CN104232514A - Bacillus subtilis and application thereof - Google Patents
Bacillus subtilis and application thereof Download PDFInfo
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- CN104232514A CN104232514A CN201410398397.4A CN201410398397A CN104232514A CN 104232514 A CN104232514 A CN 104232514A CN 201410398397 A CN201410398397 A CN 201410398397A CN 104232514 A CN104232514 A CN 104232514A
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Abstract
The invention discloses bacillus subtilis. The bacillus subtilis with the preservation number of CGMCC No.7542 is preserved in the China General Microbiological Culture Collection Center on April 28, 2013. The invention further provides an application of the bacillus subtilis in strawberry root rot. The bacillus subtilis B85 has a very good inhibition effect on strawberry root rot, grey mould fruit rot of strawberry, strawberry anthracnose and other pathogenic fungi, and also has a very good preventing and controlling effect on other plant diseases. The bacillus subtilis has a high proliferation speed, can be artificially cultured, is easy to operate, facilitates production and application, has the advantages of strong stress resistance and the like, especially has an important meaning to prevention and control of fungi caused strawberry root rot, is simple in culture condition and easy to preserve, is convenient for industrial production, and has excellent development and application prospects.
Description
Technical field
The invention belongs to plant protection art, particularly relate to a kind of subtilis (Bacillus subtilis) B85 and application thereof.
Background technology
In recent years, along with the public is to the common concern of chemical pesticide security in food and environment, and the forbidding of some dangerous chemical agricultural chemicals, 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 introduces to impel natural beneficial microorganism colony to increase to the useful external source Antagonistic Fungi of plant or by adjusting ambient in the plant natural ecosystem be broken, and show biological and ecological methods to prevent plant disease, pests, and erosion activity, reaches diseases prevention object by the interaction between microorganism.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, have a clear superiority in the product development of microbial pesticide, 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 is also classified as the Study and Development of microbial pesticide as state key tackling of key scientific and technical problems and hi-tech development plan, and government of Jiangsu Province is also using the important component part of microbial pesticide initiative as biological husbantry.
Biological pesticide (biopesticides), contain living microorganism (fungi, bacterium and virus) according to the definition of Copping and Menn, insect plant disease nematode, botanical pesticide, microbial pesticide, secondary metabolites from microorganisms (microbiotic) etc., for preventing and treating various insect and disease; It is threaten little to human health that biological pesticide (particularly microbial pesticide) has unique advantage, safer to environment, and this is one of problem of being concerned about most of the whole society just.Therefore, since entering 21 century, national governments and plant protection service person pay attention to the development and application of biological pesticide more, become study hotspot.
The characteristic of microbial pesticide imparts agricultural sustainable development strategy, is protecting agriculture ecotope, ensures food safety and provide material base and technical support; Microbial pesticide industry is also one of new growing point with development potentiality in the future economy simultaneously.The policy system promoting 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 have very vital role.
Strawberry (Fragaria ananassa Duchesne) be a kind of integrate cultivation and view and admire perennial root, per nnial herb.In cultivated strawberry district, root rot, gray mold and anthrax hinder Strawberry industry development bottleneck.Wherein the harm of Strawberry Root Rot (Fusarium oxsporum) is the most serious, causes butt rot, strawberry directly can be caused dead, cause very large financial loss.Mostly adopt chemical agent to spray plant surface to the control of this disease, cause soil pesticide residue in various degree and pollution problem, affect the healthy of quality product and people.From preserving the ecological environment and the angle of food safety, strawberry production needs low toxicity, it is few to pollute, and controls Diseases of Strawberry, reached the object of strawberry safe edible to the microbial preparation of human-body safety.Also the development of biological husbantry development and government, social and biological pesticide industry is met.
Summary of the invention
Goal of the invention: in order to overcome the deficiency of the existing Prevention Technique of Strawberry Root Rot, the first object of the present invention is to provide a kind of subtilis (Bacillus subtilis) B85 Pestalotiopsis photiniae to antagonistic action.
The second object of the present invention is the application providing a kind of subtilis B85.
Technical scheme: in order to solve above-mentioned first object, the technical solution adopted in the present invention is: a kind of subtilis, its Classification And Nomenclature is subtilis (Bacillus subtilis), in China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC) preservation, 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.7542, and preservation date is on 04 28th, 2013.
In order to solve the second above-mentioned object, the technical solution adopted in the present invention is: the application of described subtilis B85 in control Strawberry Root Rot.
Have particular application as: be inoculated in liquid fermentation medium by subtilis (Bacillus subtilis) B85, cultivate 48-72 hour at 28 DEG C of-35 DEG C of bottom fermentations, the fermented liquid obtained is as the microbial inoculum of control Pestalotiopsis photiniae.
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 application of subtilis (Bacillus subtilis) B85 that present invention also offers in control grey mould fruit rot of strawberry.
Have particular application as: be inoculated in liquid fermentation medium by subtilis (Bacillus subtilis) B85, cultivate 48-72 hour at 28 DEG C of-35 DEG C of bottom fermentations, the fermented liquid obtained is as the microbial inoculum of control Pestalotiopsis photiniae.
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 application of subtilis (Bacillus subtilis) B85 that present invention also offers in control Strawberry anthracnose.
Have particular application as: be inoculated in liquid fermentation medium by subtilis (Bacillus subtilis) B85, cultivate 48-72 hour at 28 DEG C of-35 DEG C of bottom fermentations, the fermented liquid obtained is as the microbial inoculum of control Strawberry anthracnose.
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 application of subtilis (Bacillus subtilis) B85 that present invention also offers in control Herba Dendrobii gray mold.
Have particular application as: be inoculated in liquid fermentation medium by subtilis (Bacillus subtilis) B85, cultivate 48-72 hour at 28 DEG C of-35 DEG C of bottom fermentations, the fermented liquid obtained is as the microbial inoculum of control Herba Dendrobii gray mold.
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.
Subtilis (Bacillus subtilis) B85 that present invention also offers is preventing and treating the application in wheat scab.
Have particular application as: be inoculated in liquid fermentation medium by subtilis (Bacillus subtilis) B85, cultivate 48-72 hour at 28 DEG C of-35 DEG C of bottom fermentations, the fermented liquid obtained is as the microbial inoculum preventing and treating wheat scab.
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 subtilis (Bacillus subtilis) B85 described in the present invention is as follows:
Described subtilis (Bacillus subtilis) B85 bacterium colony on beef-protein medium flat board is larger, oyster white, the general 4-6mm of diameter, surface elevation, has gauffer, edge is more regular, thalline is shaft-like, the blunt circle in two ends, tool peritrichous, size is 2.5735-4.78 × 0.53-0.80 μm, average 3.35 × 0.70 μm; Gemma two ends are raw, and oval, size is 0.45-0.69 × 0.46-0.62 μm, average 0.59 × 0.55 μm.
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 subtilis is as shown in SEQ ID No.1.By surveyed 16SrDNA sequence by BLAST comparison, be accredited as subtilis (Bacillus subtilis).
Beneficial effect: compared with prior art, advantage of the present invention is:
(1) subtilis (Bacillus subtilis) B85 is inhibited to various plants pathogenic bacteria, plays very important effect, such as kind and One's name is legion in antagonistic bacterium is to biocontrol of plant disease; To the mode of action variation of 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 Strawberry Root Rot; Also be ensure Chinese medicine security-critical means.
(2) subtilis (Bacillus subtilis) B85 culture condition simply, is easily preserved, and is easy to suitability for industrialized production, has good development prospect.
Accompanying drawing explanation
The aspect graph of subtilis (Bacillus subtilis) B85 in Fig. 1 a the present invention;
Fig. 1 b is subtilis (B.subtilis) B85 gemma aspect graph in the present invention (at one end or two ends have refractive body);
Fig. 2 a be in the present invention subtilis (B.subtilis) B85 to the restraining effect of Pestalotiopsis photiniae (Fusarium oxsporum);
Fig. 2 b be in the present invention subtilis (B.subtilis) B85 to the restraining effect of Botrytis cinerea germ (Botrytis fabae);
Fig. 2 c be in the present invention subtilis (B.subtilis) B85 to the restraining effect of Strawberry anthracnose bacterium (Colletotrichum gloeosporiodes);
Fig. 2 d be in the present invention subtilis (B.subtilis) B85 to the restraining effect of Herba Dendrobii ash arrhizus bacteria (Botryotinia fuckeliana);
Fig. 2 e be in the present invention subtilis (B.subtilis) B85 to the restraining effect of fusarium graminearum (Fusarium asiaticum);
Fig. 3 a adopts subtilis (B.subtilis) B85 to the controlling experiment of Strawberry Root Rot in the present invention.
Fig. 3 b is the strawberry (contrast) not adopting the process of subtilis (B.subtilis) B85 bacterium liquid in the present invention.
Embodiment
The application of subtilis of the present invention (Bacillus subtilis) B85 is elaborated below in conjunction with experimental example.
Experimental example 1
Subtilis (Bacillus subtilis) B85 is to the dull and stereotyped bacteriostatic action of Strawberry Root Rot (Fusarium oxsporum), Botrytis cinerea germ (Botrytis fabae), Strawberry anthracnose bacterium (Colletotrichum gloeosporiodes), Herba Dendrobii ash arrhizus bacteria (Botryotinia fuckeliana) and fusarium graminearum (Fusarium asiaticum).
By subtilis (Bacillus subtilis) the B85 bacterial strain streak inoculation by LB slant activation in the dull and stereotyped side of 90mm PDA, again the punch tool of Pestalotiopsis photiniae, Botrytis cinerea germ, Strawberry anthracnose bacterium, Herba Dendrobii ash arrhizus bacteria and fusarium graminearum PDA plate culture diameter 5mm is beaten and get the bacterium block of homogeneity with amount, be inoculated in dull and stereotyped opposite side.Cultivate 14 days and 28 days for 30 ± 1 DEG C, observe bacteriostasis, and record the width (as table 1) of inhibition zone, experiment effect is as shown in Fig. 2 a to Fig. 2 e.Test-results shows, subtilis (Bacillus subtilis) B85 all has good antagonistic action to 5 kinds of pathogenic bacterias, judges to draw up effect with the diameter occupying culture dish, with aseptic PDA nahlock in contrast.
Subtilis (Bacillus subtilis) B85 and different pathogenic fungies carry out after antagonistic experiment cultivates 28 days, inhibition zone width different (table 2) therebetween.
Table 1 B85 is to the restraining effect of Pestalotiopsis photiniae and Botrytis cinerea cause of disease bacterium 14 days and 28 days
Note: the numeral in table 1 gets the mean value of 5 PDA flat boards
Experimental example 2: the dull and stereotyped bacteriostatic action of subtilis (Bacillus subtilis) B85 meta-bolites Herba Hyperici elodeoidis certain kind of berries root rot (Fusarium oxsporum), Botrytis cinerea bacterium (Botrytis cinerea), strawberry anthrax-bacilus (Colletotrichum gloeosporiodes), fusarium graminearum (Fusarium asiaticum).
The fermented liquid bacterial filter cultivating subtilis (Bacillus subtilis) B85 of 3 days filters, and the filter sizes of bacterial filter is 0.25 μm; The punch tool of PDA flat board (diameter 90mm) the culture diameter 5mm of 5 kind of plant pathogenic fungies is beaten and gets the bacterium block of homogeneity with amount, be inoculated in the dull and stereotyped side of PDA; Again the punch tool of PDA dull and stereotyped opposite side diameter 5mm is beaten and get the nahlock of homogeneity with amount, and nahlock is taken out; The 100 μ L bacteria-free filtrates of subtilis (Bacillus subtilis) B85 after filtration are connected in the dull and stereotyped opposite side cavity of PDA again, cultivate 14 days and 28 days for 30 ± 1 DEG C, observe the fungistatic effect of B85.Fungi-proofing for contrast not deliver a child, after contrast mycelia covers with flat board, observe antagonistic activity (see table 2).Test-results shows, subtilis (Bacillus subtilis) B85 meta-bolites all has strong antagonistic action to 5 kinds of pathogenic bacterias.
Table 2 B85 fermented liquid is to the restraining effect of 5 kinds of Pestalotiopsis photiniae and Botrytis cinerea cause of disease bacterium 14 days and 28 days
Fig. 2 a is that subtilis (Bacillus subtilis) B85 fermented liquid is to the restraining effect of Pestalotiopsis photiniae (Fusarium oxsporum);
Fig. 2 b subtilis (Bacillus subtilis) B85 fermented liquid is to the restraining effect of Botrytis cinerea germ (Botrytis fabae);
Fig. 2 c subtilis (Bacillus subtilis) B85 fermented liquid is to the restraining effect of Strawberry anthracnose bacterium (Colletotrichum gloeosporiodes);
Fig. 2 d subtilis (Bacillus subtilis) B85 fermented liquid is to the restraining effect of Herba Dendrobii ash arrhizus bacteria (Botryotinia fuckeliana);
Fig. 2 e subtilis (Bacillus subtilis) B85 fermented liquid is to the restraining effect of fusarium graminearum (Fusarium asiaticum).
Experimental example 3: whether checking subtilis (Bacillus subtilis) B85 has inhibiting test to Pestalotiopsis photiniae.
1, experimental installation
Bechtop, PRX-250A type intelligence growth cabinet, training English board DHZ-DA TCYQ constant temperature oscillator.
2, experimental principle
Before cultivated strawberry, after the root clear water of Strawberry Seedlings is cleaned, drying, be placed in the diluent of subtilis (B.subtilis) B85 and soak 2-3 minute, take out airing, subtilis B85 can stick to continued growth breeding on strawberry root, produce meta-bolites, thus suppress the growth of Pestalotiopsis photiniae.
3, experimental technique:
1) cultivation of subtilis (B.subtilis) B85 bacterium liquid: be inoculated into by subtilis (B.subtilis) B85 in the triangular flask of the LB substratum containing 500mL, cultivates 60 hours for 35 DEG C by 120 turns.
2) to carry disease germs the preparation of strawberry root: the Strawberry Seedlings of cultivation is put into subtilis (B.subtilis) B85 bacterium liquid (2.2 hundred million thalline/mL) prepared and soak about 2-3 minute, take out and dry (root table face is anhydrous) and cultivate.
3) contrast: Strawberry Seedlings is put into sterilized water and soak to take out for about 2 minutes and dry and cultivate.
4) antagonistic experiment: by step 2) with step 3) in soaked Strawberry Seedlings dry to not dripping, plant in pattern plastic warmhouse booth respectively, within 8 weeks, carry out observations as shown in Fig. 3 a to Fig. 3 b.
Result shows: please make text description to result.
Here is specific embodiments of the invention
Embodiment 1: the preparation method of subtilis (Bacillus subtilis) B85 microbial inoculum: subtilis (B.subtilis) B85 is inoculated in fermention medium, in 500mL triangular flask, liquid amount is 300mL, 28 DEG C-35 DEG C, shaking speed 120 revs/min, 48 hours, the fermented liquid obtained was the original bacteria liquid of control Strawberry Root Rot.
Wherein the formula of fermention medium is: 1000mL distilled water, extractum carnis 2g, peptone 5g, sodium-chlor 4g, pH6.5.
Embodiment 2: the preparation method of subtilis (B.subtilis) B85 microbial inoculum: subtilis B85 is inoculated in fermention medium, in 500mL triangular flask, liquid amount is 300mL, 28 DEG C-35 DEG C, shaking speed 160 revs/min, 72 hours, the fermented liquid obtained was the original bacteria liquid of control Strawberry Root Rot.
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 subtilis (B.subtilis) B85 microbial inoculum: subtilis B85 is inoculated in fermention medium, in 500mL triangular flask, liquid amount is 300mL, 28 DEG C-35 DEG C, shaking speed 150 revs/min, 55 hours, the fermented liquid obtained was the original bacteria liquid of control Strawberry Root Rot.
Wherein the formula of fermention medium is: 1000mL distilled water, extractum carnis 3g, peptone 8g, sodium-chlor 5g, pH7.
Claims (8)
1. a subtilis, its Classification And Nomenclature is subtilis (Bacillus subtilis), be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is: CGMCC No.7542, and preservation date is on 04 28th, 2013.
2. subtilis according to claim 1 is preventing and treating the application in Strawberry Root Rot.
3. apply as claimed in claim 2, it is characterized in that: subtilis (Bacillus subtilis) is inoculated in fermention medium, cultivate 48-72 hour at 28 DEG C of-35 DEG C of bottom fermentations, the fermented liquid obtained is as the microbial inoculum of control Strawberry Root Rot.
4. apply as claimed in claim 3, it 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, pH 6.5-7.5.
5. the application of subtilis according to claim 1 in control grey mould fruit rot of strawberry.
6. the application of subtilis according to claim 1 in control Strawberry anthracnose.
7. the application of subtilis according to claim 1 in control Herba Dendrobii gray mold.
8. subtilis according to claim 1 is preventing and treating the application in wheat scab.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834195A (en) * | 2017-04-06 | 2017-06-13 | 中国科学院过程工程研究所 | A kind of screening of anti-booth vegetable grey mold bacillus subtilis and bacterial preparation process |
| CN108424860A (en) * | 2017-12-21 | 2018-08-21 | 宜春学院 | One bacillus subtilis and its screening technique and its application |
| CN109234210A (en) * | 2018-11-19 | 2019-01-18 | 江苏科技大学 | For the bacillus subtilis of antagonism grey mould fruit rot of strawberry and its preparation of biocontrol agent and application |
| CN109593682A (en) * | 2018-12-29 | 2019-04-09 | 贵州大学 | Bacillus subtilis GU MT332 and its application |
| CN113151092A (en) * | 2021-04-25 | 2021-07-23 | 中国热带农业科学院热带生物技术研究所 | Bacillus new species with strawberry anthracnose inhibition activity and application thereof |
| CN113583914A (en) * | 2021-08-27 | 2021-11-02 | 微生物肥料技术研究推广中心 | Microbial powder for preventing and treating strawberry root rot and preparation method thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834195A (en) * | 2017-04-06 | 2017-06-13 | 中国科学院过程工程研究所 | A kind of screening of anti-booth vegetable grey mold bacillus subtilis and bacterial preparation process |
| CN108424860A (en) * | 2017-12-21 | 2018-08-21 | 宜春学院 | One bacillus subtilis and its screening technique and its application |
| CN108424860B (en) * | 2017-12-21 | 2021-04-09 | 宜春学院 | Bacillus subtilis and screening method and application thereof |
| CN109234210A (en) * | 2018-11-19 | 2019-01-18 | 江苏科技大学 | For the bacillus subtilis of antagonism grey mould fruit rot of strawberry and its preparation of biocontrol agent and application |
| CN109593682A (en) * | 2018-12-29 | 2019-04-09 | 贵州大学 | Bacillus subtilis GU MT332 and its application |
| CN113151092A (en) * | 2021-04-25 | 2021-07-23 | 中国热带农业科学院热带生物技术研究所 | Bacillus new species with strawberry anthracnose inhibition activity and application thereof |
| CN113151092B (en) * | 2021-04-25 | 2023-02-28 | 中国热带农业科学院热带生物技术研究所 | Novel bacillus strain with strawberry anthracnose inhibition activity and application thereof |
| CN113583914A (en) * | 2021-08-27 | 2021-11-02 | 微生物肥料技术研究推广中心 | Microbial powder for preventing and treating strawberry root rot and preparation method thereof |
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