CN107384836A - A kind of Paenibacillus polymyxa inhibiting-bacteria preparation and preparation method thereof - Google Patents
A kind of Paenibacillus polymyxa inhibiting-bacteria preparation and preparation method thereof Download PDFInfo
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- CN107384836A CN107384836A CN201710765169.XA CN201710765169A CN107384836A CN 107384836 A CN107384836 A CN 107384836A CN 201710765169 A CN201710765169 A CN 201710765169A CN 107384836 A CN107384836 A CN 107384836A
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Abstract
The present invention relates to a kind of Paenibacillus polymyxa inhibiting-bacteria preparation and preparation method thereof.The inhibiting-bacteria preparation is that Paenibacillus polymyxa CGMCC NO.12410 ferment 36h in KL culture mediums, takes its fermentation supernatant or thalline methanolic extract as inhibiting-bacteria preparation.For CGMCC NO.12410 to be cultivated under the same terms in LB, PDB, Landy, GSC, NB, KL, TSC, the bacteriostasis of fermentation supernatant has larger difference.In culture medium of the same race, its fermentation supernatant bacteriostasis is significantly lower than the bacteriostasis of its thalline methanol extraction thing.The 36h it is demonstrated experimentally that bacterial strain CGMCC NO.12410 ferment in KL culture mediums, the antimicrobial spectrum of its thalline methanol extraction thing is wide, can suppress a variety of disease funguses.Meanwhile grey mold diseases prevention inoculation experiments are carried out on tamato fruit, the minimum bacteriostasis rate for showing thalline methanol extraction thing is 88.6%, and fungistatic effect is preferable.
Description
Technical field
The present invention relates to the biological control of plant disease, the biological control research belonged in plant protection subject is led with application
Domain.
Background technology
Anti- Zhiduo County of plant disease is prevented and treated using chemical agent in production at present.But the abuse of chemical agent draws
Played a series of ecological safety and food-safety problem, thus using beneficial microbe come control Occurrence and development of disease mutually than
Potentiality are had more down.
The biocontrol microorganisms species reported is various, wherein Paenibacillus polymyxa (Paenibacillus polymyxa), right
Plant has non-pathogenic, can not only produce the plant hormones such as auxin, the basic element of cell division and promote plant growth, can also produce
Raw polymyxins (Polymyxin), colistin (Colistin), Paenibacillin, Gavaserin and Saltavidin, Qiao
Sharp peptide rhzomorph (Jolipeptin), gatavalin (Gatavalin), circulin (Polypeptins), kill fusanin
Etc. (Fusaricidins) antibacterial substance suppresses the development of disease.
Chinese patent application 201610422377.5 (publication date on November 16th, 2016) discloses glues class gemma more one plant
Bacillus (Paenibacillus polymyxa) CGMCC NO.12410, have antibacterial broad spectrum activity, field control graw mold of tomato
Positive effect, than Sukeling preventive effect improve 57.3%.But found in putting into practice, when preparing biological control microbial inoculum, difference system
The product differentiation that Preparation Method obtains is notable, therefore, needs further to be studied for the preparation method of the microbial inoculum.
The content of the invention
In order to improve Paenibacillus polymyxa (Paenibacillus polymyxa) CGMCC NO.12410 bateriostatics
The extracted amount of matter and antibacterial ability, the invention provides a kind of extracting method of the antibacterial substance specifically for the bacterium, effectively
Ground solves above mentioned problem.
A kind of preparation method of Paenibacillus polymyxa inhibiting-bacteria preparation, it is by Paenibacillus polymyxa CGMCC NO.12410
Ferment 36h in KL culture mediums, takes its fermentation supernatant or thalline methanolic extract as inhibiting-bacteria preparation.
The invention also discloses the Paenibacillus polymyxa inhibiting-bacteria preparation as made from the above method.
The present invention further discloses described Paenibacillus polymyxa inhibiting-bacteria preparation in antagonism various plants disease fungus
Antibacterial substance application.Application particularly in graw mold of tomato is prevented and treated.
The present invention is in order to improve the extracted amount of the antibacterial substance of Paenibacillus polymyxa and antibacterial ability, the culture to it
Base and incubation time are selected.As a result after proving that bacterial strain CGMCC NO.12410 cultivate 3d in selected culture medium, culture medium
KL, PDB, Landy fermentation supernatant fungistatic effect are preferable, and bacteriostasis rate is more than 85%；Culture medium TSB, LB fermentation supernatant
There is no bacteriostatic activity；Culture medium KL, PDB, NB thalline methanol extraction thing fungistatic effect are preferable, and bacteriostasis rate is more than 80%；
Culture medium TSB and LB thalline methanol extraction thing do not have notable bacteriostatic activity, and bacteriostasis rate is below 15%.In KL culture mediums
In, after cultivating 36h, the bacteriostasis of bacterial strain CGMCC NO.12410 bacteria concentration, fermented liquid supernatant and thalline methanol extraction thing
Reach highest, be optimal incubation time.
Paenibacillus polymyxa (Paenibacillus polymyxa) CGMCC NO.12410 thalline methanol extraction thing energy
Enough suppress botrytis cinerea (Botrytis cinerea) growth, to other germs such as cucumber fusarium axysporum (Fusarium
Oxysporum), Monilinia fructicola (Monilia fructicola), rice blast fungus (Pyricularia oryzae) etc.
Also there is preferable antagonism.
Meanwhile bacterial strain CGMCC NO.12410 thalline methanol extraction things are subjected to grey mold diseases prevention inoculation in fact on tamato fruit
Test, fungistatic effect is obvious, and bacteriostasis rate reaches more than 88%.
Brief description of the drawings
Fig. 1 is influence of the different culture media to bacterial strain CGMCC NO.12410 cell concentration and bacteriostasis.
Fig. 2 is influence of the incubation time to bacterial strain CGMCC NO.12410 bacteria concentrations and bacteriostasis.
Fig. 3 is bacterial strain CGMCC NO.12410 thalline methanol extraction thing antimicrobial spectrum.(5% notable water of significant difference
Flat to be represented with a, b, c, cd, d, different letters represent significant difference.Wherein a and d differences are the most notable, and cd and d have more significant
Difference)
Fig. 4 is the methanol extraction thing of bacterial strain CGMCC NO.12410 thalline to graw mold of tomato control effect testing.In figure：Upper one
Behavior treatment group, next behavior control group
Specific embodiment
Embodiment 1：The selection of bacterial strain CGMCC NO.12410 culture mediums
(1) by bacterial strain CGMCC NO.12410 with 5% connect bacterium amount be respectively connected to NB, LB, KB, Landy, PDB, TSB,
In GSC culture mediums, often processing sets 3 repetitions, and 3d is cultivated under the conditions of 28 DEG C, 180r/min.
(2) detect its ODat600 value respectively with ultraviolet-uisible spectrophotometer and detect its bacteria concentration.
(3) take 40ml to be centrifuged with 12000rpm/min rotating speeds respectively the culture medium after culture 3d, take supernatant, thalline stays
With.Supernatant is filtered with 0.22 μm of bacterial filter.Supernatant after filter is added in PDA to mix with 20% amount and is inverted flat board, it is corresponding
Aseptic culture medium matches mixing PDA using equivalent and is inverted flat board as control, and the center of flat board is inoculated with a diameter of 9mm tomato gray mould
Bacteria cake, every group sets 3 repetitions.Flat board botrytis cinerea to be compareed covers with, investigation control and respective handling flat-plate bacterial colony diameter.
(4) mixing of thalline 4ml methanol solutions will be continued to employ, be extracted overnight under the conditions of 4 DEG C, with 12000rpm/min rotating speeds from
The heart, supernatant is taken, filtered with the small device of 0.22 μm of nylon membrane filtration.Organic filtrate is added to mix in PDA with 1% amount and is inverted flat board,
Mixing PDA is matched using equivalent with sterile methanol and is inverted flat board as control, a diameter of 9mm of center inoculation of flat board tomato ash
Mould cake, every group sets 3 repetitions.Flat board botrytis cinerea to be compareed covers with, and investigates all flat-plate bacterial colony diameters.
As a result bacterial strain CGMCC NO.12410 bacteria concentration highests in NB culture mediums are proved, OD at 600nm value is
1.66；Secondly it is GSC, Landy, KL, PDB culture medium, OD at 600nm value is more than 1.00；In TSB and LB culture mediums
In increases slowly, OD at 600nm value is below 0.35.Culture medium KL, Landy, PDB fermentation supernatant shows higher
Bacteriostatic activity, bacteriostasis rate are respectively 85.9%, 81.9%, 88.6%.Other culture mediums show weaker bacteriostatic activity or even not had
There is bacteriostatic activity.Culture medium NB, PDB, KL thalline methanolic extract shows higher bacteriostatic activity, and bacteriostasis rate is respectively
84.7%th, 85.2%, 80.5%.From the point of view of comprehensive fungistatic effect, PDB is beneficial to bacterial strain CGMCC NO.12410 suppressions with KL culture mediums
The generation (Fig. 1) of fungus matter.Present invention selection KL culture mediums do the selection of further culture period.
Embodiment 2：The selection of incubation time
(1) by bacterial strain CGMCC NO.12410 with (500ml tapers in the 5% KL culture mediums for meeting bacterium amount access 100ml
Bottle), cultivated under the conditions of 28 DEG C, 180r/min, a sample is taken per 12h, taken 2ml every time, terminate sampling after 48h.
(2) acquirement sample is detected into its ODat600 value with ultraviolet-uisible spectrophotometer and detects its bacteria concentration.
(3) 1ml samples will be taken to be centrifuged with 12000rpm/min rotating speeds, take supernatant, thalline is continued to employ.Supernatant is thin with 0.22 μm
Bacterium filter filters.
(4) mixing of thalline 1ml methanol solutions will be continued to employ, be extracted overnight under the conditions of 4 DEG C, with 12000rpm/min rotating speeds from
The heart, supernatant is taken, filtered with the small device of 0.22 μm of nylon membrane filtration.
(5) by a diameter of 9mm tomato gray mould pure culture biscuits involvng inoculation to the center of PDA plate, after cultivating 1~2 day, put down in PDA
Plate surrounding is separately added into 50 μ L above-mentioned (3), (4) treatment fluid away from 4 diameter 9mm holes are made a call at colony edge 12mm, and often processing sets 3
Individual repetition.Its antibacterial bandwidth is measured after 3 days, often processing sets 3 repetitions.Measure its antibacterial substance and intracellular or extracellular be present.
As a result after showing that bacterial strain CGMCC NO.12410 are seeded in KL culture mediums, bacteria concentration increases rapidly, is reached in 36h
To highest, ODat600 values are 1.32, and bacteria concentration reduces over time.The antibacterial bandwidth of supernatant is along with bacteria concentration
Growth, which has also risen, reaches up to 4mm in 36h, then rapid to decline.The antibacterial bandwidth of thalline methanol extraction thing is in 36h
Reach up to 6.2mm, antibacterial bandwidth tends towards stability afterwards.So final result shows that bacterial strain CGMCC NO.12410 train in KL
The optimal incubation time supported in base is 36h (Fig. 2).
Embodiment 3：Thalline methanol extraction thing
The thalline methanol extraction thing extracted in KL culture mediums is added in PDA to mix with 1% amount and is inverted flat board, respectively
Inoculated Rice sheath blight fungus, Monilinia fructicola, cucumber fusarium axysporum, Pyricularia oryzae, gaeumannomyces graminis, Sclerotinia sclerotiorum,
8 kinds of plant pathogenic fungis such as botrytis cinerea, cucumber fusarium axysporum, using methanol as control, investigate colony diameter.As a result show
Show that methanol extraction thing is best to the inhibition of Monilinia fructicola, botrytis cinerea, Sclerotinia sclerotiorum, inhibiting rate is respectively
97.7%th, 84.2%, 88.2%.It has certain fungistatic effect (Fig. 3) to other disease funguses simultaneously.
Embodiment 4：Indoor grey mold diseases prevention inoculation experiments
The consistent tomato of size maturity is chosen, diameter 9mm circular wound is done, injects 20 μ methanol extraction material conducts
Processing, control is used as using methanol.Diameter 9mm ash arrhizus bacteria bacteria cakes are connect in wound afterwards, add water moisturizing under the conditions of 25 DEG C, after 4 days
Investigate germ extension diameter.
As a result show that germ extension has 4cm or so on control group tamato fruit, and treatment group ash arrhizus bacteria does not expand substantially
Exhibition.The inhibition of methanol extraction material is obvious, inhibiting rate more than 88.6% (Fig. 4).
Claims (4)
1. a kind of preparation method of Paenibacillus polymyxa inhibiting-bacteria preparation, it is characterised in that by Paenibacillus polymyxa CGMCC
NO.12410 ferments 36h in KL culture mediums, takes its fermentation supernatant or thalline methanolic extract as inhibiting-bacteria preparation.
2. a kind of Paenibacillus polymyxa inhibiting-bacteria preparation, it is characterised in that be made by following methods：Paenibacillus polymyxa
CGMCC NO.12410 ferment 36h in KL culture mediums, take its fermentation supernatant or thalline methanolic extract as inhibiting-bacteria preparation.
3. Paenibacillus polymyxa inhibiting-bacteria preparation described in claim 2 is in the antibacterial substance of antagonism various plants disease fungus
Using.
4. application of the Paenibacillus polymyxa inhibiting-bacteria preparation in graw mold of tomato is prevented and treated described in claim 2.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110938570A (en) * | 2019-12-13 | 2020-03-31 | 北京农业生物技术研究中心 | Paenibacillus polymyxa Lla-02 and application thereof |
| CN114874931A (en) * | 2021-12-22 | 2022-08-09 | 河北省科学院生物研究所 | Compound microbial agent and preparation method and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110938570A (en) * | 2019-12-13 | 2020-03-31 | 北京农业生物技术研究中心 | Paenibacillus polymyxa Lla-02 and application thereof |
| CN110938570B (en) * | 2019-12-13 | 2021-03-05 | 北京农业生物技术研究中心 | Paenibacillus polymyxa Lla-02 and application thereof |
| CN114874931A (en) * | 2021-12-22 | 2022-08-09 | 河北省科学院生物研究所 | Compound microbial agent and preparation method and application thereof |
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