CN103937699A - Bacillus cereus bacterial strain and applications thereof - Google Patents
Bacillus cereus bacterial strain and applications thereof Download PDFInfo
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- CN103937699A CN103937699A CN201410061856.XA CN201410061856A CN103937699A CN 103937699 A CN103937699 A CN 103937699A CN 201410061856 A CN201410061856 A CN 201410061856A CN 103937699 A CN103937699 A CN 103937699A
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- bacillus cereus
- bacterial strain
- bacilluscereuszy
- cereus bacterial
- botrytis cinerea
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Abstract
The invention discloses a bacillus cereus bacterial strain (Bacilluscereuszy-1) and applications thereof. The bacillus cereus bacterial strain (Bacilluscereuszy-1) is preserved in China Center for Type Culture Collection (Wuhan university, Wuhan, China), and preservation number is No.M2014007. Germination inhibition rate of a 2-fold diluent of a fermentation aseptic filtrate of Bacilluscereuszy-1 on botrytis cinerea spores is 95.2%, and inhibition rate of a 10-fold diluent of the fermentation aseptic filtrate of Bacilluscereuszy-1 on mycelia growth is higher than that of a 40% pyrimethanil suspension, so that application prospect of the bacillus cereus bacterial strain (Bacilluscereuszy-1) in agriculture is promising. Technical route of the bacillus cereus bacterial strain (Bacilluscereuszy-1) is reasonable, reliable pilotscale experiment data and design consideration are provided for large-scale industrialized production; and establishment of foundation is provided for development of related biological products and field promotion and application.
Description
Technical field
The invention belongs to microbial technology field, relate to the application of a kind of bacterium in biological and ecological methods to prevent plant disease, pests, and erosion field, particularly the purposes of bacillus cereus bacterial strain aspect control grey mould fruit rot of strawberry.
Background technology
Strawberry nutrition is abundant, have the title of " fruit queen ", but because it organizes tender and lovely succulence, pericarp is very thin, thereby in gathering and storing and transporting, is vulnerable to damage and microbial infection causes it to rot.Wherein by Botrytis cinerea (
botrytis cinereapers ex Fr) gray mold causing is one of topmost disease in strawberry production, can make Yield of Strawberry loss reach more than 50% when serious, and this has directly affected the producer's interests, also limits further developing of Strawberry industry.For a long time, control the effective means of grey mould fruit rot of strawberry and remain use chemical bactericide, yet along with appearance and the enhancing of people to environmental consciousness of the problems such as resistance and pesticide residue, so the biological control of safety, low toxicity becomes the focus of research.Biological control is to carry out in the world the first-selected prophylactico-therapeutic measures of control of plant disease at present, this, screening beneficial microorganism separated from occurring in nature for the antagonism of pathogenic micro-organism, the inhibiting method of preventing and treating, has efficient, nontoxic, noresidue and the feature such as low-cost.From the research of existing biocontrol of plant disease, report, as the wood in biotechnological formulation is mould, in the control of gray mold, bring into play significant role with yeast.
Summary of the invention
A kind of bacillus cereus that provides is provided, makes effect and the chemical agent effect no significant difference of this bacterium control grey mould fruit rot of strawberry, and there is higher eco-friendly feature.
For achieving the above object, the invention provides a kind of bacterium that separation obtains from soil, called after bacterial strain zy-1, this bacterium is bacterium colony subcircular on NA substratum, and canescence is opaque, the large 3-7 mm of bacterium colony, the irregular expansion shape that is in edge, surface irregularity particulate state, like ground-glass appearance or wax-like, is not secreted pigment; For gram positive bacterium, shaft-like, 1.0～1.3 * 3.0～5.0 μ m, chaining, mobility, gemma is oval, and end is raw or inferior end is raw, and gemma expands not obvious; Can tolerate the cultivation of 7% NaCl, catalase is positive, glucose fermentation is positive, Starch Hydrolysis is positive, nitrate reduction is positive, gelatine liquefication is positive, positive, the semi-solid puncture test of maltose fermentation is positive, casein hydrolysis positive, VP negative, Citrate trianion are utilized negative, indole test is negative, sucrose fermentation is negative, lactose fermentation is negative, N.F,USP MANNITOL hydrolysis feminine gender.In addition, through 16SrDNA, analyze, the bacterial strain high with bacterial strain zy-1 homology is genus bacillus, in conjunction with physiological and biochemical property, identifies that it is bacillus cereus.
Bacillus cereus of the present invention on January 6th, 2014 be deposited in Chinese Typical Representative culture collection center (address: Wuhan, China Wuhan University), preserving number: CCTCC NO:M 2014007, name be called: bacillus cereus zy-1 (
bacillus cereuszy-1).
The bacteria-free filtrate of bacillus cereus zy-1 of the present invention has significant inhibition to the spore-germination of Botrytis cinerea bacterium, compared with the control, the percentage of germination that 2 times of diluents of bacterial strain zy-1 bacteria-free filtrate suppress spore is 95.2%, in addition, the inhibition to mycelial growth of 10 times of diluents of bacteria-free filtrate of zy-1 is better than 800 times of diluents of 40% phonetic mould amine suspension agent.Show that bacillus cereus of the present invention has remarkable inhibition to the spore of Botrytis cinerea bacterium and mycelia.
The bacteria-free filtrate of bacillus cereus zy-1 of the present invention shows the measurement result of botrytis cinerea excised leaf activity, with the strawberry excised leaf that zy-1 processes, has no morbidity, compared with the control, can significantly reduce the state of an illness and occur.
As can be known from the above technical solutions: bacillus cereus of the present invention (
bacillus cereuszy-1) from the horse's mouth, the wide accommodation to natural condition such as temperature, pH is strong to the tolerance of poor environment, can maintain the eubiosis, can adapt to the disease ecosystem, easily cultivate and preserve, suppress the effective of Botrytis cinerea germ, agriculturally have very large application prospect.This technological line is reasonable, can be large-scale industrial production reliable pilot scale data and design considerations are provided, realize liquid fermenting suitability for industrialized production bacillus cereus (
bacillus cereuszy-1), for exploitation associated biomolecule preparation and land for growing field crops, promote and lay the foundation.
Accompanying drawing explanation
Fig. 1 is the agarose gel electrophoresis figure of biocontrol microorganisms zy-1 bacterial strain 16SrDNA amplified fragments.M represents DNA molecular amount; 1 represents biocontrol microorganisms zy-1 sample.
Fig. 2 is that 2 times of diluents processing of bacillus cereus zy-1 bacteria-free filtrate (left figure) affect botrytis cinerea spore-germination.
10 times of diluents of Fig. 3 bacillus cereus zy-1 bacteria-free filtrate are processed (right figure) to be affected ash arrhizus bacteria mycelial growth.
The in vitro leaf section of Fig. 4 is measured the prevention effect of bacillus cereus zy-1 bacteria-free filtrate (right figure) to grey mould fruit rot of strawberry.
Embodiment
Be below specific embodiments of the invention, technical scheme of the present invention is done to further description, but protection scope of the present invention is not limited to these embodiment.Every do not deviate from the change of the present invention design or be equal to substitute include within protection scope of the present invention.
embodiment 1: the separation of bacillus cereus zy-1 of the present invention
The isolation and purification of Antagonistic Fungi: 1. take the 10 g wheatland soil samples of picking up from Zaoyang City, Hubei Province, 2. be positioned over the middle concussion suspension of sodium hexametaphosphate solution (dispersion agent) 5 min of 200 mL, 3. under the state suspending, draw in 0.1% the water agar that 20 mL add 80 mL, concussion suspension 5 min, 4., under the state suspending in stirring, draw the suspension of 0.3 mL, 5. the suspension of 0.3 mL is pressed to 10 times of gradient dilutions, be respectively 10
-1, 10
-2with 10
-3, 6. gradient dilution liquid is placed in respectively to baking oven 30 min of 80 ℃, after taking out after cooling, 7. being coated with 3 flat boards (3 flat boards of each weaker concn), is 1/10 TSA selective medium, and 8. all flat boards are placed in 22 ℃ of cultivations, after 2-3 d, select single bacterium colony, numbering is preserved.In plate, face-off is cultivated and is detected its restraining effect to Botrytis cinerea germ, selects to form the bacterial strain of antibacterial band, called after bacterial strain zy-1.
embodiment 2: the mensuration of bacillus cereus Physiology and biochemistry of the present invention and 16S rDNA sequence
Physiology and biochemistry detected result: zy-1, on NA plate culture medium, cultivates 36 h for 28 ℃, bacterium colony subcircular, and canescence, opaque, bacterium colony is large, 3-7 mm, the irregular diffusion type that is in edge, surface irregularity particulate state, like obscure glass or wax-like, is not secreted pigment; For gram positive bacterium, shaft-like, 1.0～1.3 * 3.0～5.0 μ m, chaining, mobility, gemma is oval, and end is raw or inferior end is raw, and gemma expands not obvious; Can tolerate the cultivation of 7%NaCl, catalase is positive, glucose fermentation is positive, Starch Hydrolysis is positive, nitrate reduction is positive, gelatine liquefication is positive, positive, the semi-solid puncture test of maltose fermentation is positive, casein hydrolysis positive, VP negative, Citrate trianion are utilized negative, indole test is negative, sucrose fermentation is negative, lactose fermentation is negative, N.F,USP MANNITOL hydrolysis feminine gender.With reference to < < uncle outstanding Bacteria Identification handbook > > (the 8th edition), < < common bacteria system identification handbook > > and < < bacillus > >, be initially identified as bacillus.
The mensuration of 16SrDNA sequence: in beef extract-peptone liquid nutrient medium, 180 28 ℃ of rpm cultivate 24 h, centrifugal collection thalline by zy-1 inoculation.Adopt the raw work Ezup pillar genome DNA extraction test kit in Shanghai (bacterium) to extract genome.The genome extracting of take is template, adopts forward primer 27bf 5 '-AGAGTTTGATCCTGGCTCAG-3 '; 1492br 5 '-GGTTACCTTGTTACGACTT-3 ' carries out pcr amplification to the 16SrDNA gene of bacterial strain.PCR condition is: 94 ℃ of 3 min; 94 ℃ of 45s, 54 ℃ of 30s, 72 ℃ of 1 min 30sec, 35 circulations; 72 ℃ are extended 5 min, and 4 ℃ stop.PCR product is served the order-checking of extra large handsome Bioisystech Co., Ltd, and length is 1466 bp(Fig. 1).Sequencing result in Ebiocloud with
bacillus cereussimilarity is 99.7%.
In conjunction with colonial morphology, Physiology and biochemistry and Molecular Identification, by this bacterial strain be designated as bacillus cereus (
bacillus cereuszy-1).
embodiment 3: the test of bacillus cereus of the present invention to the spore germination of Botrytis cinerea germ and mycelial growth
1.the preparation of bacillus cereus culturing filtrate: bacterial strain zy-1 is after 28 ℃ of shaking culture 3 d, and fermented liquid centrifugal (12000 r/min, 20 min), gets biofilter for supernatant liquor (0.22 μ m) degerming, acquisition without fermented liquid.
Adopt depression slide to send out method and carry out spore germination test.Botrytis cinerea germ is connected on PDA flat board, cultivates 5d at 23 ℃, after it produces spore, with sterilized water, spore is eluted to make concentration be 10
5individual/ml spore suspension, draws 50 μ L and joins in 500 μ L centrifuge tubes without fermented liquid, then draws the spore suspension 50 μ L that prepare, and makes liquid and spore suspension balanced mix even.Draw above-mentioned mixed solution 40 μ L and drip on depression slide, then frame is put in the culture dish with shallow seated groundwater, adds a cover moisturizing and is incubated in the incubator of 23 ℃.Every processing repeats 4 times and measures.Blank is done in processing with NA substratum.Test-results is in Table 1.As seen from Table 1,2 times of diluents of bacillus cereus zy-1 bacteria-free filtrate suppress spore germination rates 95.2%, along with the increase of extension rate is reducing the inhibiting rate of spore-germination.
table 1 bacillus cereus zy-1 bacteria-free filtrate affects Botrytis cinerea germ spore-germination
3.adopt the test of mycelial growth rate assay method.Botrytis cinerea germ is connected on PDA flat board, cultivates 3d at 23 ℃, adopt, respectively by ready without fermented liquid 1000 μ L and 200 μ L, equivalent adds in PDA substratum, pour into (Ф 6cm) in sterilizing culture dish, preculture good for the same circumference of examination bacterial classification colony edge on punch tool (ф 5mm) beat and get bacterium cake, mycelia faces down and is inoculated in culture dish central authorities, every processing repeats for 3 times, is placed in dark culturing in 23 ℃ of incubators, take NA substratum as contrast.When contrasting bacterium colony size and approach culture dish edge, measure and respectively process colony diameter (mm), compare calculate bacterium colony expansion growth inhibition ratio with control treatment, test-results is in Table 2.As seen from Table 2, bacillus cereus zy-1 bacteria-free filtrate (10 times of diluents) inhibition is similar or slightly excellent compared with conventional chemical medicament, and safe (see figure 3).
Bacterium colony expansion growth inhibition ratio calculation formula:
the inhibition of table 2 bacillus cereus zy-1 bacteria-free filtrate to Botrytis cinerea germ
embodiment 4: the mensuration of bacillus cereus of the present invention to botrytis cinerea excised leaf activity
Adopt Leaf method to measure the activity of bacillus cereus zy-1: to get the From Strawberry Leaves of same leaf age, with sterilized water, repeatedly rinse 3 times, after indoor drying, spray zy-1 without fermented liquid, after bacterium liquid is naturally dry, get Botrytis cinerea bacterium bacterium cake, mycelia faces down and is inoculated in From Strawberry Leaves central authorities.Be placed in the large culture dish of 18cm of water agar, be placed under 23 ℃ of scattered lights and cultivate and observe, the NA substratum same treatment of take is contrast.Every processing 3 slice, thin pieces repeat.Test-results is shown in Fig. 4.Excised leaf toxicity measurement result shows, spray zy-1 and have no morbidity without fermented liquid, and contrast has obvious scab.
Claims (2)
- A bacillus cereus zy-1 ( bacillus cereuszy-1), the preserving number that it is characterized in that described bacterial classification is CCTCC NO:M 2014007.
- 2. bacillus cereus zy-1 as claimed in claim 1 is in the purposes suppressing aspect grey mould fruit rot of strawberry.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107646872A (en) * | 2017-09-18 | 2018-02-02 | 石河子大学 | Bacillus cereus prevents and treats cotton disease and its somatotrophic application |
| CN110616165A (en) * | 2019-05-14 | 2019-12-27 | 陕西省微生物研究所 | Bacillus cereus MA23, microbial inoculum, preparation method and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101289649A (en) * | 2007-04-16 | 2008-10-22 | 山东理工大学 | Tomato gray mold antagonistic strain B-04-glu |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101289649A (en) * | 2007-04-16 | 2008-10-22 | 山东理工大学 | Tomato gray mold antagonistic strain B-04-glu |
Non-Patent Citations (1)
| Title |
|---|
| 李桂霞等: "番茄灰霉病高效拮抗菌株鉴定及抗菌活性研究", 《山东农业科学》, 28 February 2007 (2007-02-28), pages 69 - 72 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107646872A (en) * | 2017-09-18 | 2018-02-02 | 石河子大学 | Bacillus cereus prevents and treats cotton disease and its somatotrophic application |
| CN107646872B (en) * | 2017-09-18 | 2020-08-04 | 石河子大学 | Application of bacillus cereus in preventing and treating cotton diseases and promoting growth of cotton diseases |
| CN110616165A (en) * | 2019-05-14 | 2019-12-27 | 陕西省微生物研究所 | Bacillus cereus MA23, microbial inoculum, preparation method and application |
| CN110616165B (en) * | 2019-05-14 | 2022-06-28 | 陕西省微生物研究所 | Bacillus cereus MA23, microbial inoculum, preparation method and application |
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