CN102816713B - Bacillus amyloliquefaciens and its application - Google Patents
Bacillus amyloliquefaciens and its application Download PDFInfo
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- CN102816713B CN102816713B CN201210192469.0A CN201210192469A CN102816713B CN 102816713 B CN102816713 B CN 102816713B CN 201210192469 A CN201210192469 A CN 201210192469A CN 102816713 B CN102816713 B CN 102816713B
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Abstract
The invention discloses a bacillus amyloliquefaciens. Its classification name is (Bacillus amyloliquefaciens)B293. The bacillus amyloliquefaciens is preserved at the common microorganisms center of the CCCCM. Its collection number is CGMCC No.6081 and its collection date is April, 28th, 2012. The bacillus amyloliquefaciens is mainly used for biological control of strawberry botrytis. The bacillus amyloliquefaciens B293 has an effect of inhibiting a plurality of pathogenic fungi on strawberries and poplars. The antagonistic bacterium has a good effect of preventing and treating other plant diseases, is rapid to reproduce, can be artificially cultured, is easy to operate and convenient for production and application, has strong resistance ability, and especially is of great significance for preventing and treating strawberry botrytis caused by fungi. The antagonistic bacterium requires a simple culture condition, is easy to preserve, is easy for industrial production, and has a good development and application prospect.
Description
Technical field
The invention belongs to plant protection field, relate in particular to a kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293 and at control grey mould fruit rot of strawberry, suppress the growth of strawberry anthrax-bacilus, suppress Botryodiplodia theo-bromae growth, suppress the growth of consor vesicle shell, suppress poplar Phomopsis bacteria growing, suppress the application in not high pair of spore bacteria growing.
Background technology
In recent years, along with the common concern of the public to chemical pesticide security in food and environment, and the forbidding of some dangerous chemical pesticides, impel people to seek plant disease control new way safely and effectively, utilize beneficial microorganism controlling plant diseases increasingly to come into one's own.It is in the plant natural ecosystem being broken, to introduce to plant useful external source Antagonistic Fungi or by adjusting ambient to impel natural beneficial microorganism colony to increase, and shows biological and ecological methods to prevent plant disease, pests, and erosion activity, by the interaction between microorganism, reaches diseases prevention object.At present, the biological and ecological methods to prevent plant disease, pests, and erosion of Plant diseases has become various countries' research emphasis.
The Microbial resources abundant species that nature is potential, aspect the product development of microbial pesticide, having a clear superiority in, therefore a large amount of manpower and financial resources is all dropped in countries in the world, research, exploitation have the novel microorganism agricultural chemicals of independent intellectual property right, and drive the development in an all-round way of other high-tech industries with this.China also classifies the development of microbial pesticide and exploitation as state key tackling of key scientific and technical problems and hi-tech development plan as, and Jiangsu Province government is an important component part using microbial pesticide initiative as biological husbantry also.
Biological pesticide (biopesticides), according to the definition of Copping and Menn, comprised living microorganism (fungi, bacterium and virus), insect is planted disease nematode, botanical pesticide, microbial pesticide, secondary metabolites from microorganisms (microbiotic) etc., be used for preventing and treating various insects and disease, it is little, environmentally safe to human health threat that biological pesticide (particularly microbial pesticide) has unique advantage, one of this problem that whole society is concerned about most just.Therefore, since entering 21 century, national governments and the common people pay attention to the development and application of biological pesticide more, have become study hotspot.
The characteristic of microbial pesticide has been given agricultural sustainable development strategy, for protection agroecological environment, ensures food safety and provides material base and technical support; Microbial pesticide industry is also one of new growing point having in the future economy development potentiality simultaneously.The policy system that promotes microbial pesticide industry development is formulated in the industry development of microorganisms agricultural chemicals for government department's science, realize development proposal adjustment, promote recycling economy and sustainable society development strategy, ensure the coordinated development of our province economic construction and agroecological environment, and build our province farm produce safety system etc. and all will there is very important theory significance and practice significance.
Strawberry (Fragaria ananassa Duchesne) is a kind of perennial root, perennial herb resource plant that integrates cultivation and view and admire.In cultivated strawberry district, gray mold, anthrax and verticillium are to hinder Strawberry industry development bottleneck.Wherein the harm of grey mould fruit rot of strawberry (Botrytis cinerea Pers.ex Fr.) is the most serious, annual strawberry, endangers stem, petiole, blade vegetative period, and stipule, at later stage harm flower and fruit, directly has influence on Yield of Strawberry, causes very large financial loss.To the tradition control of this disease, mostly adopt chemical agent to spray plant surface, cause soil pesticide residue and pollution problem in various degree, affect the healthy of quality product and people.From preserving the ecological environment and the angle of food safety, strawberry production need to have low toxicity, it is few to pollute, and the microbial preparation of human-body safety is controlled to Diseases of Strawberry, reached the object of strawberry safe edible.Also meet the development of biological husbantry development and government, society and biological pesticide industry.
Summary of the invention
Goal of the invention: in order to overcome the deficiencies in the prior art, the first object of the present invention is to provide a kind of bacillus amyloliquefaciens Botrytis cinerea bacterium to antagonistic action.
The second object of the present invention is to provide a kind of bacillus amyloliquefaciens to grow, suppress consor vesicle shell and grow, suppress the application in poplar Phomopsis bacteria growing, not high pair of spore bacteria growing of inhibition at control grey mould fruit rot of strawberry, the growth of inhibition strawberry anthrax-bacilus, inhibition Botryodiplodia theo-bromae.
Technical scheme: in order to solve above-mentioned the first object, the technical solution adopted in the present invention is: a kind of many bacillus amyloliquefaciens, its Classification And Nomenclature is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293, (abbreviation CGMCC) preservation at China Committee for Culture Collection of Microorganisms's common micro-organisms center, depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode is 100101, its deposit number is: CGMCC No.6081, preservation date is on 04 28th, 2012.
In order to solve the second above-mentioned object, the technical solution adopted in the present invention is:
The application of described bacillus amyloliquefaciens in control grey mould fruit rot of strawberry.
The application of described bacillus amyloliquefaciens in suppressing the growth of strawberry anthrax-bacilus.
The application of described bacillus amyloliquefaciens in suppressing Botryodiplodia theo-bromae growth.
The application of described bacillus amyloliquefaciens in suppressing the growth of consor vesicle shell.
The application of described bacillus amyloliquefaciens in suppressing poplar Phomopsis bacteria growing.
The application of described bacillus amyloliquefaciens in suppressing not high pair of spore bacteria growing.
Have particular application as: bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293 is inoculated in fermention medium, at 28 ℃ of-35 ℃ of bottom fermentations, cultivates 48-72 hour, the fermented liquid obtaining is as microbial inoculum.
The formula of fermention medium described in the present invention is: 1000mL distilled water, extractum carnis 2-5g, peptone 5-10g, sodium-chlor 4-5g, pH6.5-7.5.
The physiologically active feature of bacillus amyloliquefaciens described in the present invention (Bacillus amyloliquefaciens) B293 is as follows:
Described bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293 bacterium colony on beef-protein medium flat board is larger, oyster white, the general 4-6mm of diameter, surface elevation, dry, have gauffer, edge is more regular, and thalline is shaft-like, the blunt circle in two ends, tool peritrichous, size is 1.59-4.25 * 0.60-0.72 μ m, average 2.77 * 0.67 μ m; Gemma two ends are raw, ellipse, and size is 0.59-1.01 * 0.40-0.61 μ m, average 0.84 * 0.50 μ m.
Gramstaining shows as the positive; Casein hydrolysis, Starch Hydrolysis, gelatine liquefication are positive, and V.P reaction is variable, and indole reaction is negative; Carbohydrate acidogenic fermentation.
The 16SrDNA sequence of bacillus amyloliquefaciens is as shown in SEQ ID No.1.Surveyed 16SrDNA sequence is compared by BLAST, identified that it is many bacillus amyloliquefaciens (Bacillus amyloliquefaciens).
Beneficial effect: compared with prior art, advantage of the present invention is:
(1) bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293 is inhibited to various plants pathogenic bacteria, plays very important effect in to biocontrol of plant disease, for example kind and One's name is legion at antagonistic bacterium; Mode of action variation to pathogenic bacteria; Reproduction speed is fast; Can artificial culture, easy handling, is convenient to production application; The advantages such as high-output stress-resistance, especially significant to the control of fungus-caused grey mould fruit rot of strawberry; Also be to guarantee to eat raw fruit security-critical means.
(2) bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293 culture condition simply, is easily preserved, and is easy to suitability for industrialized production, has good development prospect.
Accompanying drawing explanation
Fig. 1 a is the aspect graph of bacillus amyloliquefaciens in the present invention (Bacillus amyloliquefaciens) B293;
Fig. 1 b is bacillus amyloliquefaciens in the present invention (Bacillus amyloliquefaciens) B293 gemma aspect graph (at one end or two ends refractive body)
Fig. 2 a is that bacillus amyloliquefaciens in the present invention (Bacillus amyloliquefaciens) B293 is to Botrytis cinerea bacterium Botrytis cinera Pers.ex Fr.) restraining effect (10 days);
Fig. 2 b is the restraining effect (10 day) of bacillus amyloliquefaciens in the present invention (Bacillus amyloliquefaciens) B293 to strawberry anthrax-bacilus Colletotrichum fragariae A.N.Brooks;
Fig. 2 c is that bacillus amyloliquefaciens in the present invention (Bacillus amyloliquefaciens) B293 is to the Botryodiplodia theo-bromae [restraining effect (10 days) of Lasiodiplodia theobromae (Pat.) Griffon & Maubl.;
Fig. 2 d is the restraining effect (10 day) of bacillus amyloliquefaciens in the present invention (Bacillus amyloliquefaciens) B293 to consor vesicle shell (Dothiorella gregaria Sacc.);
Fig. 2 e is that bacillus amyloliquefaciens in the present invention (Bacillus amyloliquefaciens) B293 is to the restraining effect of poplar Phomopsis bacterium (Phomopsis sp.) (10 days);
Fig. 2 f is the restraining effect (10 day) of bacillus amyloliquefaciens Bacillus amyloliquefaciens B293 to not high pair of spore bacterium [Apiocarpella macrospore (Speg.) H. & P.Syd] in the present invention;
Fig. 3 a is the control test (note: 4 strains on both sides are to process strawberry with B289 of bacillus amyloliquefaciens in the present invention (Bacillus amyloliquefaciens) B293; Middle 4 strains are control treatment);
Fig. 3 b is that bacillus amyloliquefaciens in the present invention (Bacillus amyloliquefaciens) B293 bacterium liquid is processed strawberry.
Embodiment
Below in conjunction with experimental example, elaborate the application of bacillus amyloliquefaciens of the present invention (Bacillus amyloliquefaciens) B293.
Experimental example 1: bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293 is to Botrytis cinerea bacterium (Botrytis cinera Pers.ex Fr.), strawberry anthrax-bacilus (Colletotrichum fragariae A.N.Brooks), Botryodiplodia theo-bromae [Lasiodiplodia theobromae (Pat.) Griffon & Maubl.], consor vesicle shell (Dothiorella gregaria Sacc.), the dull and stereotyped bacteriostatic action of poplar Phomopsis bacterium (Phomopsis sp.) and not high pair of spore bacterium [Apiocarpella macrospore (Speg.) H. & P.Syd].
To use bacillus amyloliquefaciens (Bacillus amyloliquefaciens) the B293 bacterial strain streak inoculation of LB slant activation in the dull and stereotyped side of PDA, cause germ (4 kinds cause the germ of willow ulcer to be: Botryodiplodia theo-bromae, consor vesicle shell, poplar Phomopsis bacterium and not high pair of spore bacterium) the PDA plate culture of willow ulcer to beat and get homogeneity with the bacterium piece of amount with the punch tool of diameter 5mm Botrytis cinerea bacterium, anthrax-bacilus and 4 kinds again, be inoculated in dull and stereotyped opposite side.Cultivate for 28 ± 1 ℃ and observe bacteriostasis in 10 days, and record its width (as table 1), experiment effect is as shown in Fig. 2 a to Fig. 2 f.Test-results shows, bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293 all has antagonistic action to 6 kinds of pathogenic bacterias, to occupy the diameter judgement of the culture dish effect of drawing up, with aseptic PDA nahlock in contrast.
Table 1 B293 is to 6 kinds of strawberries and the willow pathogenic bacteria restraining effect of 10 days
Experimental example 2: bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293 meta-bolites is to strawberry anthrax-bacilus (Colletotrichum fragariae A.N.Brooks), Botrytis cinerea bacterium (Botrytis cinera Pers.ex Fr.), Botryodiplodia theo-bromae [Lasiodiplodia theobromae (Pat.) Griffon & Maubl.], consor vesicle shell (Dothiorella gregaria Sacc.), the dull and stereotyped bacteriostatic action of poplar Phomopsis bacterium (Phomopsis sp.) and not high pair of spore bacterium [Apiocarpella macrospore (Speg.) H. & P.Syd].
The fermented liquid of cultivating bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293 of 3 days filters with bacterial filter, and the filter membrane aperture of bacterial filter is 0.25 μ m; The PDA plate culture of 6 kind of plant pathogenic fungies is beaten and got homogeneity with the bacterium piece of amount with the punch tool of diameter 5mm, be inoculated in the dull and stereotyped side of PDA; Again the dull and stereotyped opposite side of PDA is beaten and got homogeneity with the nahlock of amount with the punch tool of diameter 5mm, and by nahlock point of draw; The 100 μ L bacteria-free filtrates of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293 after filtering are connected in the dull and stereotyped opposite side of PDA cavity, cultivate 10 days for 28 ℃, observe.Take does not deliver a child fungi-proofingly covers with after flat board wait contrasting mycelia as contrast, observes antagonistic activity (in Table 2).Test-results shows, bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293 meta-bolites all has strong antagonistic action to 6 kinds of pathogenic bacterias.
Table 2B293 is to 6 kinds of strawberries and the willow pathogenic bacteria restraining effect of 10 days
Experimental example 3: whether checking bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293 has inhibiting test to Botrytis cinerea bacterium.
1, experimental installation: incubator, shaking table, Bechtop;
2, experimental principle:
Before cultivated strawberry, strawberry root is placed in the nutrient solution of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293, take out, drain, bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293 can stick to the continued growth on strawberry root, produce meta-bolites, thereby suppress the growth of Botrytis cinerea bacterium.
3, experimental technique:
1) cultivation of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293 bacterium liquid: bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293 is inoculated in the triangular flask of the LB substratum that contains 50mL, 120 turn, and cultivate 60 hours for 35 ℃.
2) the carry disease germs preparation of strawberry root: soak to take out for approximately 2 minutes and dry to till not dripping selecting to put into cultured bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293 bacterium liquid for the Strawberry Seedlings of cultivation, cultivate.
3) preparation of contrast strawberry root: soak to take out for approximately 2 minutes and dry to till not dripping selecting to put into sterilized water for the Strawberry Seedlings of cultivation, cultivate.
4) sprinkling of B293 fermenting agent: after Strawberry Seedlings cultivation survives in 1 week, with B293 fermenting agent spray solution strawberry, wherein B293 fermenting agent solution is that to add the water of 10 times of stoste weight in B293 fermenting agent stoste formulated.
5) antagonistic experiment: by step 2) with step 3) in soaked Strawberry Seedlings dry to till not dripping, carrying out respectively booth plants, within 4 weeks, carry out observations as shown in Fig. 3 a to Fig. 3 b, in Fig. 3 a, show, the right and left has the eugonic large Strawberry Seedlings of 7 strain to process with B293, middle 3 strains contrasts, above 1 strain growth very little of a line, below a line two strain deads position of remaining heatable adobe sleeping platform only.
4, experimental result
1) effect of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293 to Botrytis cinerea bacterium: as shown in Figure 2 a;
2) effect of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293 to strawberry anthrax-bacilus: as shown in Figure 2 b;
3) effect of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293 to Botryodiplodia theo-bromae: as shown in Figure 2 c;
4) effect of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293 to consor vesicle shell: as shown in Figure 2 d;
5) restraining effect of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293 to poplar Phomopsis bacterium (Phomopsis sp.); As shown in Figure 2 e;
6) restraining effect of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293 to not high pair of spore bacterium [Apiocarpella macrospore (Speg.) H. & P.Syd]: as shown in Fig. 2 f.
From picture above, can find out, bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293 has good restraining effect to the growth of Botrytis cinerea bacterium, anthrax-bacilus and 4 kinds of dothiorella gregaria bacterium.
Specific embodiments of the invention below:
Embodiment 1: the preparation method of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293 microbial inoculum: bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293 is inoculated in fermention medium, pH6.5, in 250ml triangular flask, liquid amount is 150ml, 28 ℃-35 ℃, 120 revs/min of shaking speed, 48 hours, the fermented liquid obtaining was fermenting agent.
Wherein the formula of fermention medium is: 1000mL distilled water, extractum carnis 2g, peptone 5g, sodium-chlor 5g, pH6.5.
Embodiment 2: the preparation method of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293 microbial inoculum: bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293 is inoculated in fermention medium, pH7.5, in 250ml triangular flask, liquid amount is 150ml, 28 ℃-35 ℃, 160 revs/min of shaking speed, 72 hours, the fermented liquid obtaining was fermenting agent.
Wherein the formula of fermention medium is: 1000mL distilled water, extractum carnis 5g, peptone 10g, sodium-chlor 5g, pH7.5.
Embodiment 3: the preparation method of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293 microbial inoculum: bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293 is inoculated in fermention medium, pH 7, in 250ml triangular flask, liquid amount is 150ml, 28 ℃-35 ℃, 150 revs/min of shaking speed, 55 hours, the fermented liquid obtaining was fermenting agent.
Wherein the formula of fermention medium is: 1000mL distilled water, extractum carnis 3g, peptone 8g, sodium-chlor 5g, pH7.
Claims (9)
1. a bacillus amyloliquefaciens B293, its Classification And Nomenclature is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is: CGMCC No.6081, preservation date is on 04 28th, 2012.
2. the application of bacillus amyloliquefaciens B293 claimed in claim 1 in control grey mould fruit rot of strawberry.
3. the application of bacillus amyloliquefaciens B293 claimed in claim 1 in suppressing the growth of strawberry anthrax-bacilus.
4. the application of bacillus amyloliquefaciens B293 claimed in claim 1 in suppressing Botryodiplodia theo-bromae growth.
5. the application of bacillus amyloliquefaciens B293 claimed in claim 1 in suppressing the growth of consor vesicle shell.
6. the application of bacillus amyloliquefaciens B293 claimed in claim 1 in suppressing poplar Phomopsis bacteria growing.
7. the application of bacillus amyloliquefaciens B293 claimed in claim 1 in suppressing not high pair of spore bacteria growing.
8. according to the application described in any one in claim 2 to 7, it is characterized in that, bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B293 is inoculated in fermention medium, at 28 ℃ of-35 ℃ of bottom fermentations, cultivates 48-72 hour, the fermented liquid obtaining is as microbial inoculum.
9. application according to claim 8, is characterized in that, the formula of described fermention medium is: 1000mL distilled water, extractum carnis 2-5g, peptone 5-10g, sodium-chlor 4-5g, pH6.5-7.5.
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| CN103602606B (en) * | 2013-06-24 | 2015-02-18 | 江苏农林职业技术学院 | Bacillus amyloliquefaciens and application thereof in preventing and treating of plant fruit rot diseases |
| CN104450546A (en) * | 2013-11-28 | 2015-03-25 | 辽宁省农业科学院 | SY-17 strain capable of preventing and controlling pepper soil-borne diseases caused by fusarium and biologically controllable microbial agent thereof |
| CN104371944B (en) * | 2014-01-26 | 2017-11-03 | 北京绿环国际科技有限公司 | One bacillus amyloliquefaciens and its application |
| CN104560837B (en) * | 2015-02-02 | 2017-10-03 | 青岛蔚蓝生物集团有限公司 | A kind of bacillus amyloliquefaciens and its application |
| CN105532735A (en) * | 2015-11-21 | 2016-05-04 | 安徽省农业科学院植物保护与农产品质量安全研究所 | Bactericidal and insecticidal composition containing Bacillus amyloliquefaciens and matrine |
| CN105439657B (en) * | 2015-12-17 | 2018-08-24 | 山西省农业科学院生物技术研究中心 | A kind of preparation method of the special biologic organic fertilizer of resisting repeated stubbles of strawberry |
| CN108795827B (en) * | 2018-07-06 | 2020-08-18 | 四川农业大学 | Bacillus megaterium BM22, and water dispersible granule and application thereof |
| CN112458011B (en) * | 2020-11-23 | 2022-05-13 | 江苏科技大学 | Bacillus amyloliquefaciens ZJK1 and application thereof |
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