacillus subtilis PTS-394 wettable powder for preventing and treating crop soil-borne disease |
| CN106942276A (en) * | 2017-03-16 | 2017-07-14 | 河北省农林科学院植物保护研究所 | Bacillus subtilis BAB 1 prevents and treats the application of cucurbits powdery mildew |
| CN110129235A (en) * | 2019-05-28 | 2019-08-16 | 青岛力力惠生物科技股份有限公司 | A kind of heat-resisting plant lactobacillus and its application in agricultural production |
| CN112195120A (en) * | 2020-09-27 | 2021-01-08 | 基因赛奥(大连)生物科技发展有限公司 | Biocontrol strain G2C3 for preventing and treating powdery mildew of strawberries and application thereof |
| CN113881588A (en) * | 2021-06-01 | 2022-01-04 | 河北科技师范学院 | Bacillus megaterium and application thereof |
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|---|---|
| CN104403970B (en) | 2016-10-19 |
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Effective date of registration: 20161214 Address after: Miao road Laoshan District 266061 Shandong city of Qingdao province Shandong No. 29 high building 12A07 Patentee after: Qingdao Vland Biotech Group Co., Ltd. Patentee after: Qingdao Hexie Biotechnology Co. Ltd. Address before: Miao road Laoshan District 266061 Shandong city of Qingdao province Shandong No. 29 high building 12A07 Patentee before: Qingdao Vland Biotech Group Co., Ltd. |
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Address after: 251700 west of Daji road and south of Balizhuang, Hefang sub district office, Huimin County, Binzhou City, Shandong Province Patentee after: SHANDONG WEILAN LVYUAN BIO-TECH Co.,Ltd. Patentee after: QINGDAO HEXIE BIOTECHNOLOGY Co.,Ltd. Patentee after: QINGDAO VLAND BIOTECH GROUP Co.,Ltd. Address before: 266061 Shandong 12A07 Shandong High Speed Building, 29 Miaoling Road, Laoshan District, Qingdao City, Shandong Province Patentee before: QINGDAO VLAND BIOTECH GROUP Co.,Ltd. Patentee before: QINGDAO HEXIE BIOTECHNOLOGY Co.,Ltd. Patentee before: SHANDONG WEILAN LVYUAN BIO-TECH Co.,Ltd. |
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